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Sample records for platelets induces rejection

  1. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  2. [Protein kinase C activation induces platelet apoptosis].

    PubMed

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  3. Identification of platelet-derived growth factor A and B chains in human renal vascular rejection.

    PubMed Central

    Alpers, C. E.; Davis, C. L.; Barr, D.; Marsh, C. L.; Hudkins, K. L.

    1996-01-01

    Platelet-derived growth factor (PDGF) exists as a dimer composed of two homologous but distinct peptides termed PDGF-A and -B chains, and may exist as AA, AB, and BB isoforms. The PDGF-B chain has been implicated as a mediator of renal vascular rejection by virtue of up-regulated expression of its receptor, PDGF beta-receptor, in affected arteries. A role for PDGF-A chain in mediating intimal proliferation has been suggested in human atherosclerosis (Rekhter MD, Gordon D: Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques? Circ Res 1994, 75:410), but no studies of this molecule in human renal allograft injury have been reported to date. We used two polyclonal antisera to detect expression of PDGF-A chain and one monoclonal antibody to detect PDGF-B chain by immunohistochemistry in fixed, paraffin-embedded tissue from 1) normal adult kidneys, 2) a series of renal transplant biopsies chosen to emphasize features of vascular rejection, and 3) allograft nephrectomies. Immunohistochemistry was correlated with in situ hybridization on adjacent, formalin fixed tissue sections from nephrectomies utilizing riboprobes made from PDGF-A and -B chain cDNA. PDGF-A chain is widely expressed by medial smooth muscle cells of normal and rejecting renal arterial vessels of all sizes by immunohistochemistry and in situ hybridization. PDGF-A chain is also expressed by a population of smooth muscle cells (shown by double immunolabeling with an antibody to alpha-smooth muscle actin) comprising the intima in chronic vascular rejection. In arteries demonstrating acute rejection, up-regulated expression of PDGF-A chain by endothelial cells was detected by both immunohistochemistry and in situ hybridization. In contrast, PDGF-B chain was identified principally in infiltrating monocytes within the rejecting arteries, similar to its localization in infiltrating monocytes in human atherosclerosis. Although less

  4. CD8+ T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia

    PubMed Central

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP. PMID:27321376

  5. CD8(+) T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia.

    PubMed

    Qiu, Jihua; Liu, Xuena; Li, Xiaoqing; Zhang, Xu; Han, Panpan; Zhou, Hai; Shao, Linlin; Hou, Yu; Min, Yanan; Kong, Zhangyuan; Wang, Yawen; Wei, Yu; Liu, Xinguang; Ni, Heyu; Peng, Jun; Hou, Ming

    2016-01-01

    In addition to antiplatelet autoantibodies, CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8(+) T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8(+) T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8(+) T cells than those without cytotoxicity and controls. In vitro, CD8(+) T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8(+) splenocytes were used. Platelets co-cultured with these CD8(+) splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8(+) splenocytes. These findings suggest that CD8(+) T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP. PMID:27321376

  6. Platelets

    MedlinePlus

    ... are related to immunity and fighting infection. Platelet Production Platelets are produced in the bone marrow, the ... platelet destruction and also decreased bone marrow platelet production. These problems are caused by autoantibodies. Antibodies are ...

  7. Effects of Suilysin on Streptococcus suis-Induced Platelet Aggregation

    PubMed Central

    Zhang, Shengwei; Wang, Junping; Chen, Shaolong; Yin, Jiye; Pan, Zhiyuan; Liu, Keke; Li, Lin; Zheng, Yuling; Yuan, Yuan; Jiang, Yongqiang

    2016-01-01

    Blood platelets play important roles during pathological thrombocytopenia in streptococcal toxic shock syndrome (STSS). Streptococcus suis (S. suis) an emerging human pathogen, can cause STSS similarly to S. pyogenes. However, S. suis interactions with platelets are poorly understood. Here, we found that suilysin (SLY), different from other bacterial cholesterol-dependent cytolysins (CDCs), was the sole stimulus that induced platelet aggregation. Furthermore, the inside-out activation of GPIIb/IIIa of platelets mediated SLY-induced platelet aggregation. This process was triggered by Ca2+ influx that depend on the pore forming on platelets by SLY. Additionally, although SLY induced α-granule release occurred via the MLCK-dependent pathway, PLC-β-IP3/DAG-MLCK and Rho-ROCK-MLCK signaling were not involved in SLY-induced platelet aggregation. Interestingly, the pore dependent Ca2+ influx was also found to participate in the induction of platelet aggregation with pneumolysin (PLY) and streptolysin O (SLO), two other CDCs. It is possible that the CDC-mediated platelet aggregation we observed in S. suis is a similar response mechanism to that used by a wide range of bacteria. These findings might lead to the discovery of potential therapeutic targets for S. suis-associated STSS. PMID:27800304

  8. Adenovirus type 3 induces platelet activation in vitro.

    PubMed

    Jin, Ying-Yu; Yu, Xiu-Nan; Qu, Zhang-Yi; Zhang, Ai-Ai; Xing, Yu-Ling; Jiang, Li-Xin; Shang, Lei; Wang, Ying-Chen

    2014-01-01

    In the present study, we aimed to investigate platelet activation induced by adenovirus type 3 (HAdV3) in vitro. Platelet-rich plasma (PRP) or whole blood was incubated with or without HAdV at various concentrations. Platelet aggregation, platelet counting, fibrinogen and expression of platelet membrane antigens (CD41a and CD62P) were determined following incubation with HAdV for different periods of time. The results demonstrated that HAdV at the concentrations of 109-1011 vp/ml enhanced adenosine diphosphate (ADP) or ristocetin-induced platelet aggregation, however did not alter the platelet count. Infection with HAdVs also reduced fibrinogen level. P-selectin and CD41a appeared rapidly on the surface after platelets were incubated with HAdVs in vitro for 30 min. In conclusion, HAdVs may induce activation of platelets and lead to a pre-thrombotic state of peripheral blood. This finding may aid in the development of measures to prevent severe HAdV infection.

  9. Platelet membranes induce airway smooth muscle cell proliferation.

    PubMed

    Svensson Holm, Ann-Charlotte B; Bengtsson, Torbjörn; Grenegård, Magnus; Lindström, Eva G

    2011-01-01

    The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localized in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS assay and the results confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GP-ASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

  10. Platelet-leukocyte interaction in adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Shinoda, H.

    1991-01-01

    1. Platelet-activating factor (PAF, 10 nM) did not induce platelet adhesion to endothelial cells cultured in monolayer but it induced their adhesion to protein-coated plastic. However, PAF induced a marked platelet adhesion to endothelial cells when polymorphonuclear leukocytes (PMNs) were present. Lyso-PAF had no effect. 2. Phase-contrast microscopic examination showed that single platelets rather than their aggregates adhered to the endothelial cell surface around aggregating and adhering PMNs. 3. Significant platelet adhesion was induced by PAF at concentrations higher that 0.01 nM with the maximal response at 10 nM. Platelet adhesion occurred within minutes after PAF addition, reaching a maximum approximately after 30 min. Platelet adhesion also occurred significantly at a PMN:platelet ratio of 1:800, and linearly up to 1:50. 4. The PAF-induced platelet adhesion was suppressed by three structurally unrelated PAF antagonists, WEB 2086, ONO 6240 and BN 52021, in a concentration-dependent manner. 5. PAF also increased PMN adhesion to endothelial cell monolayers, which was further augmented by the presence of platelets. 6. The present study demonstrates that PAF induces platelet adhesion to endothelial cells in vitro when PMNs are present and that there is a close interaction between platelets and PMNs in their adhesion to endothelial cells. The present study further suggests that PMNs could play a central role in platelet adhesion to vascular endothlium in certain pathological conditions. Images Figure 2 PMID:1884095

  11. Point-of-care platelet function tests: detection of platelet inhibition induced by nonopioid analgesic drugs.

    PubMed

    Scharbert, Gisela; Gebhardt, Kristina; Sow, Zacharia; Duris, Monika; Deusch, Engelbert; Kozek-Langenecker, Sibylle

    2007-12-01

    Detection of platelet inhibition is of clinical relevance in the preinterventional risk-benefit assessment in chronic low-back-pain patients scheduled for invasive pain therapy. We evaluated the sensitivity of various point-of-care platelet function tests for the detection of platelet inhibition induced by nonopioid analgesic drugs. After Institutional Review Board approval and informed consent, citrated whole blood from 40 patients with chronic unspecific low back pain was investigated before and 30 min after intravenous infusion of the study medication consisting of diclofenac 75 mg (plus orphenadrin 30 mg; Neodolpasse; Fresenius Kabi Austria GmbH, Austria), parecoxib 40 mg (Dynastat; Pharmacia Europe EEIG, UK), paracetamol 1 g (Perfalgan; Bieffe Medital S.P.A., Italy), or normal saline in a randomized, cross-over, double-blinded, placebo-controlled study. Platelet function was assessed using the PFA-100 platelet function analyzer and thromboelastometry, as well as impedance aggregometry (in the last 17 patients recruited after it became commercially available). Sensitivity for detecting diclofenac-induced platelet inhibition was 85% for the PFA-100 using epinephrine as agonist and 94% for arachidonic acid-induced impedance aggregometry. ADP-induced platelet function tests, as well as cytochalasin D-modified thromboelastometry were unreliable. All tests had a low incidence of false-positive test results after normal saline. Paracetamol and parecoxib had no significant platelet inhibiting effect. The PFA-100 using epinephrine as agonist and arachidonic acid-induced impedance aggregometry are recommended for the detection of cyclooxygenase-I-inhibiting effects of nonsteroidal anti-inflammatory drugs such as diclofenac. Our findings confirm that a single rescue dose of paracetamol and parecoxib has no antiplatelet effect. PMID:17982319

  12. Hydrogen peroxide is involved in collagen-induced platelet activation.

    PubMed

    Pignatelli, P; Pulcinelli, F M; Lenti, L; Gazzaniga, P P; Violi, F

    1998-01-15

    In this study, we investigated whether (1) collagen-induced platelet aggregation is associated with a burst of H2O2, (2) this oxidant species is involved in the activation of platelets, and (3) the pathways of platelet activation are stimulated by H2O2. Collagen-induced platelet aggregation was associated with production of H2O2, which was abolished by catalase, an enzyme that destroys H2O2. H2O2 production was not observed when ADP or thrombin were used as agonists. Catalase inhibited dose-dependently thromboxane A2 production, release of arachidonic acid from platelet membrane, and Inositol 1,4,5P3 (IP3) formation. In aspirin-treated platelets stimulated with high concentrations of collagen, catalase inhibited platelet aggregation, calcium mobilization, and IP3 production. This study suggests that collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating the arachidonic acid metabolism and phospholipase C pathway.

  13. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Nezu, A.; Shinoda, H.; Minami, M.

    1996-01-01

    1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified. PMID:8789382

  14. Activated human platelets induce factor XIIa-mediated contact activation.

    PubMed

    Bäck, Jennie; Sanchez, Javier; Elgue, Graciela; Ekdahl, Kristina Nilsson; Nilsson, Bo

    2010-01-01

    Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation. PMID:19878657

  15. Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation.

    PubMed

    Berger, Markus; Reck, José; Terra, Renata M S; Beys da Silva, Walter O; Santi, Lucélia; Pinto, Antônio F M; Vainstein, Marilene H; Termignoni, Carlos; Guimarães, Jorge A

    2010-10-01

    The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.

  16. von Willebrand factor binds to platelets and induces aggregation in platelet-type but not type IIB von Willebrand disease.

    PubMed Central

    Miller, J L; Kupinski, J M; Castella, A; Ruggeri, Z M

    1983-01-01

    Platelet-type von Willebrand disease (vWD) and pseudo-vWD are two recently described intrinsic platelet defects characterized by enhanced ristocetin-induced agglutination in platelet-rich plasma. A similar finding is also typical of type IIB vWD, where it has been related to a von Willebrand factor (vWF) rather than a platelet abnormality. Platelet aggregation induced by unmodified human vWF in the absence of other stimuli has been reported in pseudo-vWD. In this study we demonstrate that vWF induces aggregation in platelet-type but not type IIB vWD. Aggregation is observed when normal plasma cryoprecipitate or purified vWF are added to platelet-rich plasma. Cryoprecipitate also aggregates washed platelets, although at higher concentrations than required for platelet-rich plasma. Purified vWF, however, induces significant aggregation of washed platelets only when plasma is added. EDTA inhibits vWF-induced aggregation. Its effect can be overcome by calcium but much less effectively by magnesium ions. Unstimulated platelets in platelet-rich plasma from patients with platelet-type but not type IIB vWD bind 125I-vWF in a specific and saturable manner. All different sized multimers of vWF become associated with platelets. Both aggregation and binding exhibit a similar vWF concentration dependence, suggesting that a correlation exists between these two events. Removal of ADP by appropriate consuming systems is without effect upon such binding or upon vWF-induced aggregation. Thrombin-induced 125I-vWF binding to washed platelets is normal in platelet-type as well as type IIB vWD. These results demonstrate that a specific binding site for unmodified human vWF is exposed on unstimulated platelets in platelet-type vWD. The relatively high vWF concentrations required for aggregation and binding may explain the lack of significant in vivo aggregation and thrombocytopenia in these patients. Moreover, these studies provide additional evidence that platelet-type and type IIB v

  17. Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner.

    PubMed

    Watson, Ben R; White, Nathan A; Taylor, Kirk A; Howes, Joanna-Marie; Malcor, Jean-Daniel M; Bihan, Dominique; Sage, Stewart O; Farndale, Richard W; Pugh, Nicholas

    2016-01-01

    Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.

  18. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding. PMID:25994029

  19. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

  20. Platelets, acting in part via P-selectin, mediate cytomegalovirus-induced microvascular dysfunction.

    PubMed

    Khoretonenko, Mikhail V; Brunson, Jerry L; Senchenkov, Evgeny; Leskov, Igor L; Marks, Christian R; Stokes, Karen Y

    2014-12-15

    Cytomegalovirus (CMV) infects a majority of the population worldwide. It has been implicated in cardiovascular disease, induces microvascular dysfunction, and synergizes with hypercholesterolemia to promote leukocyte and platelet recruitment in venules. Although platelets and platelet-associated P-selectin contribute to cardiovascular disease inflammation, their role in CMV-induced vascular responses is unknown. We assessed the role of platelets in CMV-induced microvascular dysfunction by depleting platelets and developing bone marrow chimeric mice deficient in platelet P-selectin. Wild-type and chimeric mice received mock or murine (m)CMV intraperitoneally. Five weeks later, some mice were switched to a high-cholesterol diet (HC) to investigate the synergism between mCMV and HC. Arteriolar vasodilation and recruitment of leukocytes and donor platelets in venules were measured at 11wk. mCMV with or without HC caused significant endothelial dysfunction in arterioles. Platelet depletion restored normal vasodilation in mCMV-HC but not mCMV-ND mice, whereas protection was seen in both groups for platelet P-selectin chimeras. Only mCMV + HC elevated leukocyte and platelet recruitment in venules. Leukocyte adhesion was reduced to mock levels by acute platelet depletion but was only partially decreased in platelet P-selectin chimeras. Platelets from mCMV-HC mice and, to a lesser extent, mCMV-ND but not mock-HC mice showed significant adhesion in mCMV-HC recipients. Our findings implicate a role for platelets, acting through P-selectin, in CMV-induced arteriolar dysfunction and suggest that the addition of HC leads to a platelet-dependent, inflammatory infiltrate that is only partly platelet P-selectin dependent. CMV appeared to have a stronger activating influence than HC on platelets and may represent an additional therapeutic target in vulnerable patients.

  1. In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

    PubMed

    Fent, János; Bihari, Péter; Vippola, Minnamari; Sarlin, Essi; Lakatos, Susan

    2015-08-01

    Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood.

  2. Platelets are relevant mediators of renal injury induced by primary endothelial lesions.

    PubMed

    Schwarzenberger, Claudia; Sradnick, Jan; Lerea, Kenneth M; Goligorsky, Michael S; Nieswandt, Bernhard; Hugo, Christian P M; Hohenstein, Bernd

    2015-06-01

    Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown.

  3. Platelets are relevant mediators of renal injury induced by primary endothelial lesions

    PubMed Central

    Schwarzenberger, Claudia; Sradnick, Jan; Lerea, Kenneth M.; Goligorsky, Michael S.; Nieswandt, Bernhard; Hugo, Christian P. M.

    2015-01-01

    Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown. PMID:25834071

  4. Platelet size and density affect shear-induced thrombus formation in tortuous arterioles

    NASA Astrophysics Data System (ADS)

    Chesnutt, Jennifer K. W.; Han, Hai-Chao

    2013-10-01

    Thrombosis accounts for 80% of deaths in patients with diabetes mellitus. Diabetic patients demonstrate tortuous microvessels and larger than normal platelets. Large platelets are associated with increased platelet activation and thrombosis, but the physical effects of large platelets in the microscale processes of thrombus formation are not clear. Therefore, the objective of this study was to determine the physical effects of mean platelet volume (MPV), mean platelet density (MPD) and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A computational model of the transport, shear-induced activation, collision, adhesion and aggregation of individual platelets was used to simulate platelet interactions and thrombus formation in tortuous arterioles. Our results showed that an increase in MPV resulted in a larger number of activated platelets, though MPD and level of tortuosity made little difference on platelet activation. Platelets with normal MPD yielded the lowest amount of mural thrombus. With platelets of normal MPD, the amount of mural thrombus decreased with increasing level of tortuosity but did not have a simple monotonic relationship with MPV. The physical mechanisms associated with MPV, MPD and arteriole tortuosity play important roles in platelet activation and thrombus formation.

  5. Peer Rejection Cues Induce Cardiac Slowing after Transition into Adolescence

    ERIC Educational Resources Information Center

    Gunther Moor, Bregtje; Bos, Marieke G. N.; Crone, Eveline A.; van der Molen, Maurits W.

    2014-01-01

    The present study examined developmental and gender differences in sensitivity to peer rejection across the transition into adolescence by examining beat-by-beat heart rate responses. Children between the ages of 8 and 14 years were presented with unfamiliar faces of age-matched peers and were asked to predict whether they would be liked by the…

  6. Agonist-induced platelet procoagulant activity requires shear and a Rac1-dependent signaling mechanism

    PubMed Central

    Delaney, Michael Keegan; Liu, Junling; Kim, Kyungho; Shen, Bo; Stojanovic-Terpo, Aleksandra; Zheng, Yi; Cho, Jaehyung

    2014-01-01

    Activated platelets facilitate blood coagulation by exposing phosphatidylserine (PS) and releasing microvesicles (MVs). However, the potent physiological agonists thrombin and collagen poorly induce PS exposure when a single agonist is used. To obtain a greater procoagulant response, thrombin is commonly used in combination with glycoprotein VI agonists. However, even under these conditions, only a percentage of platelets express procoagulant activity. To date, it remains unclear why platelets poorly expose PS even when stimulated with multiple agonists and what the signaling pathways are of soluble agonist-induced platelet procoagulant activity. Here we show that physiological levels of shear present in blood significantly enhance agonist-induced platelet PS exposure and MV release, enabling low doses of a single agonist to induce full-scale platelet procoagulant activity. PS exposed on the platelet surface was immediately released as MVs, revealing a tight coupling between the 2 processes under shear. Using platelet-specific Rac1−/− mice, we discovered that Rac1 plays a common role in mediating the low-dose agonist-induced procoagulant response independent of platelet aggregation, secretion, and the apoptosis pathway. Platelet-specific Rac1 function was not only important for coagulation in vitro but also for fibrin accumulation in vivo following laser-induced arteriolar injury. PMID:25079357

  7. Crocin prevents sesamol-induced oxidative stress and apoptosis in human platelets.

    PubMed

    Thushara, Ram M; Hemshekhar, Mahadevappa; Paul, Manoj; Shanmuga Sundaram, Mahalingam; Shankar, Rohith L; Kemparaju, Kempaiah; Girish, Kesturu S

    2014-10-01

    Recent studies have reported the platelet proapoptotic propensity of plant-derived molecules such as, resveratrol, thymoquinone, andrographolide and gossypol. Meanwhile, there were also reports of phytochemicals such as cinnamtannin B1, which shows antiapoptotic effect towards platelets. Platelets are mainly involved in hemostasis, thrombosis and wound healing. However, altered platelet functions can have serious pathological outcomes that include cardiovascular diseases. Platelets are sensitive to external and internal stimuli including therapeutic and dietary components. The anuclear platelets do undergo apoptosis via mitochondrial pathway. However, exaggerated rate of platelet apoptosis could lead to thrombocytopenia and other bleeding disorders. The present study deals with ameliorative efficacy of crocin on sesamol-induced platelet apoptosis. The antiapoptotic property of crocin and the proapoptotic tendency of sesamol in platelets were previously demonstrated. Therefore, it was interesting to see how these two compounds would interact and wield their effects on human platelets. Crocin effectively inhibited sesamol-induced oxidative stress on platelets, which was evidenced by the measurement of endogenously generated reactive oxygen species, particularly hydrogen peroxide, and changes in thiol levels. Further, crocin abrogated sesamol-induced biochemical events of apoptosis in platelets, which include intracellular calcium mobilization, changes in mitochondrial membrane integrity, cytochrome c release, caspase activity and phosphatidylserine externalization. Even though sesamol has proapoptotic effects on platelets, its anti-platelet activity cannot be neglected. Thus, the study proposes that sesamol could be supplemented with crocin, an approach that could not only abolish the toxic effects of sesamol on platelets, but also enhance the quality of treatment due to their synergistic action. PMID:24705676

  8. Crocin prevents sesamol-induced oxidative stress and apoptosis in human platelets.

    PubMed

    Thushara, Ram M; Hemshekhar, Mahadevappa; Paul, Manoj; Shanmuga Sundaram, Mahalingam; Shankar, Rohith L; Kemparaju, Kempaiah; Girish, Kesturu S

    2014-10-01

    Recent studies have reported the platelet proapoptotic propensity of plant-derived molecules such as, resveratrol, thymoquinone, andrographolide and gossypol. Meanwhile, there were also reports of phytochemicals such as cinnamtannin B1, which shows antiapoptotic effect towards platelets. Platelets are mainly involved in hemostasis, thrombosis and wound healing. However, altered platelet functions can have serious pathological outcomes that include cardiovascular diseases. Platelets are sensitive to external and internal stimuli including therapeutic and dietary components. The anuclear platelets do undergo apoptosis via mitochondrial pathway. However, exaggerated rate of platelet apoptosis could lead to thrombocytopenia and other bleeding disorders. The present study deals with ameliorative efficacy of crocin on sesamol-induced platelet apoptosis. The antiapoptotic property of crocin and the proapoptotic tendency of sesamol in platelets were previously demonstrated. Therefore, it was interesting to see how these two compounds would interact and wield their effects on human platelets. Crocin effectively inhibited sesamol-induced oxidative stress on platelets, which was evidenced by the measurement of endogenously generated reactive oxygen species, particularly hydrogen peroxide, and changes in thiol levels. Further, crocin abrogated sesamol-induced biochemical events of apoptosis in platelets, which include intracellular calcium mobilization, changes in mitochondrial membrane integrity, cytochrome c release, caspase activity and phosphatidylserine externalization. Even though sesamol has proapoptotic effects on platelets, its anti-platelet activity cannot be neglected. Thus, the study proposes that sesamol could be supplemented with crocin, an approach that could not only abolish the toxic effects of sesamol on platelets, but also enhance the quality of treatment due to their synergistic action.

  9. Drug-Antibody-Platelet Interaction in Quinine- and Quinidine-induced Thrombocytopenia

    PubMed Central

    Christie, Douglas J.; Aster, Richard H.

    1982-01-01

    Binding of quinine- and quinidine-dependent antibodies to platelets was studied using an electroimmunoassay to measure platelet-bound IgG. Antibodies from four patients with drug-induced thrombocytopenia differed significantly in their interaction with platelets: association constants for binding to platelets at high drug concentrations ranged from 0.29 to 2.6 × 107 M−1, the maximum number of antibody molecules bound ranged from 36,000 to 161,000/platelet, the amount of drug necessary to achieve half-maximum binding of antibodies to platelets ranged from 2 to 60 μM, and only one of the antibodies cross-reacted with the stereoisomer of the drug to which the patient was sensitized. Binding of the antibodies to platelets was enhanced at the highest achievable molar ratio of drug:antibody, 10,000:1, rather than being inhibited, as would be expected in a conventional, hapten-dependent reaction. The drug-antibody-platelet reaction was unaffected by Factor VIII/von Willebrand protein, nonspecifically aggregated IgG, or heat-labile complement components. After pretreatment with tritiated quinine, platelets retained several hundred thousand molecules of drug each, but failed to bind detectable amounts of antibody. However, platelets treated simultaneously with quinine-dependent antibody and tritiated quinine retained significantly more drug after repeated washes than platelets treated with drug and normal serum. These findings support the proposition that in quinine- and quinidine-induced thrombocytopenia, drug and antibody combine first in the soluble phase to form a complex, which then binds with high affinity to a receptor on the platelet surface (innocent bystander reaction), and demonstrate that these antibodies are heterogeneous in respect to the amount of drug required to promote their binding to platelets, the number of platelet receptors they recognize, and their binding affinities. PMID:6215430

  10. Granule stores from cellubrevin/VAMP-3 null mouse platelets exhibit normal stimulus-induced release.

    PubMed

    Schraw, Todd D; Rutledge, Tara W; Crawford, Garland L; Bernstein, Audrey M; Kalen, Amanda L; Pessin, Jeffery E; Whiteheart, Sidney W

    2003-09-01

    It is widely accepted that the platelet release reaction is mediated by heterotrimeric complexes of integral membrane proteins known as SNAREs (SNAP receptors). In an effort to define the precise molecular machinery required for platelet exocytosis, we have analyzed platelets from cellubrevin/VAMP-3 knockout mice. Cellubrevin/VAMP-3 has been proposed to be a critical v-SNARE for human platelet exocytosis; however, data reported here suggest that it is not required for platelet function. Upon stimulation with increasing concentrations of thrombin, collagen, or with thrombin for increasing time there were no differences in secretion of [3H]-5HT (dense core granules), platelet factor IV (alpha granules), or hexosaminidase (lysosomes) between null and wild-type platelets. There were no gross differences in bleeding times nor in agonist-induced aggregation measured in platelet-rich plasma or with washed platelets. Western blotting of wild-type, heterozygous, and null platelets confirmed the lack of cellubrevin/VAMP-3 in nulls and showed that most elements of the secretion machinery are expressed at similar levels. While the secretory machinery in mice was similar to humans, mice did express apparently higher levels of synaptobrevin/VAMP-2. These data show that the v-SNARE, cellubrevin/VAMP-3 is not a requirement for the platelet release reaction in mice.

  11. Platelet–cancer interactions: mechanisms and pharmacology of tumour cell-induced platelet aggregation

    PubMed Central

    Jurasz, Paul; Alonso-Escolano, David; Radomski, Marek W

    2004-01-01

    During haematogenous metastasis, cancer cells migrate to the vasculature and interact with platelets resulting in tumour cell-induced platelet aggregation (TCIPA). We review: The biological and clinical significance of TCIPA; Molecular mechanisms involved in platelet aggregation by cancer cells; Strategies for pharmacological regulation of these interactions. We conclude that pharmacological regulation of platelet–cancer cell interactions may reduce the impact of TCIPA on cancer biology. PMID:15492016

  12. Platelets induce apoptosis via membrane-bound FasL

    PubMed Central

    Schleicher, Rebecca I.; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O’Reilly, Lorraine; Meuth, Sven G.; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank

    2015-01-01

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL△m/△m) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre+ FasLfl/fl mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis. PMID:26232171

  13. Platelets induce apoptosis via membrane-bound FasL.

    PubMed

    Schleicher, Rebecca I; Reichenbach, Frank; Kraft, Peter; Kumar, Anil; Lescan, Mario; Todt, Franziska; Göbel, Kerstin; Hilgendorf, Ingo; Geisler, Tobias; Bauer, Axel; Olbrich, Marcus; Schaller, Martin; Wesselborg, Sebastian; O'Reilly, Lorraine; Meuth, Sven G; Schulze-Osthoff, Klaus; Gawaz, Meinrad; Li, Xuri; Kleinschnitz, Christoph; Edlich, Frank; Langer, Harald F

    2015-09-17

    After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL(△m/△m)) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre(+) FasL(fl/fl) mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

  14. Epicatechin and catechin modulate endothelial activation induced by platelets of patients with peripheral artery disease.

    PubMed

    Carnevale, R; Loffredo, L; Nocella, C; Bartimoccia, S; Bucci, T; De Falco, E; Peruzzi, M; Chimenti, I; Biondi-Zoccai, G; Pignatelli, P; Violi, F; Frati, G

    2014-01-01

    Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets.

  15. Epicatechin and Catechin Modulate Endothelial Activation Induced by Platelets of Patients with Peripheral Artery Disease

    PubMed Central

    Carnevale, R.; Loffredo, L.; Nocella, C.; Bartimoccia, S.; Bucci, T.; De Falco, E.; Peruzzi, M.; Chimenti, I.; Biondi-Zoccai, G.; Pignatelli, P.; Violi, F.; Frati, G.

    2014-01-01

    Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets. PMID:25180068

  16. Epicatechin and catechin modulate endothelial activation induced by platelets of patients with peripheral artery disease.

    PubMed

    Carnevale, R; Loffredo, L; Nocella, C; Bartimoccia, S; Bucci, T; De Falco, E; Peruzzi, M; Chimenti, I; Biondi-Zoccai, G; Pignatelli, P; Violi, F; Frati, G

    2014-01-01

    Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets. PMID:25180068

  17. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

    SciTech Connect

    Svensson Holm, Ann-Charlotte B.; Bengtsson, Torbjoern; Grenegard, Magnus; Lindstroem, Eva G.

    2012-03-10

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  18. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

    PubMed

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-07-01

    In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

  19. Inducing mitophagy in diabetic platelets protects against severe oxidative stress.

    PubMed

    Lee, Seung Hee; Du, Jing; Stitham, Jeremiah; Atteya, Gourg; Lee, Suho; Xiang, Yaozu; Wang, Dandan; Jin, Yu; Leslie, Kristen L; Spollett, Geralyn; Srivastava, Anup; Mannam, Praveen; Ostriker, Allison; Martin, Kathleen A; Tang, Wai Ho; Hwa, John

    2016-01-01

    Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in "prepackaging" the complex mitophagy machinery in a short-lived normal platelet support a critical role, in anticipation of exposure to oxidative stress. PMID:27221050

  20. Platelet activation during exercise induced asthma: effect of prophylaxis with cromoglycate and salbutamol.

    PubMed Central

    Johnson, C E; Belfield, P W; Davis, S; Cooke, N J; Spencer, A; Davies, J A

    1986-01-01

    Peak expiratory flow (PEF) and plasma concentrations of platelet factor 4 and beta thromboglobulin were measured before and after exercise in nine asthmatic patients and 12 non-asthmatic volunteers. Exercise was preceded by administration in random order of either placebo, salbutamol 200 micrograms, or sodium cromoglycate 2 mg from a pressurised inhaler. In control subjects there were minimal changes in PEF and plasma concentrations of platelet factor 4 and beta thromboglobulin. In the asthmatic patients the typical changes in PEF were seen on exercise; plasma concentrations of platelet factor 4 and beta thromboglobulin rose significantly in parallel, the rise preceding the fall in PEF. The changes in peak flow and platelet activation induced by exercise were attenuated by prior administration of salbutamol or cromoglycate. These results indicate that exercise induced asthma is associated with a rise in platelet release products similar to that observed in antigen induced asthma. PMID:2943049

  1. Platelet clearance via shear-induced unfolding of a membrane mechanoreceptor

    PubMed Central

    Deng, Wei; Xu, Yan; Chen, Wenchun; Paul, David S.; Syed, Anum K.; Dragovich, Matthew A.; Liang, Xin; Zakas, Philip; Berndt, Michael C.; Di Paola, Jorge; Ware, Jerry; Lanza, Francois; Doering, Christopher B.; Bergmeier, Wolfgang; Zhang, X. Frank; Li, Renhao

    2016-01-01

    Mechanisms by which blood cells sense shear stress are poorly characterized. In platelets, glycoprotein (GP)Ib–IX receptor complex has been long suggested to be a shear sensor and receptor. Recently, a relatively unstable and mechanosensitive domain in the GPIbα subunit of GPIb–IX was identified. Here we show that binding of its ligand, von Willebrand factor, under physiological shear stress induces unfolding of this mechanosensory domain (MSD) on the platelet surface. The unfolded MSD, particularly the juxtamembrane ‘Trigger' sequence therein, leads to intracellular signalling and rapid platelet clearance. These results illustrate the initial molecular event underlying platelet shear sensing and provide a mechanism linking GPIb–IX to platelet clearance. Our results have implications on the mechanism of platelet activation, and on the pathophysiology of von Willebrand disease and related thrombocytopenic disorders. The mechanosensation via receptor unfolding may be applicable for many other cell adhesion receptors. PMID:27670775

  2. Activated platelets release sphingosine 1-phosphate and induce hypersensitivity to noxious heat stimuli in vivo

    PubMed Central

    Weth, Daniela; Benetti, Camilla; Rauch, Caroline; Gstraunthaler, Gerhard; Schmidt, Helmut; Geisslinger, Gerd; Sabbadini, Roger; Proia, Richard L.; Kress, Michaela

    2015-01-01

    At the site of injury activated platelets release various mediators, one of which is sphingosine 1-phosphate (S1P). It was the aim of this study to explore whether activated human platelets had a pronociceptive effect in an in vivo mouse model and whether this effect was based on the release of S1P and subsequent activation of neuronal S1P receptors 1 or 3. Human platelets were prepared in different concentrations (105/μl, 106/μl, 107/μl) and assessed in mice with different genetic backgrounds (WT, S1P1fl/fl, SNS-S1P1−/−, S1P3−/−). Intracutaneous injections of activated human platelets induced a significant, dose-dependent hypersensitivity to noxious thermal stimulation. The degree of heat hypersensitivity correlated with the platelet concentration as well as the platelet S1P content and the amount of S1P released upon platelet activation as measured with LC MS/MS. Despite the significant correlations between S1P and platelet count, no difference in paw withdrawal latency (PWL) was observed in mice with a global null mutation of the S1P3 receptor or a conditional deletion of the S1P1 receptor in nociceptive primary afferents. Furthermore, neutralization of S1P with a selective anti-S1P antibody did not abolish platelet induced heat hypersensitivity. Our results suggest that activated platelets release S1P and induce heat hypersensitivity in vivo. However, the platelet induced heat hypersensitivity was caused by mediators other than S1P. PMID:25954148

  3. Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits low density lipoprotein-induced signaling in platelets.

    PubMed

    Relou, Ingrid A M; Gorter, Gertie; Ferreira, Irlando Andrade; van Rijn, Herman J M; Akkerman, Jan-Willem N

    2003-08-29

    At physiological concentrations, low density lipoprotein (LDL) increases the sensitivity of platelets to aggregation- and secretion-inducing agents without acting as an independent activator of platelet functions. LDL sensitizes platelets by inducing a transient activation of p38MAPK, a Ser/Thr kinase that is activated by the simultaneous phosphorylation of Thr180 and Tyr182 and is an upstream regulator of cytosolic phospholipase A2 (cPLA2). A similar transient phosphorylation of p38MAPK is induced by a peptide mimicking amino acids 3359-3369 in apoB100 called the B-site. Here we report that the transient nature of p38MAPK activation is caused by platelet endothelial cell adhesion molecule 1 (PECAM-1), a receptor with an immunoreceptor tyrosine-based inhibitory motif. PECAM-1 activation by cross-linking induces tyrosine phosphorylation of PECAM-1 and a fall in phosphorylated p38MAPK and cPLA2. Interestingly, LDL and the B-site peptide also induce tyrosine phosphorylation of PECAM-1, and studies with immunoprecipitates indicate the involvement of c-Src. Inhibition of the Ser/Thr phosphatases PP1/PP2A (okadaic acid) makes the transient p38MAPK activation by LDL and the B-site peptide persistent. Inhibition of Tyr-phosphatases (vanadate) increases Tyr-phosphorylated PECAM-1 and blocks the activation of p38MAPK. Together, these findings suggest that, following a first phase in which LDL, through its B-site, phosphorylates and thereby activates p38MAPK, a second phase is initiated in which LDL activates PECAM-1 and induces dephosphorylation of p38MAPK via activation of the Ser/Thr phosphatases PP1/PP2A. PMID:12775720

  4. Major depression induces oxidative stress and platelet hyperaggregability.

    PubMed

    Ormonde do Carmo, Monique B O; Mendes-Ribeiro, Antônio Cláudio; Matsuura, Cristiane; Pinto, Vivian L; Mury, Wanda V; Pinto, Nathalia O; Moss, Monique B; Ferraz, Marcos Rochedo; Brunini, Tatiana M C

    2015-02-01

    We have previously demonstrated an impairment of intraplatelet L-arginine-nitric oxide-cGMP pathway in major depression (MD) associated to platelet dysfunction. Here, we evaluated arginase pathway and phosphodiesterase 5 (PDE5) expression in platelets, systemic and intraplatelet oxidative status in untreated MD patients, and their effects on platelet aggregation. Blood samples were collected from 22 treatment naive MD patients (31 ± 2 yr) and 27 healthy subjects (33 ± 2 yr). MD patients presented with an activation of platelet arginase II, which competes with L-arginine for the production of nitric oxide (NO). An increase in protein carbonylation, overexpression of NADPH oxidase and PDE5, an enzyme that inactivates cGMP, was observed in platelets from MD patients compared to controls. In this context, platelet hyperaggregability was found in MD patients. On the other hand, antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase activities in serum and in platelets did not differ between groups. The increased activation of intraplatelet arginase and platelet aggregability, in addition to an overexpression of PDE5 and oxidative stress may contribute to alterations in L-arginine-NO-cGMP pathway and in platelet function, and consequently to the increased thrombotic risk in MD. PMID:25560770

  5. gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation.

    PubMed

    Lovely, Rehana S; Rein, Chantelle M; White, Tara C; Jouihan, Sari A; Boshkov, Lynn K; Bakke, Antony C; McCarty, Owen J; Farrell, David H

    2008-11-01

    The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity. PMID:18989528

  6. Platelet-activating factor-induced increases in glucose kinetics

    SciTech Connect

    Lang, C.H.; Dobrescu, C.; Hargrove, D.M.; Bagby, G.J.; Spitzer, J.J. )

    1988-02-01

    Platelet-activating factor (PAF) is a postulated mediator of many of the early hemodynamic effects of endotoxin. The aim of the present study was to determine whether in vivo administration of PAF could produce alterations in whole-body glucose metabolism that would mimic those seen during endotoxemia. Glucose kinetics were assessed in chronically catheterized conscious rats by the constant infusion of (6-{sup 3}H)- and (U-{sup 14}C)glucose before and for 4 h after either a bolus injection or a constant infusion of PAF. The bolus injection of PAF elevated the rate of glucose appearance (R{sub a}; 44%) for 1.5 h. The lower PAF infusion rate decreased blood pressure 11% to 104 mmHg, whereas the higher infusion rate decreased pressure 34% to 77 mmHg. Both PAF infusion rates produced elevations in plasma glucose and glucose R{sub a} throughout the 4-h infusion period in a dose-related manner. The PAF infusions also induced dose-related increases in plasma glucagon and catecholamine levels throughout the infusion period. Because the constant infusion of PAF did stimulate many of the hemodynamic and metabolic alterations produced by endotoxin, this study provides additional support for the potential importance of PAF as a mediator of the early hemodynamic and metabolic sequela of endotoxin shock. Furthermore, the PAF-induced changes in glucose metabolism appear to be mediated by the resultant elevation in plasma catecholamines.

  7. Environmental stress on diving-induced platelet activation.

    PubMed

    Bosco, G; Yang, Z J; Savini, F; Nubile, G; Data, P G; Wang, J P; Camporesi, E M

    2001-01-01

    Platelet activation has been suggested to play an important role in the pathogenesis of prethrombotic states and thus may be responsible for decompression illness during compressed air (scuba) diving. To investigate the effect of physical, mental, and environmental stress on platelet activation during immersion in ice-cold water, we examined 10 male breath-hold divers (BHD), 10 elite BHD (eBHD), and 10 scuba divers during immersion in an ice-covered lake at moderate altitude. Platelet activation was examined by surface expression of activation-dependent glycoproteins CD62p, CD63, and CD42a with flow cytometry 10 min before and 1 min and again 24 h after diving. Plasma epinephrine level was also measured. In addition, the relationship between the activated platelets and the epinephrine level was evaluated. The percentage of platelet activation increased from 2.1 +/- 0.4 to 5.7 +/- 0.3, 1.8 +/- 0.3 to 12.9 +/- 0.8, and 3.7 +/- 0.9 to 31.2 +/- 0.8 in BHD, eBHD, and scuba divers, respectively. The percentage of platelet activation returned to pre-immersion levels in BHD and eBHD divers 24 h after diving, but was still higher in scuba divers. A positive relationship exists between the plasma epinephrine level and the percentage of the platelet activation. This study suggests that physical and mental stress enhance platelet activation during diving in ice-cold water.

  8. A note on the use of Quin2 in studying shear-induced platelet aggregation.

    PubMed

    Giorgio, T D; Hellums, J D

    1986-02-01

    Quin2, a calcium ion chelator which can penetrate plasma membranes, was used to study the role of intracellular calcium ion concentration in mediating shear-induced platelet activation. Washed platelet suspensions were subjected to various levels of uniform, known shear stress in a cone and plate viscometer in the absence of added agonists. Additional samples were aggregated in response to chemical platelet agonists in a conventional aggregometer. The aggregometer response of Quin2-containing platelets to collagen, thrombin and ADP exhibited increased lag time and reduced maximum rate of aggregation in comparison to controls. However, the extent of aggregation of the Quin2-containing platelets eventually reached the same level as that of the controls. Very different results were obtained for aggregation by shear stress in the viscometer. Shear-induced aggregation was significantly suppressed by Quin2 treatment at both short (30 seconds) and long (300 seconds) times of exposure to the shear field. Shear-induced dense granular release and cellular lysis were unaltered by Quin2 treatment at 30 second exposure times, but both were significantly increased by Quin2 treatment at 300 second exposure times. These results suggest that intracellular calcium ion mobilization is an important early step in shear-induced platelet activation. Additionally, Quin2 appears to have effects resulting in increased platelet fragility. Thus, the findings raise questions on the suitability of Quin2 as an intracellular calcium ion probe in studies in shear fields. PMID:3705013

  9. Sunlight suppressing rejection of 280- to 320-nm UV-radiation-induced skin tumors in mice

    SciTech Connect

    Morison, W.L.; Kelley, S.P.

    1985-02-01

    Repeated exposure of female C3H/HeNCR- mice to sunlight prevented the normal immunologic rejection of a UV-induced tumor. This systemic immunologic alteration was transferred to syngeneic lethally X-irradiated animals with lymphoid cells from mice exposed to sunlight. The lymphoid cells also were able to suppress the capacity of lymphoid cells from normal animals to reject a UV-induced tumor. The 295- to 320-nm wave band appeared to be responsible for this immunosuppressive effect of sunlight because suppression was prevented by filtration of the radiation through Mylar and by application of a sunscreen containing para-aminobenzoic acid. These observations may have importance in understanding the pathogenesis of sunlight-induced skin cancer in humans.

  10. Investigation of stress-induced (100) platelet formation and surface exfoliation in plasma hydrogenated Si

    SciTech Connect

    Di Zengfeng; Wang Yongqiang; Nastasi, Michael; Rossi, Francois; Lee, Jung-Kun; Shao, Lin; Thompson, Phillip E.

    2007-12-10

    We have studied the mechanisms underlying stress-induced platelet formation during plasma hydrogenation. The stress is purposely introduced by a buried SiGe stained layer in a Si substrate. During plasma hydrogenation, diffusing H is trapped in the region of the SiGe layer and H platelets are formed. The platelet orientation is controlled by the in-plane compressive stress, which favors nucleation and growth of platelets in the plane of stress and parallel to the substrate surface, and ultimately leads to controlled fracture along the SiGe layer. Also, the Si/SiGe/Si structure is found to be more efficient in utilizing H for platelet formation and growth compared to H ion implanted Si because there are fewer defects to trap H (e.g., V{sub n}H{sub m} and I{sub n}H{sub m}); therefore, the total H dose needed for layer exfoliation is greatly reduced.

  11. Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice.

    PubMed

    Miyakawa, Kazuhisa; Joshi, Nikita; Sullivan, Bradley P; Albee, Ryan; Brandenberger, Christina; Jaeschke, Hartmut; McGill, Mitchell R; Scott, Michael A; Ganey, Patricia E; Luyendyk, James P; Roth, Robert A

    2015-10-01

    Acetaminophen (APAP)-induced liver injury in humans is associated with robust coagulation cascade activation and thrombocytopenia. However, it is not known whether coagulation-driven platelet activation participates in APAP hepatotoxicity. Here, we found that APAP overdose in mice caused liver damage accompanied by significant thrombocytopenia and accumulation of platelets in the liver. These changes were attenuated by administration of the direct thrombin inhibitor lepirudin. Platelet depletion with an anti-CD41 antibody also significantly reduced APAP-mediated liver injury and thrombin generation, indicated by the concentration of thrombin-antithrombin (TAT) complexes in plasma. Compared with APAP-treated wild-type mice, biomarkers of hepatocellular and endothelial damage, plasma TAT concentration, and hepatic platelet accumulation were reduced in mice lacking protease-activated receptor (PAR)-4, which mediates thrombin signaling in mouse platelets. However, selective hematopoietic cell PAR-4 deficiency did not affect APAP-induced liver injury or plasma TAT levels. These results suggest that interconnections between coagulation and hepatic platelet accumulation promote APAP-induced liver injury, independent of platelet PAR-4 signaling. Moreover, the results highlight a potential contribution of nonhematopoietic cell PAR-4 signaling to APAP hepatotoxicity. PMID:26179083

  12. Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

    SciTech Connect

    Nygaard, Gyrid; Herfindal, Lars; Kopperud, Reidun; Aragay, Anna M.; Holmsen, Holm; Døskeland, Stein Ove; Kleppe, Rune; Selheim, Frode

    2014-07-04

    Highlights: • We investigated the impact of cyclic nucleotide analogues on platelet activation. • Different time dependence were found for inhibition of platelet activation. • Additive effect was found using PKA- and PKG-activating analogues. • Our results may explain some of the discrepancies reported for cNMP signalling. - Abstract: In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

  13. The role of platelet aggregation and release in fragment D-induced pulmonary dysfunction.

    PubMed Central

    Manwaring, D; Curreri, P W

    1980-01-01

    The plasma concentration of fibrinogen degradation product D (fragmentt D) is markedly incrased following major burn or traumatic injury. Purified human fragment D infused into awake, restrained, nontraumatized rabbits (100 micrograms/ml blood) causes progressive thrombocytopenia, pulmonary dysfunction, vascular leak, and interstitial neutrophilia. Rabbits treated with the antihistamine diphenhydramine (Benadryl) prior to fragment D infusion fail to develop these symptoms. This study examined platelet aggregation, platelet ATP secretion, and platelet malondialdehyde release in rabbits which received fragmen D alone or fragment D following diphenhydramine pretreatment. Platelet-rich plasma was prepared from citrated blood drawn from femoral arterial catheters at 0, 2 1/2, and 4 hours postinfusion. Platelet aggregation was stimulated with either collagen or ADP. Malondialdehyde, a byproduct of thromboxane synthesis, was measured by colorimetry. Platelet aggregation and function (stimulated with collagen) were enhanced in fragment D platelet-rich plasma, since all response times decreased. Total ATP and MDA release incresed. Diphenhydramine pretreatment inhibited fragment D-enhanced aggregation, ATP release and prostaglandin (thromboxane) synthesis. No animal pretreated with diphenhydramine exhibited thrombocytopenia or respiratory dysfunction. Stimulation of platelet aggregation and release may represent one mechanism by which fragment D induces pulmonary dysfunction. Diphenhydramine inhibits these responses and may prove therapeutic in posttraumtic pulmonary complications. PMID:7406554

  14. Platelet factors induce chemotactic migration of murine mammary adenocarcinoma cells with different metastatic capabilities.

    PubMed Central

    Sarach, M. A.; Rovasio, R. A.; Eynard, A. R.

    1993-01-01

    The chemotactic response of neoplastic cells (NC) induced by soluble platelet factors was investigated. NC suspensions isolated from murine mammary gland adenocarcinomas having different metastatic capabilities were incubated in Boyden's chambers and challenged with (1) 'Early Platelet Factors' (EP), obtained from the soluble fraction of recently collagen-activated human platelets, and (2) 'Late Platelet Factors' (LP), isolated after 24 hours incubation of the platelet aggregates. Chemotaxis was expressed as the distance travelled by NC through nitrocellulose filters. NC isolated from M3, the tumour line having the stronger metastatic potential, showed a significant chemotactic response towards LP factors, whereas NC from the M2 line exhibiting the lower metastatic behaviour, showed a chemotactic response towards EP factors. Both tumour cell lines lacked motion capability towards the well known chemoattractant peptide N-f-Met-Leu-Phe-Phe as well as to serum, plasma, collagen type I or culture medium. The different chemotactic response of both tumour lines when they were challenged by concentration gradients of factors released by early or late collagen-activated human platelets, confirm a relationship between platelet activity and metastatic capabilities and suggests that platelet chemoattractants might play a role in the metastatic dissemination of these mammary gland adenocarcinomas. Images Figure 1 PMID:8217786

  15. Heparin-induced thrombocytopenia: the role of platelets genetic polymorphisms.

    PubMed

    Pamela, Scarparo; Anna Maria, Lombardi; Elena, Duner; Giovanni, Malerba; Emanuele, Allemand; Silvia, Vettore; Carmen, Blumentritt; Andreas, Greinacher; Fabrizio, Fabris

    2013-01-01

    Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy, characterized by thrombocytopenia and an increased risk for thrombotic complications secondary to the formation of IgG antibodies (Ab), recognizing a complex of heparin (H) and PF4. Using the 4T clinical score for HIT and the presence of heparin-associated Ab assayed by enzyme-linked immunosorbent assay and heparin-induced platelet aggregation, we define the phenotype of three groups of patients: 51 H/PF4/Ab patients with antibodies and without thrombocytopenia; 50 patients with thrombocytopenia (HIT) and 53 patients with thrombosis (HITT). In these patients we studied four polymorphisms: FcγRIIA-H131R, GpIIb/IIIa-HP-1, PECAM1-L125V (in linkage-disequilibrium with S563N and R670G), and FcγRIIIA-F158V, to understand if these variations may influence the different phenotypes of the patients. There were no difference in genotype or allele frequencies between controls and the three groups of patients. Afterward, we created a genotype score for multiple risk alleles for thrombosis considering as risk genotype FcγRIIA R/R131, HPA-1a/b, and PECAM1-V/V125. These polymorphisms were overrepresented in HITT patients, ascertained by a permutation test (10 000 replicates) p = 0.0198 for the two-single-nucleotide polymorphism (SNP) model and p = 0.0119 for the three-SNP model. The calculated odds ratio for thrombosis was 4.01[CI: 2.30-6.96] in the case of the presence of two at risk genotypes and 8.002 [CI: 4.59-13.93] if all the three at risk genotypes were present. In conclusion these polymorphisms could contribute to the risk of thrombotic complications in HIT.

  16. Pharmacological characterization of nanoparticle-induced platelet microaggregation using quartz crystal microbalance with dissipation: comparison with light aggregometry

    PubMed Central

    Santos-Martinez, Maria J; Tomaszewski, Krzysztof A; Medina, Carlos; Bazou, Despina; Gilmer, John F; Radomski, Marek W

    2015-01-01

    Background Engineered nanoparticles (NPs) can induce platelet activation and aggregation, but the mechanisms underlying these interactions are not well understood. This could be due in part to use of devices that study platelet function under quasi-static conditions with low sensitivity to measure platelet microaggregation. Therefore, in this study we investigated the pharmacological pathways and regulators of NP-induced platelet microaggregation under flow conditions at nanoscale using quartz crystal microbalance with dissipation (QCM-D) and compared the data thus obtained with those generated by light aggregometry. Methods Blood was collected from healthy volunteers, and platelet-rich plasma was obtained. Thrombin receptor-activating peptide, a potent stimulator of platelet function, and pharmacological inhibitors were used to modulate platelet microaggregation in the presence/absence of silica (10 nm and 50 nm) and polystyrene (23 nm) NPs. Light aggregometry was used to study platelet aggregation in macroscale. Optical, immunofluorescence, and scanning electron microscopy were also used to visualize platelet aggregates. Results Platelet microaggregation was enhanced by thrombin receptor-activating peptide, whereas prostacyclin, nitric oxide donors, acetylsalicylic acid, and phenanthroline, but not adenosine diphosphate (ADP) blockers, were able to inhibit platelet microaggregation. NPs caused platelet microaggregation, an effect not detectable by light aggregometry. NP-induced microaggregation was attenuated by platelet inhibitors. Conclusion NP-induced platelet microaggregation appears to involve classical proaggregatory pathways (thromboxane A2-mediated and matrix metalloproteinase-2-mediated) and can be regulated by endogenous (prostacyclin) and pharmacological (acetylsalicylic acid, phenanthroline, and nitric oxide donors) inhibitors of platelet function. Quartz crystal microbalance with dissipation, but not light aggregometry, is an appropriate method for

  17. Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury.

    PubMed

    Sugiyama, Michael G; Gamage, Asela; Zyla, Roman; Armstrong, Susan M; Advani, Suzanne; Advani, Andrew; Wang, Changsen; Lee, Warren L

    2016-02-01

    Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α5β1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have

  18. Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury.

    PubMed

    Sugiyama, Michael G; Gamage, Asela; Zyla, Roman; Armstrong, Susan M; Advani, Suzanne; Advani, Andrew; Wang, Changsen; Lee, Warren L

    2015-12-04

    Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α5β1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have

  19. Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species†

    PubMed Central

    Yuan, Hengjie; Houck, Katie L.; Tian, Ye; Bharadwaj, Uddalak; Hull, Ken; Zhou, Zhou; Zhou, Mingzhao; Wu, Xiaoping; Tweardy, David J.; Romo, Daniel; Fu, Xiaoyun; Zhang, Yanjun; Zhang, Jianning; Dong, Jing-fei

    2015-01-01

    Background Piperlongumine (PL) is a compound isolated from the piper longum plant. It possesses anti-cancer activities through blocking the transcription factor STAT3 and by inducing reactive oxygen species (ROS) in cancer, but not normal cells. It also inhibits platelet aggregation induced by collagen, but the underlying mechanism is not known. Objective We conducted in vitro experiments to test the hypothesis that PL regulates a non-transcriptional activity of STAT3 to specifically reduce the reactivity of human platelets to collagen. Results PL dose-dependently blocked collagen-induced platelet aggregation, calcium influx, CD62p expression and thrombus formation on collagen with a maximal inhibition at 100 μM. It reduced platelet microvesiculation induced by collagen. PL blocked the activation of JAK2 and STAT3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets pretreated with a STAT3 inhibitor. Although PL induced ROS production in platelets; quenching ROS using excessive reducing agents: 20 μM GSH and 0.5 mM L-Cysteine, did not block the inhibitory effects. The NADPH oxidase inhibitor Apocynin also had no effect. Conclusions PL inhibited collagen-induced platelet reactivity by targeting the JAK2-STAT3 pathway. We also provide experimental evidence that PL and collagen induce different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants in redox signals. PMID:26645674

  20. Platelet Aggregation and Mental Stress Induced Myocardial Ischemia: Results from the REMIT Study

    PubMed Central

    Jiang, Wei; Boyle, Stephen H.; Ortel, Thomas L.; Samad, Zainab; Velazquez, Eric J.; Harrison, Robert W.; Wilson, Jennifer; Kuhn, Cynthia; Williams, Redford B.; O’Connor, Christopher M.; Becker, Richard C.

    2015-01-01

    BACKGROUND Mental stress-induced myocardial ischemia (MSIMI) is common in patients with ischemic heart disease (IHD) and associated with a poorer cardiovascular prognosis. Platelet hyperactivity is an important factor in acute coronary syndrome. This study examined associations between MSIMI and resting and mental stress-induced platelet activity. METHODS Eligible patients with clinically stable IHD underwent a battery of 3 mental stress tests during the recruitment phase of REMIT (Responses of Myocardial Ischemia to Escitalopram Treatment) study. MSIMI was assessed by echocardiography and electrocardiography. Ex vivo platelet aggregation in response to ADP, epinephrine, collagen, serotonin, and combinations of serotonin plus ADP, epinephrine, and collagen were evaluated as was platelet serotonin transporter expression. RESULTS Of the 270 participants who completed mental stress testing, and had both resting and post-stress platelet aggregation evaluation, 43.33% (N=117) met criteria for MSIMI and 18.15% (N=49) had normal left ventricular response to stress (NLVR). The MSIMI group, relative to the NLVR groups, demonstrated heightened mental stress-induced aggregation responses, as measured by area under the curve, to collagen 10 μM (6.95[5.54] vs. −14.23[8.75].; p=0.045), epinephrine 10 μM (12.84[4.84] vs. −6.40[7.61].; p=0.037) and to serotonin 10 μM plus ADP 1 μM (6.64[5.29] vs. −27.34[8.34]; p < .001). The resting platelet aggregation and serotonin transporter expression, however, were not different between the two groups. CONCLUSIONS These findings suggest that the dynamic change of platelet aggregation caused by mental stress may underlie MSIMI. While the importance of these findings requires additional investigation, they raise concern given the recognized relationship between mental stress-induced platelet hyperactivity and cardiovascular events in patients with IHD. PMID:25819856

  1. Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

    PubMed

    Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

    2011-11-01

    The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

  2. Stacking fault induced tunnel barrier in platelet graphite nanofiber

    SciTech Connect

    Lan, Yann-Wen E-mail: ywlan@phys.sinica.edu.tw; Chang, Yuan-Chih; Chang, Chia-Seng; Chen, Chii-Dong E-mail: ywlan@phys.sinica.edu.tw; Chang, Wen-Hao; Li, Yuan-Yao

    2014-09-08

    A correlation study using image inspection and electrical characterization of platelet graphite nanofiber devices is conducted. Close transmission electron microscopy and diffraction pattern inspection reveal layers with inflection angles appearing in otherwise perfectly stacked graphene platelets, separating nanofibers into two domains. Electrical measurement gives a stability diagram consisting of alternating small-large Coulomb blockade diamonds, suggesting that there are two charging islands coupled together through a tunnel junction. Based on these two findings, we propose that a stacking fault can behave as a tunnel barrier for conducting electrons and is responsible for the observed double-island single electron transistor characteristics.

  3. Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase.

    PubMed

    Blair, Price; Rex, Sybille; Vitseva, Olga; Beaulieu, Lea; Tanriverdi, Kahraman; Chakrabarti, Subrata; Hayashi, Chie; Genco, Caroline A; Iafrati, Mark; Freedman, Jane E

    2009-02-13

    Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam(3)CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin alpha(IIb)beta(3), generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam(3)CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

  4. Correlation between the In Vitro Functionality of Stored Platelets and the Cytosolic Esterase-Induced Fluorescence Intensity with CMFDA.

    PubMed

    Wang, Jiexi; Yi, Xiaoyang; Liu, Minxia; Zhou, Qian; Ren, Suping; Wang, Yan; Yang, Chao; Zhou, Jianwei; Han, Ying

    2015-01-01

    It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.

  5. Prevention of ischemia-induced myocardial platelet deposition by exogenous prostacyclin

    SciTech Connect

    Aherne, T.; Price, D.C.; Yee, E.S.; Hsieh, W.R.; Ebert, P.A.

    1986-07-01

    The antithrombotic effects of prostacyclin infusion on myocardial platelet deposition were studied in a canine model during and after global ischemia. Eleven isolated heart preparations were subjected to 1 hour of cardioplegic arrest under moderate hypothermia (27 to 28/sup 0/C), including a control group (n = 7) and a prostacyclin-treated group (n = 4). The hearts of four other dogs were continuously perfused for 180 minutes. Platelet deposition was measured at 15 minute intervals throughout the 3 hour study. Serial full-thickness myocardial biopsy specimens were analyzed for activity of /sup 111/In-labeled platelets with /sup 99m/Tc-labeled erythrocyte correction for tissue blood content. The pattern of platelet distribution was determined by scintiscans of each heart, taken with a gamma camera at the end of the 60 minute reperfusion period. Substantial myocardial platelet deposition was found in the control hearts after ischemia but not in the prostacyclin-treated group (p less than 0.05). Furthermore, prostacyclin infusion had a significant disaggregatory effect on intracoronary platelet deposits when the precardioplegic and postcardioplegic biopsy specimens were analyzed (p less than 0.05). Three hours of continuous perfusion did not increase tissue /sup 111/In-labeled platelet activity. Ex vivo images showed platelet deposition to be a diffuse patchy process with significantly more /sup 111/In activity in the endocardium than in the epicardium after global ischemia (p less than 0.05). These data show the potent antithrombotic properties of prostacyclin in preventing and disaggregating ischemia-induced intracoronary platelet deposition during and after cardioplegic arrest.

  6. Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis.

    PubMed

    Chang, Yao-Wen; Hsieh, Pei-Wen; Chang, Yu-Tsui; Lu, Meng-Hong; Huang, Tur-Fu; Chong, Kowit-Yu; Liao, Hsiang-Ruei; Cheng, Ju-Chien; Tseng, Ching-Ping

    2015-12-15

    Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective. PMID:26528756

  7. Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis

    PubMed Central

    Chang, Yu-Tsui; Lu, Meng-Hong; Huang, Tur-Fu; Chong, Kowit-Yu; Liao, Hsiang-Ruei; Cheng, Ju-Chien; Tseng, Ching-Ping

    2015-01-01

    Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective. PMID:26528756

  8. [Cyclooxygenase inhibitors in some dietary vegetables inhibit platelet aggregation function induced by arachidonic acid].

    PubMed

    Wang, Xin-Hua; Shao, Dong-Hua; Liang, Guo-Wei; Zhang, Ru; Xin, Qin; Zhang, Tao; Cao, Qing-Yun

    2011-10-01

    The study was purposed to investigate whether the cyclooxygenase inhibitors from some dietary vegetables can inhibit platelet aggregation function by the arachidonic acid (AA). The vegetable juice was mixed with platelet rich plasma (PRP), and asprin was used as positive control. The maximum ratio of platelet aggregation induced by AA was measured on the aggregometer; heme and cyclooxygenase-1 (COX(1)) or cyclooxygenase-2 (COX(2)) were added to test tubes containing COX reaction buffer, the mixture was vortex-mixed and exposed to aspirin or vegetable juice, followed by addition of AA and then hydrochloric acid (1 mol/L) was added to stop the COX reaction, followed by chemical reduction with stannous chloride solution. The concentration of COX inhibitors was detected by the enzyme immunoassay kit; vegetable juice (aspirin as positive control) was mixed with whole blood, which was followed by the addition of AA, and then the reaction was stopped by adding indomethacin, centrifuged, then the supernatant was collected, and the plasma thromboxane B(2) (TXB(2)) was measured by radioimmunoassay. The results showed that spinach juice, garlic bolt juice, blanched garlic leave juice and Chinese leek juice could inhibit by 80% human platelet aggregation induced by AA. 4 kinds of vegetables were all found a certain amount of cyclooxygenase inhibitors, which COX(1) and COX(2) inhibitor concentrations of spinach were higher than that of aspirin; 4 vegetable juice could significantly reduce the human plasma concentrations of TXB(2) induced by AA (p < 0.05). It is concluded that 4 kinds of raw vegetables containing cyclooxygenase inhibitors inhibit the production of TXA(2) and thus hinder platelet aggregation. Raw spinach, garlic bolt, blanched garlic and chinese leek inhibit significantly AA-induced human platelet aggregation in vitro. 4 kinds of vegetables may have a good potential perspective of anti-platelet aggregation therapy or prevention of thrombosis.

  9. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    SciTech Connect

    Winocour, P.D.; Colwell, J.A.

    1985-05-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.

  10. Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia.

    PubMed

    Tutwiler, Valerie; Madeeva, Daria; Ahn, Hyun Sook; Andrianova, Izabella; Hayes, Vincent; Zheng, X Long; Cines, Douglas B; McKenzie, Steven E; Poncz, Mortimer; Rauova, Lubica

    2016-01-28

    Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

  11. [THE INFLUENCE OF HYDROGEN SULFIDE ON COLLAGEN-INDUCED AGGREGATION OF HUMAN PLATELETS].

    PubMed

    Petrova, I V; Trubacheva, O A; Mangataeva, O S; Suslova, T E; Kovalev, I V; Gusakova, S V

    2015-10-01

    Study the impact of hydrogen sulfide on collagen-induced platelet aggregation from healthy donors and patients with type 2 diabetes. In healthy individuals, in contrast to patients with type 2 diabetes, NaHS significantly inhibited platelet aggregation. Activators of cAMP signaling (forskolin and phosphodiesterase inhibitor) significantly reduced platelet aggregation in both groups of examinees. NO-synthase inhibitors increased platelet aggregation in healthy volunteers, but not in patients with type 2 diabetes. The presence of H2S donor did not alter the extent of platelet aggregation at high concentrations of cAMP or decreased production of nitric oxide. It is assumed that the antiplatelet effect of H2S is not associated with the effect on the signal system, mediated cAMP or nitric oxide. Change H2S-dependent regulation of platelet aggregation in patients with type 2 diabetes is caused by disorders have been reported with this disease: the increase of intracellular calcium ion concentration, oxidative damage to proteins, hyperhomocysteinemia, glycosylation of key proteins involved in this process.

  12. Relationship between ADP-induced platelet-fibrin clot strength and anti-platelet responsiveness in ticagrelor treated ACS patients

    PubMed Central

    Li, Dan-Dan; Wang, Xu-Yun; Xi, Shao-Zhi; Liu, Jia; Qin, Liu-An; Jing, Jing; Yin, Tong; Chen, Yun-Dai

    2016-01-01

    Background Ticagrelor provides enhanced antiplatelet efficacy but increased risk of bleeding and dyspnea. This study aimed to display the relationship between ADP-induced platelet-fibrin clot strength (MAADP) and clinical outcomes in acute coronary syndrome (ACS) patients treated by ticagrelor. Methods Consecutive Chinese-Han patients with ACS who received maintenance dose of ticagrelor on top of aspirin were recruited. After 5-day ticagrelor maintenance treatment, MAADP measured by thrombelastography (TEG) were recorded for the evaluation of ticagrelor anti-platelet reactivity. Pre-specified cutoffs of MAADP > 47 mm for high on-treatment platelet reactivity (HTPR) and MAADP < 31 mm for low on-treatment platelet reactivity (LTPR) were applied for evaluation. The occurrences of primary ischemic cardiovascular events (including a composite of cardiac death, non-fatal myocardial infarction and stroke), the Thrombolysis in Myocardial Infarction (TIMI) defined bleeding events, and ticagrelor related dyspnea were recorded after a follow-up of three months. Results Overall, 176 ACS patients (Male: 79.55%, Age: 59.91 ± 10.54 years) under ticagrelor maintenance treatment were recruited. The value of MAADP ranged from 4.80% to 72.90% (21.27% ± 12.07% on average), with the distribution higher skewed towards the lower values. Using the pre-specific cutoffs for HTPR and LTPR, seven patients (3.98%) were identified as HTPR and 144 patients (81.82%) as LTPR. After a follow-up of three months in 172 patients, major cardiovascular events occurred in no patient, but TIMI bleeding events in 81 (47.09%) with major bleedings in three patients. All patients with major bleedings were classified as LTPR. Ticagrelor related dyspnea occurred in 31 (18.02%) patients, with 30 (21.28%) classified as LTPR and no one as HTPR (P = 0.02). Conclusions In ticagrelor treated ACS patients, MAADP measured by TEG might be valuable for the prediction of major bleeding and ticagrelor related dyspnea

  13. Inhibitors of ex vivo aggregation of human platelets induced by decompression, during reduced barometric pressure.

    PubMed

    Murayama, M; Kumaroo, K K

    1986-05-15

    It has been shown experimentally ex vivo that human platelet aggregation is induced by decompression (reduced pressure) produced by various means, i.e., reduced barometric pressure, reduced hydrostatic pressure, and reduced hydrodynamic pressure due to Bernoulli's principle. We report here that the spontaneous platelet aggregation induced by reduced barometric pressure (253 torr for three hours) is inhibited by 1:10(7) diluted Japanese herbal plant oil (JHP) and also by two of its major constituents, menthone and menthol with the median inhibitory concentration (IC50) in the millimolar range. These drugs gave essentially similar results when collagen and ADP were used as aggregating agents. Inhibitor concentrations were determined by microscopic examination of platelets in wet preparations when the aggregating stimulus was reduced pressure and by optical aggregometry when collagen and ADP were the aggregating agents. Potential usefulness of these compounds in the prevention of decompression syndrome (DCS) and acute mountain sickness (AMS) are discussed.

  14. Aprotinin reduces cardiopulmonary bypass-induced blood loss and inhibits fibrinolysis without influencing platelets.

    PubMed

    Orchard, M A; Goodchild, C S; Prentice, C R; Davies, J A; Benoit, S E; Creighton-Kemsford, L J; Gaffney, P J; Michelson, A D

    1993-11-01

    Cardiopulmonary bypass (CPB) induces a bleeding defect which leads to enhanced blood loss. A double-blind study was carried out comparing aprotinin with placebo in patients undergoing re-operation for heart valve replacement. The results confirm that aprotinin is effective at reducing such loss. In the placebo treated group, significant increases were observed, during CPB, in the plasma concentrations of fibrinolytic activity, tissue plasminogen activator antigen, D-dimer, and beta-thromboglobulin. Platelet counts fell within 5-10 min of the patients going onto CPB, but this could be accounted for by the dilutional effect of the extracorporeal circuit. Inhibition of responsiveness of platelets, as judged by aggregometry, was significant only at the end of bypass when collagen was the agonist and after protamine reversal when ristocetin was the agonist. CPB did not enhance the release, into the circulation, of glycocalicin (a proteolytic fragment of glycoprotein Ib). In the aprotinin-treated group, the formation of fibrin degradation products as measured by D-dimer was inhibited. However, aprotinin did not influence the change in platelet count, suppress beta-thromboglobulin release from platelets, prevent the inhibition of platelet function or influence the concentration of plasma glycocalicin during the study period. These observations confirm that CPB leads to a fibrinolytic state and less responsive platelets. This study also indicates that aprotinin-induced reduction in blood loss is associated with inhibition of plasmin-mediated fibrin digestion and that the mechanism by which aprotinin reduces blood loss is not via protection of platelets during CPB.

  15. Platelet participation in the pathogenesis of dermonecrosis induced by Loxosceles gaucho venom.

    PubMed

    Tavares, F L; Peichoto, M E; Marcelino, J R; Barbaro, K C; Cirillo, M C; Santoro, M L; Sano-Martins, I S

    2016-06-01

    Loxosceles gaucho spider venom induces in vitro platelet activation and marked thrombocytopenia in rabbits. Herein, we investigated the involvement of platelets in the development of the dermonecrosis induced by L. gaucho venom, using thrombocytopenic rabbits as a model. L. gaucho venom evoked a drop in platelet and neutrophil counts 4 h after venom injection. Ecchymotic areas at the site of venom inoculation were noticed as soon as 4 h in thrombocytopenic animals but not in animals with initial normal platelet counts. After 5 days, areas of scars in thrombocytopenic animals were also larger, evidencing the marked development of lesions in the condition of thrombocytopenia. Histologically, local hemorrhage, collagen fiber disorganization, and edema were more severe in thrombocytopenic animals. Leukocyte infiltration, predominantly due to polymorphonuclears, was observed in the presence or not of thrombocytopenia. Thrombus formation was demonstrated by immunohistochemistry at the microvasculature, and it occurred even under marked thrombocytopenia. Taken together, platelets have an important role in minimizing not only the hemorrhagic phenomena but also the inflammatory and wound-healing processes, suggesting that cutaneous loxoscelism may be aggravated under thrombocytopenic conditions.

  16. IL-1α induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs

    PubMed Central

    Nagasaki, Mika; Kunishima, Shinji; Sawaguchi, Akira; Sakata, Asuka; Sakaguchi, Hiroyasu; Ohmori, Tsukasa; Manabe, Ichiro; Italiano, Joseph E.; Ryu, Tomiko; Takayama, Naoya; Komuro, Issei; Kadowaki, Takashi; Nagai, Ryozo

    2015-01-01

    Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture–dependent thrombopoiesis and increased platelet counts. IL-1α–IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs. PMID:25963822

  17. Direct Adaptive Rejection of Vortex-Induced Disturbances for a Powered SPAR Platform

    NASA Technical Reports Server (NTRS)

    VanZwieten, Tannen S.; Balas, Mark J.; VanZwieten, James H.; Driscoll, Frederick R.

    2009-01-01

    The Rapidly Deployable Stable Platform (RDSP) is a novel vessel designed to be a reconfigurable, stable at-sea platform. It consists of a detachable catamaran and spar, performing missions with the spar extending vertically below the catamaran and hoisting it completely out of the water. Multiple thrusters located along the spar allow it to be actively controlled in this configuration. A controller is presented in this work that uses an adaptive feedback algorithm in conjunction with Direct Adaptive Disturbance Rejection (DADR) to mitigate persistent, vortex-induced disturbances. Given the frequency of a disturbance, the nominal DADR scheme adaptively compensates for its unknown amplitude and phase. This algorithm is extended to adapt to a disturbance frequency that is only coarsely known by including a Phase Locked Loop (PLL). The PLL improves the frequency estimate on-line, allowing the modified controller to reduce vortex-induced motions by more than 95% using achievable thrust inputs.

  18. Lesion-induced accumulation of platelets promotes survival of adult neural stem / progenitor cells.

    PubMed

    Kazanis, Ilias; Feichtner, Martina; Lange, Simona; Rotheneichner, Peter; Hainzl, Stefan; Öller, Michaela; Schallmoser, Katharina; Rohde, Eva; Reitsamer, Herbert A; Couillard-Despres, Sebastien; Bauer, Hans-Christian; Franklin, Robin J M; Aigner, Ludwig; Rivera, Francisco J

    2015-07-01

    The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury.

  19. Lesion-induced accumulation of platelets promotes survival of adult neural stem / progenitor cells.

    PubMed

    Kazanis, Ilias; Feichtner, Martina; Lange, Simona; Rotheneichner, Peter; Hainzl, Stefan; Öller, Michaela; Schallmoser, Katharina; Rohde, Eva; Reitsamer, Herbert A; Couillard-Despres, Sebastien; Bauer, Hans-Christian; Franklin, Robin J M; Aigner, Ludwig; Rivera, Francisco J

    2015-07-01

    The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury. PMID:25819103

  20. Endothelial cell apoptosis induced by bacteria-activated platelets requires caspase-8 and -9 and generation of reactive oxygen species.

    PubMed

    Kuckleburg, Christopher J; Tiwari, Raksha; Czuprynski, Charles J

    2008-02-01

    A common feature of severe sepsis is vascular inflammation and damage to the endothelium. Because platelets can be directly activated by bacteria and endotoxin, these cells may play an important role in determining the outcome of sepsis. For example, inhibiting platelet interactions with the endothelium has been shown to attenuate endothelial cell damage and improve survival during sepsis. Although not entirely understood, the interactions between bacteria-activated platelets and the endothelium may play a key role in the vascular pathology of bacterial sepsis. Haemophilus somnus is a bacterial pathogen that causes diffuse vascular inflammation and endothelial damage. In some cases H. somnus infection results in an acute and fatal form of vasculitis in the cerebral microvasculature known as thrombotic meningoencephalitis (TME). In this study, we have characterized the mechanisms involved in endothelial cell apoptosis induced by activated platelets. We observed that direct contact between H. somnus-activated platelets and endothelial cells induced significant levels of apoptosis; however, Fas receptor activation on bovine endothelial cells was not able to induce apoptosis unless protein synthesis was disrupted. Endothelial cell apoptosis by H. somnus-activated platelets required activation of both caspase-8 and caspase-9, as inhibitors of either caspase inhibited apoptosis. Furthermore, activated platelets induced endothelial cell production of reactive oxygen species (ROS) and disrupting ROS activity in endothelial cells significantly inhibited apoptosis. These findings suggest that bacterial activation of platelets may contribute to endothelial cell dysfunction observed during sepsis, specifically by inducing endothelial cell apoptosis.

  1. Flow cytometric analysis of material-induced platelet activation in a canine model: elevated microparticle levels and reduced platelet life span.

    PubMed

    Gemmell, C H; Yeo, E L; Sefton, M V

    1997-11-01

    Assessment of material-induced platelet activation is important given that it is thought to be a major mechanism of biomaterials thrombogenicity. We monitored, by flow cytometry, platelet microparticle (MP) levels in the circulation during the connection of polyvinyl alcohol (PVA) hydrogel and polyethylene (PE) test segments (3.18 mm ID, 20 and 50 cm L) to our chronically shunted beagle dogs. We report that circulating microparticle levels were dependent on test segment material, length, and time. The connection of 50-cm lengths of PVA hydrogel test segments led to MP levels two to three times greater than background at 48 h, while the connection of polyethylene test segments did not lead to elevated microparticle levels. MP levels were near background 24 h after removal of the PVA test segment. To determine platelet life span during the connection of test segments, platelets were labeled in vivo with biotin and their disappearance monitored flow cytometrically. While platelet life span for shunted dogs (no test segment) was 4.7 +/- 0.2 days, the connection of PVA hydrogel test segments led to a platelet life span of < 2 days.

  2. Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

    SciTech Connect

    Burroughs, S.F.; Johnson, G.J. )

    1990-04-01

    beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of (14C)-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with (3H)-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist (3H)-U46619 and antagonist (3H)-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ((Ca2+)i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in (Ca2+)i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.

  3. High Fat Diet Induces Adhesion of Platelets to Endothelium in Two Models of Dyslipidemia

    PubMed Central

    Gonzalez, Jaime; Donoso, Wendy; Díaz, Natalia; Albornoz, María Eliana; Huilcaman, Ricardo; Morales, Erik

    2014-01-01

    Cardiovascular diseases (CVD) represent about 30% of all global deaths. It is currently accepted that, in the atherogenic process, platelets play an important role, contributing to endothelial activation and modulation of the inflammatory phenomenon, promoting the beginning and formation of lesions and their subsequent thrombotic complications. The objective of the present work was to study using immunohistochemistry, the presence of platelets, monocytes/macrophages, and cell adhesion molecules (CD61, CD163, and CD54), in two stages of the atheromatous process. CF-1 mice fed a fat diet were used to obtain early stages of atheromatous process, denominated early stage of atherosclerosis, and ApoE−/− mice fed a fat diet were used to observe advanced stages of atherosclerosis. The CF-1 mice model presented immunostaining on endothelial surface for all three markers studied; the advanced atherosclerosis model in ApoE−/− mice also presented granular immunostaining on lesion thickness, for the same markers. These results suggest that platelets participate in atheromatous process from early stages to advance d stages. High fat diet induces adhesion of platelets to endothelial cells in vivo. These findings support studying the participation of platelets in the formation of atheromatous plate. PMID:25328689

  4. The Role of Platelet Factor 4 in Radiation-Induced Thrombocytopenia

    SciTech Connect

    Lambert, Michele P.; Xiao Liqing; Nguyen, Yvonne; Kowalska, M. Anna; Poncz, Mortimer

    2011-08-01

    Purpose: Factors affecting the severity of radiation-induced thrombocytopenia (RIT) are not well described. We address whether platelet factor 4 (PF4; a negative paracrine for megakaryopoiesis) affects platelet recovery postradiation. Methods and Materials: Using conditioned media from irradiated bone marrow (BM) cells from transgenic mice overexpressing human (h) PF4 (hPF4+), megakaryocyte colony formation was assessed in the presence of this conditioned media and PF4 blocking agents. In a model of radiation-induced thrombocytopenia, irradiated mice with varying PF4 expression levels were treated with anti-hPF4 and/or thrombopoietin (TPO), and platelet count recovery and survival were examined. Results: Conditioned media from irradiated BM from hPF4+ mice inhibited megakaryocyte colony formation, suggesting that PF4 is a negative paracrine released in RIT. Blocking with an anti-hPF4 antibody restored colony formation of BM grown in the presence of hPF4+ irradiated media, as did antibodies that block the megakaryocyte receptor for PF4, low-density lipoprotein receptor-related protein 1 (LRP1). Irradiated PF4 knockout mice had higher nadir platelet counts than irradiated hPF4+/knockout litter mates (651 vs. 328 x 106/mcL, p = 0.02) and recovered earlier (15 days vs. 22 days, respectively, p <0.02). When irradiated hPF4+ mice were treated with anti-hPF4 antibody and/or TPO, they showed less severe thrombocytopenia than untreated mice, with improved survival and time to platelet recovery, but no additive effect was seen. Conclusions: Our studies show that in RIT, damaged megakaryocytes release PF4 locally, inhibiting platelet recovery. Blocking PF4 enhances recovery while released PF4 from megakaryocytes limits TPO efficacy, potentially because of increased release of PF4 stimulated by TPO. The clinical value of blocking this negative paracrine pathway post-RIT remains to be determined.

  5. Heparin-induced thrombocytopenia: when a low platelet count is a mandate for anticoagulation.

    PubMed

    Ortel, Thomas L

    2009-01-01

    Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by the development of antibodies to platelet factor 4 (PF4) and heparin. The thrombocytopenia is typically moderate, with a median platelet count nadir of approximately 50 to 60 x 10(9) platelets/L. Severe thrombocytopenia has been described in patients with HIT, and in these patients antibody levels are high and severe clinical outcomes have been reported (eg, disseminated intravascular coagulation with microvascular thrombosis). The timing of the thrombocytopenia in relation to the initiation of heparin therapy is critically important, with the platelet count beginning to drop within 5 to 10 days of starting heparin. A more rapid drop in the platelet count can occur in patients who have been recently exposed to heparin (within the preceding 3 months), due to preformed anti-heparin/PF4 antibodies. A delayed form of HIT has also been described that develops within days or weeks after the heparin has been discontinued. In contrast to other drug-induced thrombocytopenias, HIT is characterized by an increased risk for thromboembolic complications, primarily venous thromboembolism. Heparin and all heparin-containing products should be discontinued and an alternative, non-heparin anticoagulant initiated. Alternative agents that have been used effectively in patients with HIT include lepirudin, argatroban, bivalirudin, and danaparoid, although the last agent is not available in North America. Fondaparinux has been used in a small number of patients with HIT and generally appears to be safe. Warfarin therapy should not be initiated until the platelet count has recovered and the patient is systemically anticoagulated, and vitamin K should be administered to patients receiving warfarin at the time of diagnosis of HIT. PMID:20008202

  6. Ovariectomy aggravated sodium induced hypertension associated with altered platelet intracellular Ca2+ in Dahl rats.

    PubMed

    Otsuka, K; Ohno, Y; Sasaki, T; Yamakawa, H; Hayashida, T; Suzawa, T; Suzuki, H; Saruta, T

    1997-12-01

    Our purpose was to determine the effect of ovariectomy on intracellular Ca2+ mobilization and platelet aggregation in sodium induced hypertension. At the age of 12 weeks ovariectomy or sham operation was performed in female Dahl-Iwai salt sensitive rats on a 0.3% NaCl diet. Four weeks later we assessed the effects of ovariectomy and an 8% NaCl diet on agonist induced intracellular Ca2+ mobilization in fura-2 loaded platelets and platelet aggregation. Ovariectomy enhanced the increase of systolic blood pressure and heart to body weight ratio on an 8% NaCl diet. However, thrombin evoked intracellular Ca2+ was not correlated with systolic blood pressure (r = -0.338, P = .17), and was lowered by sodium loading and ovariectomy (360+/-23 to 285+/-9, 296+/-10 nmol/L, P < .05). Furthermore, the ionomycin induced intracellular calcium fraction in the absence of external Ca2+ that reflected internal Ca2+ discharge capacity was reduced in ovariectomized rats compared with sham operated rats on an 8% NaCl diet (648+/-15 v 768+/-35 nmol/L, P < .05). The internal Ca2+ discharge capacity was inversely correlated with systolic blood pressure (r = -0.506, P = .03). In addition to the decreased internal Ca2+ discharge capacity, intracellular Ca2+-independent platelet aggregation by phorbol 12-myristate 13-acetate, a protein kinase C activator, was significantly enhanced in hypertensive rats. We concluded that ovariectomy enhanced sodium induced hypertension associated with the decreased internal Ca2+ discharge capacity and increased platelet aggregation in Dahl-Iwai salt-sensitive rats.

  7. Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa.

    PubMed

    Al-Tamimi, Mohammad; Grigoriadis, George; Tran, Huy; Paul, Eldho; Servadei, Patricia; Berndt, Michael C; Gardiner, Elizabeth E; Andrews, Robert K

    2011-04-01

    This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

  8. Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa.

    PubMed

    Al-Tamimi, Mohammad; Grigoriadis, George; Tran, Huy; Paul, Eldho; Servadei, Patricia; Berndt, Michael C; Gardiner, Elizabeth E; Andrews, Robert K

    2011-04-01

    This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation. PMID:21252089

  9. Carotid endarterectomy in patients with heparin-induced platelet activation: comparative efficacy of aspirin and iloprost (ZK36374).

    PubMed

    Kappa, J R; Cottrell, E D; Berkowitz, H D; Fisher, C A; Sobel, M; Ellison, N; Addonizio, V P

    1987-05-01

    Patients with heparin-induced platelet activation who are reexposed to heparin may have recurrent thrombocytopenia, intravascular thrombosis, arterial emboli, or sudden death. To permit carotid endarterectomy in two patients with confirmed heparin-induced platelet activation, we compared the efficacies of aspirin and iloprost, a stable analogue of prostacyclin, in preventing heparin-induced platelet activation. In the first patient, although aspirin prevented both in vitro heparin-induced platelet aggregation (70% without and 7.5% with aspirin) and 14C serotonin release (48% without and 0% with aspirin), intraoperative administration of heparin resulted in an increase in plasma levels of platelet factor 4 from 8 to 260 ng/ml and beta-thromboglobulin levels from 29 to 39 ng/ml. In addition, the circulating platelet count decreased from 221,000 to 174,000 microliters, and 15% spontaneous platelet aggregation was observed. Fortunately, fibrinopeptide A levels remained less than 10 ng/ml intraoperatively, and no thrombotic complications occurred. In the second patient, aspirin did not prevent heparin-induced platelet aggregation in vitro (65% without and 41% with aspirin); however, iloprost (0.01 mumol/L) prevented both in vitro heparin-induced platelet aggregation (59.5% without and 0.0% with iloprost) and 14C serotonin release (56.7% without and 0.0% with iloprost). Therefore, a continuous infusion of iloprost was begun before administration of heparin and was continued until 20 minutes after reversal of heparin with protamine. After intraoperative administration of heparin, plasma levels of platelet factor 4 increased from 19 to 200 ng/ml, and beta-thromboglobulin levels increased from 56 to 76 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2437338

  10. Endothelial expression of E-selectin is induced by the platelet-specific chemokine platelet factor 4 through LRP in an NF-κB–dependent manner

    PubMed Central

    Yu, Guangyao; Rux, Ann H.; Ma, Peihong; Bdeir, Khalil; Sachais, Bruce S.

    2005-01-01

    The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4), a platelet-specific chemokine released on platelet activation, has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin RNA is time and dose dependent. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. PF4 induces E-selectin expression by activation of transcriptional activity. Activation of nuclear factor-κB is critical for PF4-induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. Further, we have identified the low-density lipoprotein receptor-related protein as the cell surface receptor mediating this effect. These results demonstrate that PF4 is able to increase expression of E-selectin by endothelial cells and represents another potential mechanism by which platelets may participate in atherosclerotic lesion progression. PMID:15591119

  11. Platelet lipidomic.

    PubMed

    Dolegowska, B; Lubkowska, A; De Girolamo, L

    2012-01-01

    Lipids account for 16-19 percent dry platelet matter and includes 65 percent phospholipids, 25 percent neutral lipids and about 8 percent glycosphingolipids. The cell membrane that surrounds platelets is a bilayer that contains different types phospholipids symmetrically distributed in resting platelets, such as phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine, and sphingomyelin. The collapse of lipid asymmetry is exposure of phosphatidylserine in the external leaflet of the plasma bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. During activation, this asymmetrical distribution becomes disrupted, and PS and PE become exposed on the cell surface. The transbilayer movement of phosphatidylserine is responsible for the platelet procoagulant activity. Exposure of phosphatidylserine is a flag for macrophage recognition and clearance from the circulation. Platelets, stored at room temperature for transfusion for more than 5 days, undergo changes collectively known as platelet storage lesions. Thus, the platelet lipid composition and its possible modifications over time are crucial for efficacy of platelet rich plasma therapy. Moreover, a number of substances derived from lipids are contained into platelets. Eicosanoids are lipid signaling mediators generated by the action of lipoxygenase and include prostaglandins, thromboxane A2, 12-hydroxyeicosatetraenoic acid. Isoprostanes have a chemical structure similar to this of prostanoids, but are differently produced into the particle, and are ligands for prostaglandins receptors, exhibiting biological activity like thromboxane A2. Endocannabinoids are derivatives from arachidonic acid which could reduce local pain. Phospholipids growth factors (sphingolipids, lysophosphatidic acid, platelet-activating factor) are involved in tissue

  12. Platelets prevent IFN-alpha/beta-induced lethal hemorrhage promoting CTL-dependent clearance of lymphocytic choriomeningitis virus.

    PubMed

    Iannacone, Matteo; Sitia, Giovanni; Isogawa, Masanori; Whitmire, Jason K; Marchese, Patrizia; Chisari, Francis V; Ruggeri, Zaverio M; Guidotti, Luca G

    2008-01-15

    We found that mice infected with different isolates of lymphocytic choriomeningitis virus (LCMV) develop a mild hemorrhagic anemia, which becomes severe and eventually lethal in animals depleted of platelets or lacking integrin beta3. Lethal hemorrhagic anemia is mediated by virus-induced IFN-alpha/beta that causes platelet dysfunction, mucocutaneous blood loss and suppression of erythropoiesis. In addition to the life-threatening hemorrhagic anemia, platelet-depleted mice fail to mount an efficient cytotoxic T lymphocyte (CTL) response and cannot clear LCMV. Transfusion of functional platelets into these animals reduces hemorrhage, prevents death and restores CTL-induced viral clearance in a manner partially dependent on CD40 ligand (CD40L). These results indicate that, upon activation, platelets expressing integrin beta3 and CD40L are required for protecting the host against the induction of an IFN-alpha/beta-dependent lethal hemorrhagic diathesis and for clearing LCMV infection through CTLs.

  13. The use of quartz crystal microbalance with dissipation (QCM-D) for studying nanoparticle-induced platelet aggregation

    PubMed Central

    Santos-Martinez, Maria Jose; Inkielewicz-Stepniak, Iwona; Medina, Carlos; Rahme, Kamil; D’Arcy, Deirdre M; Fox, Daniel; Holmes, Justin D; Zhang, Hongzhou; Radomski, Marek Witold

    2012-01-01

    Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D) has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast and scanning helium ion microscopy. PMID:22275839

  14. Mouse model of alloimmune-induced vascular rejection and transplant arteriosclerosis.

    PubMed

    Enns, Winnie; von Rossum, Anna; Choy, Jonathan

    2015-05-17

    Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

  15. Platelet function: aggregation by PAF or sequestration in lung is not modified during immediate or late allergen-induced bronchospasm in man.

    PubMed

    Hemmendinger, S; Pauli, G; Tenabene, A; Pujol, J L; Bessot, J C; Eber, M; Cazenave, J P

    1989-05-01

    Among the mediators involved in the pathophysiologic mechanisms that underly the reactions of the acute and delayed phases of bronchospasm induced by allergens in man, platelet-activating factor (PAF) could play an important role, in particular by its effects on platelets. In animals, inhalation or injection of PAF causes a platelet-dependent bronchoconstriction that is blocked by prior administration of an antiplatelet antiserum and accompanied by platelet accumulation in the pulmonary vessels. In man, inhalation of PAF causes a bronchospasm and induces a bronchial hyperreactivity. Abnormalities of platelet aggregation and the secretion into plasma of platelet factor 4 and beta-thromboglobulin have been described in patients with asthma during induced bronchospasm. Platelet functions have been studied in 15 patients with asthma before and after allergen bronchial provocation tests. There was no difference between platelet counts, plasma concentrations of platelet factor 4 and beta-thromboglobulin, and platelet aggregation induced by several agonists (adrenaline, arachidonic acid, or PAF) before and immediately after the allergen bronchial provocation test. There was no platelet pulmonary sequestration as studied with 111Indium-labeled platelets during 24 hours after the antigen challenge, and the life span of circulating platelets was normal. Our results do not support an important direct role for PAF in the pathophysiology of asthma. It is still possible that the current methodology is too insensitive to detect amounts of PAF in the circulation or that PAF is acting locally. PMID:2523922

  16. Three-dimentional simulation of flow-induced platelet activation in artificial heart valves

    NASA Astrophysics Data System (ADS)

    Hedayat, Mohammadali; Asgharzadeh, Hafez; Borazjani, Iman

    2015-11-01

    Since the advent of heart valve, several valve types such as mechanical and bio-prosthetic valves have been designed. Mechanical Heart Valves (MHV) are durable but suffer from thromboembolic complications that caused by shear-induced platelet activation near the valve region. Bio-prosthetic Heart Valves (BHV) are known for better hemodynamics. However, they usually have a short average life time. Realistic simulations of heart valves in combination with platelet activation models can lead to a better understanding of the potential risk of thrombus formation in such devices. In this study, an Eulerian approach is developed to calculate the platelet activation in three-dimensional simulations of flow through MHV and BHV using a parallel overset-curvilinear immersed boundary technique. A curvilinear body-fitted grid is used for the flow simulation through the anatomic aorta, while the sharp-interface immersed boundary method is used for simulation of the Left Ventricle (LV) with prescribed motion. In addition, dynamics of valves were calculated numerically using under-relaxed strong-coupling algorithm. Finally, the platelet activation results for BMV and MHV are compared with each other.

  17. Effect of diabetic duration on hemorheological properties and platelet aggregation in streptozotocin-induced diabetic rats

    PubMed Central

    Yeom, Eunseop; Byeon, Hyeokjun; Lee, Sang Joon

    2016-01-01

    Diabetes mellitus with abnormal glucose concentration is associated with changes in hemorheological properties, endothelial function, and platelets hyperactivity. Disturbances may significantly be responsible for diabetes-related vascular complications. In this study, hemorheological and hemodynamic properties were measured according to diabetic duration after streptozotocin treatment in rats. For ex vivo measurements, an extracorporeal model was adopted. Flow rate and blood viscosity were measured using a microfluidic device. Erythrocyte aggregation and morphological parameters of erythrocytes were measured by modified erythrocyte sedimentation rate and the phase-contrast holography under in vitro conditions. The platelet aggregation and mean pressure in the femoral artery were estimated under ex vivo conditions. Hemorheological properties including blood viscosity, erythrocyte aggregation and shape parameters for the control group are significantly different with those for diabetic groups. The changes with respect to diabetic duration were relatively unnoticeable. However, the platelet aggregation is strongly dependent on the diabetic duration. Based on these results, hyperglycemia exposure may induce hemorheological variations in early stages of diabetes mellitus. High platelet aggregation may become more pronounced according to the diabetic duration caused by variations in hemorheological properties resulting in endothelial dysfunction. This study would be helpful in understanding the effects of diabetic duration on biophysical properties. PMID:26898237

  18. Critical temperature ranges of hypothermia-induced platelet activation: possible implications for cooling patients in cardiac surgery.

    PubMed

    Straub, Andreas; Breuer, Melanie; Wendel, Hans P; Peter, Karlheinz; Dietz, Klaus; Ziemer, Gerhard

    2007-04-01

    Cooling of the patient is routinely applied in cardiac surgery to protect organs against ischemia. Hypothermia induces activation of platelets, but the effects of temperatures such as used during cardiac surgery are not well described. To investigate this in an in-vitro study heparinized whole blood was incubated at different temperatures (37 degrees C, 34.5 degrees C, 32 degrees C, 29.5 degrees C, 27 degrees C, 24.5 degrees C, 22 degrees C, 19.5 degrees C and 17 degrees C). The effect of these temperatures on aggregation, P-selectin expression, GP IIb/IIIa activation and platelet microparticle (PMP) formation of unstimulated and ADP-stimulated platelets of 36 subjects was evaluated in flow cytometry. A four-parametric logistic model was fitted to depict the temperature effect on platelet parameters. Lower temperatures increased aggregates, P-selectin expression, and GP IIb/IIIa activation. The number of PMPs decreases with hypothermia. Additional experiments revealed a slight influence of heparin on platelet P-selectin expression but excluded an effect of this anticoagulant on the other evaluated parameters. Threshold temperatures, which mark 5% changes of platelet parameters compared to values at 37 degrees C, were calculated. On ADP-stimulated platelets the thresholds for P-selectin expression and GP IIb/IIa activation are 34.0 degrees C and 36.4 degrees C, respectively, and lie in the temperature range routinely applied in cardiac surgery. Hypothermia-induced platelet activation may develop in most patients undergoing cardiac surgery, possibly resulting in thromboembolic events, coagulation defects, and proinflammatory leukocyte bridging by P-selectin bearing platelets and PMPs. These findings suggest that pharmacological protection of platelets against hypothermia-induced damage may be beneficial during cardiac surgery.

  19. Acquired dysfunction due to the circulation of "exhausted" platelets.

    PubMed

    Pareti, F I; Capitanio, A; Mannucci, L; Ponticelli, C; Mannucci, P M

    1980-08-01

    An acquired platelet functional defect was found to be present in eight patients who presented with various clinical conditions--three with renal allograft rejection, three with the hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, one with acute consumption coagulopathy due to an incompatible transfusion and one with systemic lupus erythematosus. They showed defective platelet aggregation and reduced levels of adenine nucleotides and serotonin with abnormal uptake and storage of the amine. The bleeding time was more prolonged than predicted from the platelet count. These abnormalities were strikingly similar to those occurring in patients with congenital storage pool deficiency. The acquired defect is thought to be related to the presence in the circulation of "exhausted" platelets following their in vivo exposure to inducers of the release reaction such as damaged endothelium, thrombin and immune complexes. The bleeding tendency of the underlying diseases might be aggravated by the impairment of platelet function. PMID:7405945

  20. Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

    PubMed

    Kageyama, Yasunari; Doi, Tomoaki; Akamatsu, Shigeru; Kuroyanagi, Gen; Kondo, Akira; Mizutani, Jun; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Ogura, Shinji

    2013-10-01

    We previously reported that the collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets. It has been shown that Rac, which belongs to the Rho family of small GTPases, is involved in the collagen-induced platelet aggregation. In this study, we investigated the role of Rac in the collagen-stimulated release of PDGF-AB and sCD40L in human platelets. Human blood was donated from healthy volunteers and platelet-rich plasma was obtained from the blood samples. The samples were then treated with 1.0 µg/ml collagen for 0, 1, 3, or 5 min and Rac1 activity was determined using the Rac1 Activation Assay kit. We found that collagen stimulated the activation of Rac in human platelets in a time-dependent manner. However, pre-treatment with NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, reduced the collagen-induced platelet aggregation. NSC23766 markedly attenuated not only the collagen-induced p44/p42 MAP kinase phosphorylation, but also the phosphorylation of HSP27 at three serine residues (Ser-15, Ser-78 and Ser-82). In addition, the collagen‑induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. These results strongly suggest that Rac regulates the collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets, resulting in the stimulation of PDGF-AB secretion and the release of sCD40L.

  1. Plasma Fibrinogen Is a Natural Deterrent to Amyloid β–Induced Platelet Activation and Neuronal Toxicity

    PubMed Central

    Sonkar, Vijay K; Kulkarni, Paresh P; Chaurasia, Susheel N; Dash, Ayusman; Jauhari, Abhishek; Parmar, Devendra; Yadav, Sanjay; Dash, Debabrata

    2016-01-01

    Alzheimer’s disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons and deposition of amyloid β (Aβ) in the form of extracellular plaques. Aβ is considered to have a critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid precursor protein that releases Aβ into circulation. We have recently demonstrated that the Aβ active fragment containing amino acid sequence 25–35 (Aβ25–35) is highly thrombogenic in nature and elicits strong aggregation of washed human platelets in a RhoA-dependent manner. In this study, we evaluated the influence of fibrinogen on Aβ-induced platelet activation. Intriguingly, Aβ failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aβ25–35, which could be reversed by increasing doses of Aβ. Fibrinogen also attenuated Aβ-induced platelet responses such as secretion, clot retraction, rise in cytosolic Ca+2 and reactive oxygen species. Fibrinogen prevented intracellular accumulation of full-length Aβ peptide (Aβ42) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aβ by preventing its interaction with cells. PMID:27262026

  2. Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction.

    PubMed

    Bank, Sarbashri; Jana, Pradipta; Maiti, Smarajit; Guha, Santanu; Sinha, A K

    2014-07-24

    The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 μM dermcidin, the platelets became resistant to the inhibitory effect of aspirin similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin antibody restored the sensitivity of the platelets to the aspirin effect. Incubation of AMI PRP pretreated with 15 μM aspirin, a stimulator of the NO synthesis, resulted in the increased production of NO in the platelets that removed the bound dermcidin by 40% from the high affinity binding sites of AMI platelets. When the same AMI PRP was retreated with 10 μM aspirin, the aggregation of platelets was completely inhibited by NO synthesis.

  3. Manipulation of oxygenation and flow-induced shear stress can increase the in vitro yield of platelets from cord blood.

    PubMed

    Lasky, Larry C; Sullenbarger, Brent

    2011-11-01

    A method to produce clinically useful platelets in vitro would help overcome the frequent shortages, donor deferrals, disease transmission, and alloimmunization with volunteer donor-derived platelets. Using CD34 positively selected cord blood cells, we investigated ways to increase platelet quality and yield in a three-dimensional modular perfusion bioreactor system. We found a two- to threefold increase in platelet numbers produced only when the early phases of the culture process were carried out at 5% oxygen, versus when 20% oxygen was used throughout the culture period (p<0.05), and much more than when 5% oxygen was used throughout. When the medium was routed through the cell-scaffold construct, versus when it flowed under and over the construct, or just intermittent feeding was used, the number of platelets increased two- to threefold (p<0.05), and enhanced collagen-induced aggregation. The 5% oxygen early in the culture process mimics the marrow adjacent to the bone where early progenitors proliferate. Flow through the cell-scaffold construct creates shear forces that mimic the flow in central venous sinuses of the marrow and enhances platelet production from proplatelets. The use of altered oxygen levels and cross flow enhanced platelet numbers and quality, and will contribute to eventual in vitro platelet production for clinical use.

  4. Platelet dysfunction induced by parenteral carbenicillin and ticarcillin. Studies of the dose-response relationship and mechanism of action in dogs.

    PubMed Central

    Johnson, G. J.; Rao, G. H.; White, J. G.

    1978-01-01

    Sequential studies of platelet function were performed in dogs receiving continuous intravenous carbenicillin (CARB) or ticarcillin (TIC). Dose- and time-dependent platelet dysfunction was uniformly observed during the administration of CARB or TIC, 250 to 1000 mg/kg/24 hr. ADP-induced primary and secondary platelet aggregation was markedly inhibited within 24 to 48 hours in dogs receiving 750 or 1000 mg/kg/24 hr, but maximum impairment of aggregation did not occur until 3 to 5 days in dogs receiving 250 or 500 mg/kg/24 hr. Platelet glass bead column retention was abnormal in all dogs studied, and platelet factor 3 availability was impaired in 91%. Collagen-induced platelet aggregation was consistently impaired and the bleeding time was prolonged only during the infusion of greater than or equal to 750 mg/kg/24 hr. Plasma fibrinogen concentrations and thrombin times remained normal. CARB and TIC infusions resulted in inhibition of 14C-serotonin release and slightly decreased platelet ADP, while serotonin, ATP, and ultrastructure remained unchanged. The mutual correction of abnormal platelet aggregation by mixing CARB or TIC platelets with aspirin-treated platelets suggested that CARB and TIC inhibited the platelet release reaction by a mechanism other than inhibition of platelet cyclo-oxygenase. The platelet inhibitory properties of CARB and TIC demonstrated in this study suggest that they may be useful antithrombotic agents. PMID:645824

  5. Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets

    SciTech Connect

    Bruene, B.; Molina Y Vedia, L.; Lapetina, E.G. )

    1990-05-01

    {alpha}-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by {alpha}-thrombin was clearly distinct from the {alpha} subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively G{sub i{alpha}} and G{sub s{alpha}}; the 47-kDa protein that is phosphorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

  6. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of /sup 125/I-fibrinogen to rat platelets

    SciTech Connect

    McGregor, L.; Toor, B.; McGregor, J.L.; Renaud, S.; Clemetson, K.J.

    1984-03-01

    The aggregation to ADP and the binding of /sup 125/I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.

  7. Methylglyoxal induces platelet hyperaggregation and reduces thrombus stability by activating PKC and inhibiting PI3K/Akt pathway.

    PubMed

    Hadas, Karin; Randriamboavonjy, Voahanginirina; Elgheznawy, Amro; Mann, Alexander; Fleming, Ingrid

    2013-01-01

    Diabetes is characterized by a dysregulation of glucose homeostasis and platelets from patients with diabetes are known to be hyper-reactive and contribute to the accelerated development of vascular diseases. Since many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG) rather than to hyperglycemia itself, the aim of the present study was to characterize the effects of MG on platelet function. Washed human platelets were pre-incubated for 15 min with MG and platelet aggregation, adhesion on matrix-coated slides and signaling (Western blot) were assessed ex vivo. In vivo, the effect of MG on thrombus formation was determined using the FeCl3-induced carotid artery injury model. MG potentiated thrombin-induced platelet aggregation and dense granule release, but inhibited platelet spreading on fibronectin and collagen. In vivo, MG accelerated thrombus formation but decreased thrombus stability. At the molecular level, MG increased intracellular Ca(2+) and activated classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signaling. In conclusion, these findings indicate that the enhanced MG concentration measured in diabetic patients can directly contribute to the platelet dysfunction associated with diabetes characterized by hyperaggregability and reduced thrombus stability.

  8. Alleviation of viper venom induced platelet apoptosis by crocin (Crocus sativus): implications for thrombocytopenia in viper bites.

    PubMed

    Santhosh, M Sebastin; Thushara, R M; Hemshekhar, M; Sunitha, K; Devaraja, S; Kemparaju, K; Girish, K S

    2013-11-01

    Viper envenomations are characterized by prominent local and systemic manifestations including hematological alterations. Snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2) plays crucial role in the pathophysiology of hemorrhage by targeting/altering the platelets function which may result in thrombocytopenia. Platelets undergo the classic events of mitochondria-mediated apoptotic pathway due to augmented endogenous reactive oxygen species (ROS) levels. The observed anticoagulant effects during viper envenomations could be due to exacerbated platelet apoptosis and thrombocytopenia. Moreover, antivenin treatments are ineffective against the venom-induced oxidative stress; therefore, it necessitates an auxiliary therapy involving antioxidants which can effectively scavenge the endothelium-generated/endogenous ROS and protect the platelets. The present study explored the effects of viper venom on platelet apoptosis and its amelioration by a phytochemical crocin. The study evaluated the Vipera russelli venom-induced apoptotic events including endogenous ROS generation, intracellular Ca(2+) mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation and phosphatidylserine externalization which were effectively mitigated when the venom was pre-treated with crocin. The study highlights one of the less studied features of venom-induced secondary complications i.e. platelet apoptosis and sheds light on the underlying basis for venom-induced thrombocytopenia, systemic hemorrhage and in vivo anticoagulant effect.

  9. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    PubMed

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  10. A role for platelet glycoprotein Ib-IX and effects of its inhibition in endotoxemia-induced thrombosis, thrombocytopenia and mortality

    PubMed Central

    Yin, Hong; Stojanovic, Aleksandra; Xu, Weidong; Corken, Adam; Zakharov, Alexander; Qian, Feng; Pavlovic, Sasha; Krbanjevic, Aleksandar; Lyubimov, Alexander V.; Wang, Zaijie J.; Ware, Jerry; Du, Xiaoping

    2014-01-01

    Objective Poor prognosis of sepsis is associated with bacterial lipopolysaccharide (LPS)-induced intravascular inflammation, microvascular thrombosis, thrombocytopenia, and disseminated intravascular coagulation. Platelets are critical for thrombosis, and there have been increasing evidence of the importance of platelets in endotoxemia. The platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), mediates platelet adhesion to inflammatory vascular endothelium and exposed subendothelium. Thus, we have investigated the role of GPIb-IX in LPS-induced platelet adhesion, thrombosis and thrombocytopenia. Approach and Results LPS-induced mortality is significantly decreased in mice expressing a functionally deficient mutant of GPIbα. Furthermore, we have developed a micellar peptide inhibitor, MPαC, which selectively inhibits the VWF-binding function of GPIb-IX and GPIb-IX-mediated platelet adhesion under flow without affecting GPIb-IX-independent platelet activation. MPαC inhibits platelet adhesion to LPS-stimulated endothelial cells in vitro and alleviates LPS-induced thrombosis in glomeruli in mice. Importantly, MPαC reduces mortality in LPS-challenged mice, suggesting a protective effect of this inhibitor during endotoxemia. Interestingly, MPαC, but not the integrin antagonist, Integrilin, alleviated LPS-induced thrombocytopenia. Conclusion These data indicate an important role for the platelet adhesion receptor GPIb-IX in LPS-induced thrombosis and thrombocytopenia, and suggest the potential of targeting GPIb as an anti-platelet strategy in managing endotoxemia. PMID:24051142

  11. Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study.

    PubMed

    Scharbert, G; Kalb, M L; Essmeister, R; Kozek-Langenecker, S A

    2010-01-01

    The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30-34 degrees C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37 degrees C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate at five different test temperatures between 30 degrees C and 34 degrees C. Aggregation responses at 37 degrees C served as controls. At temperatures of mild and moderate hypothermia (30-34 degrees C), overall platelet aggregation was increased compared to 37 degrees C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31 degrees C and 34 degrees C and in response to adenosine diphosphate between 30 degrees C and 34 degrees C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30 degrees C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30 degrees C and 34 degrees C requires further detailed study. PMID:19954411

  12. Anti-Human Leukocyte Antigen Antibody Induced Autoimmunity: Role In Chronic Rejection

    PubMed Central

    Nath, DS; Basha, H Ilias; Mohanakumar, T

    2009-01-01

    Purpose of review We provide evidence for the role of de novo development of immune responses to self-antigens in the post-transplant period and its possible induction by alloimmunity in the pathogenesis of chronic rejection following lung, heart and kidney transplantation. This review details recent findings for the two distinct yet inter-dependent immune processes in the immune-pathogenesis of chronic rejection. Recent findings The contribution of both humoral and cell mediated allo-immune responses against mismatched donor histocompatibility antigens (HLA) in the pathogenesis of chronic rejection is well established. Recent studies have focused on development of immune responses to self-antigens during the post-transplant period and its correlation with chronic rejection. These self-antigens include myosin and vimentin in cardiac, K-alpha-1-tubulin and collagen-V in lung and angiotensin II type 1 receptor, collagen-IV and VI in kidney transplants. During the post-transplant period, the development of immune responses to self-antigens is facilitated by induction of a distinct subset of auto-reactive T-helper cells referred to as Th17 cells. Summary Following organ transplantation, tissue injury and remodeling inflicted by Abs to HLA antigens is conducive to develop autoimmunity. Antibodies (Abs) to HLA and self-antigens are detectable in the serum of transplant recipients who develop chronic rejection. Anti-HLA Abs are often present transiently but precede the development of Abs to self-antigens. PMID:19898237

  13. Normalizing Rejection.

    PubMed

    Conn, Vicki S; Zerwic, Julie; Jefferson, Urmeka; Anderson, Cindy M; Killion, Cheryl M; Smith, Carol E; Cohen, Marlene Z; Fahrenwald, Nancy L; Herrick, Linda; Topp, Robert; Benefield, Lazelle E; Loya, Julio

    2016-02-01

    Getting turned down for grant funding or having a manuscript rejected is an uncomfortable but not unusual occurrence during the course of a nurse researcher's professional life. Rejection can evoke an emotional response akin to the grieving process that can slow or even undermine productivity. Only by "normalizing" rejection, that is, by accepting it as an integral part of the scientific process, can researchers more quickly overcome negative emotions and instead use rejection to refine and advance their scientific programs. This article provides practical advice for coming to emotional terms with rejection and delineates methods for working constructively to address reviewer comments. PMID:26041785

  14. Prevention of gastric mucosal injury induced by anti-platelet drugs by famotidine.

    PubMed

    Uotani, Takahiro; Sugimoto, Mitsushige; Nishino, Masafumi; Ichikawa, Hitomi; Sahara, Shu; Yamade, Mihoko; Iwaizumi, Moriya; Yamada, Takanori; Osawa, Satoshi; Sugimoto, Ken; Umemura, Kazuo; Watanabe, Hiroshi; Miyajima, Hiroaki; Furuta, Takahisa

    2014-08-01

    Anti-platelet drug-induced gastric mucosal injury correlates with intragastric pH. Our aim was to investigate prophylaxis effects of famotidine, one of the representative histamine-2 receptor antagonists (H2RA), on gastric mucosal injury induced by dual therapy with low-dose aspirin and clopidogrel in relation to Helicobacter pylori (H. pylori) infection and CYP2C19 genotypes. This study was conducted for 20 healthy Japanese volunteers (10 H. pylori-positive and 10-negative subjects) with 100 mg aspirin plus 75 mg clopidogrel (AC) once-daily dosing and AC plus 20 mg famotidine twice-daily dosing (ACH). Mucosal injury was endoscopically assessed on day 3 and 7 and 24-hour intragastric pH and antiplatelet-function test was performed on day 7. Median pH in ACH was similar between CYP2C19 extensive metabolizer (EM) and intermediate metabolizer (IM)/poor metabolizer (PM) and was significantly higher in H. pylori-positive than negative subjects (P < .05). Mucosal injury with ACH significantly decreased in both day 3 and 7 compared with AC, irrespective with H. pylori and CYP2C19 genotypes (P < .05). Although antiplatelet effect of clopidogrel in EM was significantly higher than that in IM/PM, the additional famotidine did not affect the effect. Anti-platelet drug-induced gastric injury was alleviated by famotidine without attenuation of anti-platelet functions irrespective of H. pylori and CYP2C19 genotypes. PMID:24615745

  15. The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

    PubMed

    Jackson, Elke C G; Ortar, Giorgio; McNicol, Archie

    2013-12-01

    Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2β1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response.

  16. Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

    PubMed Central

    Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

    2011-01-01

    BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

  17. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

  18. Differential inhibition of tumour cell-induced platelet aggregation by the nicotinate aspirin prodrug (ST0702) and aspirin

    PubMed Central

    Medina, Carlos; Harmon, Shona; Inkielewicz, Iwona; Santos-Martinez, Maria Jose; Jones, Michael; Cantwell, Paula; Bazou, Despina; Ledwidge, Mark; Radomski, Marek W; Gilmer, John F

    2012-01-01

    BACKGROUND AND PURPOSE Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA. EXPERIMENTAL APPROACH TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively. KEY RESULTS Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA. CONCLUSIONS AND IMPLICATIONS Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA. PMID:22122360

  19. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  20. Platelet-activating factor induces eosinophil peroxidase release from purified human eosinophils.

    PubMed Central

    Kroegel, C; Yukawa, T; Dent, G; Chanez, P; Chung, K F; Barnes, P J

    1988-01-01

    The degranulation response of purified human eosinophils to platelet-activating factor (PAF) has been studied. PAF induced release of eosinophil peroxidase (EPO) and beta-glucuronidase from highly purified human eosinophils with an EC50 of 0.9 nM. The order of release was comparable with that induced by phorbol myristate acetate (PMA). The new specific PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-H-thieno[3,2-f] [1,2,4]triazolo-[4,3a][1,4]-diazepin-2-yl](4-morpholinyl)- 1-propane-one (WEB 2086) inhibited the PAF-induced enzyme release by human eosinophils in a dose-dependent manner. The viability of eosinophils were unaffected both by PAF and WEB 2086. The results suggest that PAF may amplify allergic and inflammatory reactions by release of preformed proteins from eosinophil granules. PMID:3410498

  1. Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

    PubMed Central

    Bonacina, F.; Barbieri, S.S.; Cutuli, L.; Amadio, P.; Doni, A.; Sironi, M.; Tartari, S.; Mantovani, A.; Bottazzi, B.; Garlanda, C.; Tremoli, E.; Catapano, A.L.; Norata, G.D.

    2016-01-01

    Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cell's derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis. PMID:26976330

  2. Characterization of platelet-activating factor-induced cutaneous edema and erythema in dogs.

    PubMed

    Watanabe, Miwa; Osada, Hironari; Shimizu, Sunao; Goto, Shun; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Ohmori, Keitaro

    2016-09-01

    OBJECTIVE To characterize platelet-activating factor (PAF)-induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions. ANIMALS 6 healthy Beagles. PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 μg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each). RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema. CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation. PMID:27580108

  3. Serotonin-induced platelet aggregation predicts the antihypertensive response to serotonin receptor antagonists.

    PubMed

    Gleerup, G; Persson, B; Hedner, T; Winther, K

    1993-01-01

    The 5-HT2-receptor antagonist ketanserin (20-40 mg b.i.d.) was administered to 62 patients of both sexes with uncomplicated primary hypertension. After 4 weeks of treatment about 50% of the patients had reached the target diastolic blood pressure of 90 mm Hg or below. Interindividual variability was large. In a retrospective analysis the variability could not be explained by sex or the dose of ketanserin. There was a weak association between age and systolic blood pressure response (r = 0.24; P = 0.06), which could be entirely accounted for by the higher base line blood pressure in the elderly patients. In one group of patients (n = 12), the ex vivo aggregation to serotonin (10(-6) M) was studied during treatment with placebo and ketanserin. Ketanserin completely inhibited 5-HT-induced aggregation in all patients. There was a close correlation between the area under the 5-HT-induced platelet aggregation curve during placebo and the subsequent reduction in diastolic blood pressure after 4 weeks of treatment with ketanserin. The present data suggest that the blood pressure response to ketanserin can be predicted from the ex vivo sensitivity of platelets to serotonin. By implication, they also support a role for serotonergic mechanisms in hypertension.

  4. Regulation of hormone-induced Ca sup 2+ mobilization in the human platelets

    SciTech Connect

    Crouch, M.F.; Lapetina, E.G. )

    1990-03-01

    {alpha}-Thrombin, {gamma}-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca{sup 2+} stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca{sup 2+} level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on {alpha}{sub 2}-adrenergic receptors, was by itself ineffective at mobilizing Ca{sup 2+} stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca{sup 2+} from cellular stores. This appeared to be due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. The current work is to characterize this protein that may be a member of the G{sub i}, GTP-binding protein family.

  5. Albumin inhibits platelet-activating factor (PAF)-induced responses in platelets and macrophages: implications for the biologically active form of PAF.

    PubMed Central

    Grigoriadis, G.; Stewart, A. G.

    1992-01-01

    1. Platelet-activating factor (PAF) binds with high affinity to albumin leading Clay et al. (1990) to suggest that the active form of PAF is the albumin-PAF complex. 2. In the present study the proposal that albumin-bound, rather than monomeric PAF, is the active form of PAF at PAF receptors was critically evaluated by examining the effect of albumin on the potency of PAF in isolated platelets and macrophages. 3. Bovine serum albumin inhibited concentration-dependently PAF-induced responses in platelets and macrophages. The most probable explanation of this finding is that BSA reduced the concentration of free PAF. 4. Thus, we conclude that free PAF, rather than the albumin-PAF complex is the active form. Consequently, local concentrations of albumin will influence profoundly the potency of endogenously released PAF. Moreover, estimates of the affinity of PAF for PAF receptors made in buffers containing BSA, underestimate the true affinity of PAF for its receptors by approximately 3 orders of magnitude. PMID:1330167

  6. Rejection episodes.

    PubMed

    Koyama, H; Cecka, J M

    1992-01-01

    Based upon analyses of 40,671 kidney transplants reported to the UNOS Scientific Renal Transplant Registry between October 1987 and August 1992: 1. Twenty-four percent of the 21,923 recipients of first cadaver grafts experienced one or more rejection episodes during their transplant hospitalization, 52% during the first 6 months. At 12 months, only 40% of patients remained rejection-free. Patients who experienced any rejection during the first 6 months had a 72% 1-year graft survival rate compared with 95% for those who remained rejection-free (p < 0.001). 2. Recipients of transplants from living donors had a significantly lower incidence of rejection episodes. There was a clear effect of histocompatibility in comparing the incidence of rejection in HLA-identical sibling transplants (8% at discharge and 32% at 1 year) with that in 1-haplotype disparate transplants (22% at discharge and 52% at 1 year, p < 0.01 at each time point). Rejections were reported for 25% of transplants from other living donors at discharge and for 56% at 1 year, similar to the figures for cadaver transplants. 3. Histocompatibility also influenced the incidence of rejection in first cadaver-donor transplants. Only 15% of recipients of 0-HLA-A,B mismatched kidneys had rejection episodes reported at discharge, compared with 26% of those who received kidneys completely mismatched for HLA-A,B antigens (p < 0.01). At 1 year, 56% of HLA-A,B matched patients remained rejection-free, whereas only 35% of those mismatched for 4 antigens had no reported rejection through the first year (p < 0.01). Considering HLA-DR antigen mismatches, 19% of the 0-antigen mismatched group had rejection episodes at discharge, versus 28% for those with 2 HLA-DR mismatches (p < 0.01), and at 1 year, the percentage who were rejection-free decreased from 48% to 40% and 34% with 0, 1, and 2 HLA-DR mismatches, respectively. 4. The incidence of rejection episodes decreased as the recipient's age increased. Patients under age

  7. Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

    NASA Astrophysics Data System (ADS)

    Wei, Yuxi; Wang, Changyun; Li, Jing; Guo, Qi; Qi, Hongtao

    2009-09-01

    We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA ( P<0.01) and increasing the synthesis of 6-keto-PGF1α, thus changing the plasma TXB2/6-keto-PGF1α balance when the platelets were activated ( P<0.01). Therefore, STP altered AA metabolism and these findings partly revealed the molecular mechanism by which STP inhibits ADP-induced platelet aggregation.

  8. Orally given gastroprotective capsaicin does not modify aspirin-induced platelet aggregation in healthy male volunteers (human phase I examination).

    PubMed

    Sandor, B; Papp, J; Mozsik, Gy; Szolcsanyi, J; Keszthelyi, Zs; Juricskay, I; Toth, K; Habon, Tamas

    2014-12-01

    Capsaicin is a well-known component of red pepper. Recent studies have shown that capsaicin could prevent gastric ulcer provoked by various NSAID-s like acetylsalicylic acid (ASA). Primary objective of this human clinical phase I trial was to investigate whether two different doses of capsaicin co-administered with ASA could alter the inhibitory effect of ASA on platelet aggregation. 15 healthy male subjects were involved in the study and treated orally with 400 μg capsaicin, 800 μg capsaicin, 500 mg ASA, 400 μg capsaicin+500 mg ASA and 800 μg capsaicin+500 mg ASA. Blood was drawn before and 1, 2, 6 and 24 hours after the drug administration. After that epinephrine induced platelet aggregation was measured by optical aggregometry. Between treatments, volunteers had a 6-day wash-out period. Our results showed that capsaicin had no effect on platelet aggregation, while as expected, ASA monotherapy resulted in a significant and clinically effective platelet aggregation inhibition (p ≤ 0.001). The combined ASA-capsaicin therapies reached equivalent effectiveness in platelet aggregation inhibition as ASA monotherapy. Our investigation proved that capsaicin did not influence the inhibitory effect of ASA on platelet aggregation, thus the capsaicin-ASA treatment would combine the antiplatelet effect of ASA with the possible gastroprotection of capsaicin.

  9. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    SciTech Connect

    Ryu, T.; Davis, J.M.; Schwartz, K.A. )

    1990-07-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.

  10. A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α2β1

    PubMed Central

    JARVIS, G. E.; BIHAN, D.; HAMAIA, S.; PUGH, N.; GHEVAERT, C. J. G.; PEARCE, A. C.; HUGHES, C. E.; WATSON, S. P.; WARE, J.; RUDD, C. E.; FARNDALE, R. W.

    2013-01-01

    Summary Background Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α2β1. Adhesion and degranulation-promoting adapter protein (ADAP) regulates αIIbβ3 in platelets and αLβ2 in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen. Objectives To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α2β1. Methods Using ADAP−/− mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP−/− platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α2β1-selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α2β1, was reduced in ADAP−/− platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP−/− platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α2β1. In addition, we found that ADAP−/− mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation. PMID:22103309

  11. Correct acceptance weighs more than correct rejection: a decision bias induced by question framing.

    PubMed

    Kareev, Yaakov; Trope, Yaacov

    2011-02-01

    We propose that in attempting to detect whether an effect exists or not, people set their decision criterion so as to increase the number of hits and decrease the number of misses, at the cost of increasing false alarms and decreasing correct rejections. As a result, we argue, if one of two complementary events is framed as the positive response to a question and the other as the negative response, people will tend to predict the former more often than the latter. Performance in a prediction task with symmetric payoffs and equal base rates supported our proposal. Positive responses were indeed more prevalent than negative responses, irrespective of the phrasing of the question. The bias, slight but consistent and significant, was evident from early in a session and then remained unchanged to the end. A regression analysis revealed that, in addition, individuals' decision criteria reflected their learning experiences, with the weight of hits being greater than that of correct rejections.

  12. Serotonin-induced mate rejection in the female cabbage butterfly, Pieris rapae crucivora

    NASA Astrophysics Data System (ADS)

    Obara, Yoshiaki; Fukano, Yuya; Watanabe, Kenta; Ozawa, Gaku; Sasaki, Ken

    2011-11-01

    Virgin female cabbage butterflies, Pieris rapae crucivora, accept and mate with courting males, whereas mated females reject them and assume the "mate refusal posture". This study tested whether the biogenic amines, serotonin (5HT), dopamine (DA), and octopamine (OA), were responsible for this change in behavior. The results showed that 2-3-day-old virgin females fed with 5HT rejected courting males significantly more frequently compared with controls fed on sucrose. In contrast, the proportions of courting males rejected by virgin females fed with either DA or OA did not differ from sucrose-fed controls. Oral application of each amine resulted in significantly increased levels of the amine applied (or its metabolite) in the brain. The results strongly suggest that 5HT or a 5HT metabolite may be responsible for the post-mating change in behavioral response of 2-3-day-old virgin females to courting males. Similar effects of 5HT treatment were observed in 6-8-day-old virgin females, but in this case the results were only marginally different from the controls, suggesting that the effect may decline with increasing female age.

  13. In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry

    PubMed Central

    Nielsen, Tine Bo; Nielsen, Morten Hjuler; Handberg, Aase

    2015-01-01

    Microvesicles (MVs) are submicron vesicles with sizes of 0.1–1.0 μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5 μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5 μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as <1 μm and positive for lactadherin binding and cell-specific markers. Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of

  14. Differential Inhibition of Human Atherosclerotic Plaque–Induced Platelet Activation by Dimeric GPVI-Fc and Anti-GPVI Antibodies

    PubMed Central

    Jamasbi, Janina; Megens, Remco T.A.; Bianchini, Mariaelvy; Münch, Götz; Ungerer, Martin; Faussner, Alexander; Sherman, Shachar; Walker, Adam; Goyal, Pankaj; Jung, Stephanie; Brandl, Richard; Weber, Christian; Lorenz, Reinhard; Farndale, Richard; Elia, Natalie; Siess, Wolfgang

    2015-01-01

    Background Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. Objectives This study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Methods Human atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. Results GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Conclusions Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences

  15. Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans.

    PubMed

    Morita, Akihiro; Isawa, Haruhiko; Orito, Yuki; Iwanaga, Shiroh; Chinzei, Yasuo; Yuda, Masao

    2006-07-01

    To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates. PMID:16759235

  16. Alpha-adrenoceptor blockade by phentolamine inhibits adrenaline-induced platelet activation in vivo without affecting resting measurements.

    PubMed

    Larsson, P T; Wallén, N H; Egberg, N; Hjemdahl, P

    1992-04-01

    1. The effects of phentolamine (500 micrograms/min) on platelet aggregability in vivo at rest and during adrenaline infusion were assessed by ex vivo filtragometry and measurements of plasma beta-thromboglobulin levels in 10 healthy male subjects. Plasma levels of von Willebrand factor antigen and free fatty acids were also measured. 2. Adrenaline induced marked and expected increases in heart rate and systolic blood pressure and decreased diastolic blood pressure when venous plasma adrenaline levels were elevated from 0.12 +/- 0.02 to 2.9 +/- 0.3 nmol/l (P less than 0.01). 3. Adrenaline caused platelet activation in vivo. Ex vivo filtragometry readings were shortened by 58 +/- 9% (P less than 0.01), plasma beta-thromboglobulin levels increased by 99 +/- 44% (P less than 0.01) and platelet counts increased by 26 +/- 6% (P less than 0.01). Plasma levels of von Willebrand factor antigen and free fatty acids increased by 53 +/- 5% and 475 +/- 113% (both P less than 0.01), respectively. 4. Phentolamine enhanced the beta-adrenergic vasodilator responses to adrenaline, as both the decrease in diastolic blood pressure and the reflexogenic increase in heart rate were enhanced (both P less than 0.01). Marked elevations in plasma noradrenaline levels were found during infusions of phentolamine and adrenaline (P less than 0.001). 5. Phentolamine did not alter platelet indices at rest, but abolished adrenaline-induced platelet activation, as filtragometry readings, plasma beta-thromboglobulin levels and platelet counts remained at, or below, resting levels. Responses of plasma levels of von Willebrand factor antigen and free fatty acids to adrenaline were not influenced by phentolamine and did not seem to influence platelet responses.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. A serotonin-induced N-glycan switch regulates platelet aggregation

    PubMed Central

    Mercado, Charles P.; Quintero, Maritza V.; Li, Yicong; Singh, Preeti; Byrd, Alicia K.; Talabnin, Krajang; Ishihara, Mayumi; Azadi, Parastoo; Rusch, Nancy J.; Kuberan, Balagurunathan; Maroteaux, Luc; Kilic, Fusun

    2013-01-01

    Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation. PMID:24077408

  18. A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation.

    PubMed

    Boukerche, H; Ruchaud-Sparagano, M H; Rouen, C; Brochier, J; Kaplan, C; McGregor, J L

    1996-02-01

    P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M(r)) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIb alpha). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825 +/- 2886, mean +/- SD) Kd = 1.5 +/- 0.5 nM) compared to resting platelets (2801 +/- 1278, mean +/- SD) (Kd = 1.5 +/- 0.6 nM), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab')2 or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell-cell contact, may play an active role in platelet-platelet interactions. PMID:8603015

  19. Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα

    PubMed Central

    Gitz, Eelo; Koopman, Charlotte D.; Giannas, Alèkos; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan-Willem N.; Gerritsen, Hans C.; Urbanus, Rolf T.

    2013-01-01

    Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation. PMID:23753027

  20. Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation.

    PubMed

    Vanassche, Thomas; Kauskot, Alexandre; Verhaegen, Jan; Peetermans, Willy E; van Ryn, Joanne; Schneewind, Olaf; Hoylaerts, Marc F; Verhamme, Peter

    2012-06-01

    Interactions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus. PMID:22437005

  1. The Heparin-Induced Thrombocytopenia and Thrombosis Syndrome: Treatment with Intraarterial Urokinase and Systemic Platelet Aggregation Inhibitors

    SciTech Connect

    Murphy, Kenneth D.; McCrohan, Gerard; DeMarta, Deborah A.; Shirodkar, Nitin B.; Kwon, Oun J.; Chopra, Paramjit S.

    1996-03-15

    We report a case of the heparin-induced thrombocytopenia and thrombosis syndrome presenting with acute ischemia of a lower limb. The patient was successfully treated by withdrawal of heparin products, intraarterial urokinase, and platelet anti-aggregation therapy consisting of Dextran and aspirin.

  2. Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC).

    PubMed

    Ohana, Ofra Malka; Ozer, Janet; Prinsloo, Isebrand; Benharroch, Daniel; Gopas, Jacob

    2015-01-01

    Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (Lewis(X)), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

  3. Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC).

    PubMed

    Ohana, Ofra Malka; Ozer, Janet; Prinsloo, Isebrand; Benharroch, Daniel; Gopas, Jacob

    2015-01-01

    Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (Lewis(X)), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system. PMID:26418972

  4. Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC)

    PubMed Central

    Ohana, Ofra Malka; Ozer, Janet; Prinsloo, Isebrand; Benharroch, Daniel; Gopas, Jacob

    2015-01-01

    Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (LewisX), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system. PMID:26418972

  5. A noble function of BAY 11-7082: Inhibition of platelet aggregation mediated by an elevated cAMP-induced VASP, and decreased ERK2/JNK1 phosphorylations.

    PubMed

    Lee, Hyun-Sub; Kim, Sung Dae; Lee, Whi Min; Endale, Mehari; Kamruzzaman, S M; Oh, Won Jun; Cho, Jae Youl; Kim, Sang Keun; Cho, Hyun-Jeong; Park, Hwa-Jin; Rhee, Man Hee

    2010-02-10

    Platelets, though anucleated, possess several transcription factors, including NF-kappaB, that exert non-genomic functions regulating platelet activation. Since platelets have not only been recognized as central players of homeostasis, but also participated in pathological conditions such as thrombosis, atherosclerosis, and inflammation, we examined rat platelet NF-kappaB expression and evaluated the effects of anti-inflammatory drug BAY 11-7082, an inhibitor of NF-kappaB activation, in platelet physiology. Western blotting revealed that rat platelets express NF-kappaB. BAY 11-7082, dose dependently, inhibited collagen- or thrombin-induced-platelet aggregation. ATP release, TXB(2) formation, P-selectin expression, and intercellular Ca(2+) concentration activated by collagen were reduced in BAY 11-7082-treated platelets. BAY 11-7082 elevated intracellular levels of cAMP, but not cGMP, and its co-incubation with cAMP-activating agent (forskolin) or its hydrolyzing enzyme inhibitor (3-isobutyl-1-methylxanthine, IBMX), synergistically inhibited collagen-induced-platelet aggregation. In addition, vasodilator-stimulated-phosphoprotein (VASP) phosphorylation was enhanced in BAY 11-7082-treated platelets, which was partially inhibited by a protein kinase A (PKA) inhibitor, H-89. Moreover, while p38 mitogen-activated protein kinase (MAPK) was not affected, BAY 11-7082 attenuated c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. In conclusion, BAY 11-7082 inhibits platelet activation, granule secretion, and aggregation, and that this effect is mediated by inhibition of JNK1 and ERK2 phosphorylations, and partially by stimulation of cAMP-dependent PKA VASP phosphorylation. The ability of BAY 11-7082 to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the drug is considered as anti-atherothrombosis, and anti-inflammatory therapy.

  6. Serotonin uptake rates in platelets from angiotensin II-induced hypertensive mice.

    PubMed

    Singh, Preeti; Fletcher, Terry W; Li, Yicong; Rusch, Nancy J; Kilic, Fusun

    2013-03-01

    Angiotensin II (Ang II) is a critical component of the renin-angiotensin system that contributes to hypertension. Although platelets in blood from hypertensive subjects have an abnormal biological profile, it is unclear if circulating Ang II influences platelet aggregation or thrombus formation. One of the abnormalities presented to the platelets during hypertension is an elevated plasma concentration of serotonin (5-HT) caused by reduced 5-HT uptake secondary to loss of the 5-HT transporter (SERT) on the platelet plasma membrane. In the current study, we evaluated in vivo platelet function after 7 days of subcutaneous Ang II infusion to establish hypertension in mice and additionally assessed the biology of isolated platelets exposed to Ang II in vitro. The administration of Ang II elevated systolic blood pressure, but markers of platelet activation including P-selectin and (PE)Jon/A staining were not changed. However, the aggregation response to collagen was reduced in isolated platelets from Ang II-infused mice, which also showed reduced 5-HT uptake by SERT. In vitro exposure of isolated platelets to Ang II also resulted in a loss of surface SERT associated with a reduced aggregation response to collagen. These abnormalities were reversed by increasing concentrations of the Ang II receptor antagonist, valsartan. Interestingly, SERT KO mice failed to fully develop hypertension in response to Ang II infusion and isolated platelets from these animals were insensitive to the anti-aggregatory influence of Ang II. Thus, Ang II blunts the aggregation responses of platelets and the mechanism underlying this action may involve a loss of SERT on the platelet plasma membrane. The latter event depletes intracellular 5-HT in platelets, an event that is associated with reduced aggregation. The widespread use of antihypertensive drugs that target the renin-angiotensin system suggest the potential clinical utility of our findings and emphasize the importance of understanding

  7. Serotonin uptake rates in platelets from angiotensin II-induced hypertensive mice

    PubMed Central

    Singh, Preeti; Fletcher, Terry W.; Li, Yicong; Rusch, Nancy J.; Kilic, Fusun

    2013-01-01

    Angiotensin II (Ang II) is a critical component of the renin-angiotensin system that contributes to hypertension. Although platelets in blood from hypertensive subjects have an abnormal biological profile, it is unclear if circulating Ang II influences platelet aggregation or thrombus formation. One of the abnormalities presented to the platelets during hypertension is an elevated plasma concentration of serotonin (5-HT) caused by reduced 5-HT uptake secondary to loss of the 5-HT transporter (SERT) on the platelet plasma membrane. In the current study, we evaluated in vivo platelet function after 7 days of subcutaneous Ang II infusion to establish hypertension in mice and additionally assessed the biology of isolated platelets exposed to Ang II in vitro. The administration of Ang II elevated systolic blood pressure, but markers of platelet activation including P-selectin and PEJon/A staining were not changed. However, the aggregation response to collagen was reduced in isolated platelets from Ang II-infused mice, which also showed reduced 5-HT uptake by SERT. In vitro exposure of isolated platelets to Ang II also resulted in a loss of surface SERT associated with a reduced aggregation response to collagen. These abnormalities were reversed by increasing concentrations of the Ang II receptor antagonist, valsartan. Interestingly, SERT KO mice failed to fully develop hypertension in response to Ang II infusion and isolated platelets from these animals were insensitive to the anti-aggregatory influence of Ang II. Thus, Ang II blunts the aggregation responses of platelets and the mechanism underlying this action may involve a loss of SERT on the platelet plasma membrane. The latter event depletes intracellular 5-HT in platelets, an event that is associated with reduced aggregation. The widespread use of antihypertensive drugs that target the renin-angiotensin system suggest the potential clinical utility of our findings and emphasize the importance of understanding the

  8. Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway.

    PubMed

    Randriamboavonjy, Voahanginirina; Schrader, Jürgen; Busse, Rudi; Fleming, Ingrid

    2004-02-01

    Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

  9. Insulin Induces the Release of Vasodilator Compounds From Platelets by a Nitric Oxide–G Kinase–VAMP-3–dependent Pathway

    PubMed Central

    Randriamboavonjy, Voahanginirina; Schrader, Jürgen; Busse, Rudi; Fleming, Ingrid

    2004-01-01

    Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS−/− mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of αIIbβ3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO–G kinase–dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase–dependent association of syntaxin 2 with vesicle-associated membrane protein 3. PMID:14744991

  10. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases

    PubMed Central

    Lu, Yu; Li, Quan; Liu, Yu-Ying; Sun, Kai; Fan, Jing-Yu; Wang, Chuan-She; Han, Jing-Yan

    2015-01-01

    Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25–5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25–100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases. PMID:26345207

  11. Cystamine immobilization on TiO 2 film surfaces and the influence on inhibition of collagen-induced platelet activation

    NASA Astrophysics Data System (ADS)

    Zhou, Yujuan; Weng, Yajun; Zhang, Liping; Jing, Fengjuan; Huang, Nan; Chen, Junying

    2011-12-01

    Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO 2 films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO 2 films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

  12. Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction.

    PubMed

    Jones, Letitia D; Jackson, Joseph W; Maggirwar, Sanjay B

    2016-01-01

    The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND.

  13. Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction

    PubMed Central

    Jones, Letitia D.; Jackson, Joseph W.; Maggirwar, Sanjay B.

    2016-01-01

    The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND. PMID:26986758

  14. Platelet activating factor induces dopamine release in PC-12 cell line

    SciTech Connect

    Bussolino, F.; Tessari, F.; Turrini, F.; Braquet, P.; Camussi, G.; Prosdocimi, M.; Bosia, A. Institut Henri Beaufour, Le Plessis Robinson )

    1988-10-01

    The ability of platelet activating factor (PAF) to stimulate dopamine release and modify calcium homeostasis in PC-12 cell line was studied. PAF-induced dopamine release is related to its molecular form, with only the R-form steric configuration ((R)PAF), but not its S-form or its 2-lyso derivative, effective at being active. In addition, PAF acts at very low concentrations in a dose-dependent manner (0.1-30 nM). Preincubation with PAF receptor antagonists (CV-3988 and BN52021) as well as the specific desensitization of PC-12 cells to (R)PAF abolish the (R)PAF-induced dopamine release. Several lines of evidence suggest that dopamine release is dependent on a (R)PAF-induced calcium influx and efflux modulation. Dopamine release by PC-12 cells challenged with (R)PAF is associated with a rapid {sup 45}Ca influx and efflux and a rise in cytoplasmic calcium concentrations ((Ca{sup 2+}){sub i}) evaluated by using the calcium indicators fura-2 and quin2. At 30 nM (R)PAF, the absence of extracellular calcium inhibits the dopamine release but not the rise of (Ca{sup 2+}){sub i} from the internal stores, suggesting the importance of calcium influx in (R)PAF-induced dopamine release. PAF, which has been reported to be synthesized by stimulated neuronal cells may thus have a physiological modulatory role on cells with neurosecretory properties.

  15. Activating stimuli induce platelet microRNA modulation and proteome reorganisation.

    PubMed

    Cimmino, Giovanni; Tarallo, Roberta; Nassa, Giovanni; De Filippo, Maria Rosaria; Giurato, Giorgio; Ravo, Maria; Rizzo, Francesca; Conte, Stefano; Pellegrino, Grazia; Cirillo, Plinio; Calabro, Paolo; Öhman, Tiina; Nyman, Tuula A; Weisz, Alessandro; Golino, Paolo

    2015-07-01

    Platelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology. PMID:25903651

  16. Circulating primers enhance platelet function and induce resistance to antiplatelet therapy

    PubMed Central

    Blair, T A; Moore, S F; Hers, I

    2015-01-01

    Background Aspirin and P2Y12 antagonists are antiplatelet compounds that are used clinically in patients with thrombosis. However, some patients are ‘resistant’ to antiplatelet therapy, which increases their risk of developing acute coronary syndromes. These patients often present with an underlying condition that is associated with altered levels of circulating platelet primers and platelet hyperactivity. Platelet primers cannot stimulate platelet activation, but, in combination with physiologic stimuli, significantly enhance platelet function. Objectives To explore the role of platelet primers in resistance to antiplatelet therapy, and to evaluate whether phosphoinositide 3-kinase (PI3K) contributes to this process. Methods and Results We used platelet aggregation, thromboxane A2 production and ex vivo thrombus formation as functional readouts of platelet activity. Platelets were treated with the potent P2Y12 inhibitor AR-C66096, aspirin, or a combination of both, in the presence or absence of the platelet primers insulin-like growth factor-1 (IGF-1) and thrombopoietin (TPO), or the Gz-coupled receptor ligand epinephrine. We found that platelet primers largely overcame the inhibitory effects of antiplatelet compounds on platelet functional responses. IGF-1-mediated and TPO-mediated, but not epinephrine-mediated, enhancements in the presence of antiplatelet drugs were blocked by the PI3K inhibitors wortmannin and LY294002. Conclusions These results demonstrate that platelet primers can contribute to antiplatelet resistance. Furthermore, our data demonstrate that there are PI3K-dependent and PI3K-independent mechanisms driving primer-mediated resistance to antiplatelet therapy. PMID:26039631

  17. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  18. Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells

    PubMed Central

    Gorbea, Enrique; Ullrich, Stephen E.

    2015-01-01

    Ultraviolet (UV) radiation-induced systemic immune suppression is a major risk factor for skin cancer induction. The migration of dermal mast cells from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced keratinocyte-derived platelet-activating factor (PAF) activates mast cell migration, in part by up regulating the expression of CXCR4 on the surface of mast cells. Others have indicated that epigenetic mechanisms regulate CXCR4 expression, so we asked whether PAF activates epigenetic mechanisms in mast cells. Human mast cells were treated with PAF and the effect on DNA methylation and/or acetylation was measured. PAF suppressed the expression of DNA methyltransferase (DNMT) 1 and 3b. On the other hand, PAF increased p300 histone acetyltransferase expression, and the acetylation of histone H3, which coincided with a decreased expression of the histone deacetylase HDAC2. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the CXCR4 promoter. Finally, inhibiting histone acetylation blocked p300 up-regulation and suppressed PAF-induced surface expression of CXCR4. Our findings suggest a novel molecular mechanism for PAF, activation of epigenetic modifications. We suggest that PAF may serve as an endogenous molecular mediator that links the environment (UV radiation) with the epigenome. PMID:26316070

  19. Differential changes in platelet reactivity induced by acute physical compared to persistent mental stress.

    PubMed

    Hüfner, Katharina; Koudouovoh-Tripp, Pia; Kandler, Christina; Hochstrasser, Tanja; Malik, Peter; Giesinger, Johannes; Semenitz, Barbara; Humpel, Christian; Sperner-Unterweger, Barbara

    2015-11-01

    Platelets are important in hemostasis, but also contain adhesion molecules, pro-inflammatory and immune-modulatory compounds, as well as most of the serotonin outside the central nervous system. Dysbalance in the serotonin pathways is involved in the pathogenesis of depressive symptoms. Thus, changes in platelet aggregation and content of bioactive compounds are of interest when investigating physiological stress-related mental processes as well as stress-related psychiatric diseases such as depression. In the present study, a characterization of platelet reactivity in acute physical and persistent mental stress was performed (aggregation, serotonin and serotonin 2A-receptor, P-selectin, CD40 ligand, matrix metalloproteinase-2 and -9 (MMP-2 and -9), platelet/endothelial adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), β-thromboglobulin (β-TG) and platelet factor 4 (PF-4). Acute physical stress increased platelet aggregability while leaving platelet content of bioactive compounds unchanged. Persistent mental stress led to changes in platelet content of bioactive compounds and serotonin 2A-receptor only. The values of most bioactive compounds correlated with each other. Acute physical and persistent mental stress influences platelets through distinct pathways, leading to differential changes in aggregability and content of bioactive compounds. PMID:26192713

  20. Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα

    PubMed Central

    Carrim, Naadiya; Arthur, Jane F.; Hamilton, Justin R.; Gardiner, Elizabeth E.; Andrews, Robert K.; Moran, Niamh; Berndt, Michael C.; Metharom, Pat

    2015-01-01

    Background Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. Objective To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. Methods and results Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. Conclusions Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically. PMID:26569550

  1. The effect of the menstrual cycle and of decompression stress on arachidonic acid-induced platelet aggregation and on intrinsic platelet thromboxane production in women compared with men.

    PubMed

    Markham, S M; Dubin, N H; Rock, J A

    1991-12-01

    Menstrual cycle variations in platelet aggregation and thromboxane production in association with sex steroids have been reported. External stimuli such as decompression sickness have been associated with clotting activity changes, specifically, increased platelet aggregation. Differences in response of platelets from women and men, when subjected to such a stress, have been observed. This study evaluated the ability of washed platelets from women in the proliferative and secretory phases of the menstrual cycle to aggregate in response to arachidonic acid and the aggregation difference between washed platelets from women and men in response to decompression stress and arachidonic acid. Additionally, platelet thromboxane production differences between the assessed platelet populations were compared. Our results indicate no difference in platelet aggregability between phases of the menstrual cycle. A significant aggregation difference between platelets from women and men was noted. Platelets from women were more sensitive to arachidonic acid aggregation. These differences were not affected by decompression stress. No difference in thromboxane B2 production was noted between the platelet populations evaluated.

  2. Oxymetazoline enhances epidermal- and platelet-derived growth factor-induced DNA synthesis.

    PubMed

    Nickenig, G; Ko, Y; Nettekoven, W; Appenheimer, M; Schiermeyer, B; Vetter, H; Sachinidis, A

    1994-01-01

    In the present study, the effect of 10(-9) to 10(-6) M epinephrine (alpha- and beta-agonist), norepinephrine (alpha- and beta 1-antagonist) isoproterenol (beta-agonist) salbutamol (beta 2-agonist), phenylephrine (alpha 1-agonist) and oxymetazoline (mainly alpha 2-agonist) on DNA synthesis in vascular smooth muscle cells (VSMCs) from rat aorta has been investigated. Our results show that only oxymetazoline induced a moderate dose-dependent elevation of [3H]thymidine incorporation into cell DNA (10(-6) M, 100-300%). Epidermal growth factor (EGF) (50 ng/ml) and platelet-derived growth factor (PDGF)-BB induced an elevation of the [3H]thymidine incorporation into cell DNA from 154 +/- 7 (basal value) to 1270 +/- 95 and 1552 +/- 178 cpm/microgram protein (mean +/- S.D., n = 3). Oxymetazoline (10(-6) M) and phenylephrine induced an increase of [3H]thymidine incorporation to 368 +/- 53 and 205 +/- 27 cpm/microgram protein, respectively. In contrast to phenylephrine, oxymetazoline caused an elevation of the PDGF-BB- and EGF-induced [3H]thymidine incorporation to 1561 +/- 143 and 2086 +/- 235 (means S.D., n = 3), respectively. In addition, EGF (1 to 50 ng/ml) induced a dose-dependent increase of [3H]thymidine incorporation from 154 +/- 7 (basal value) to 486 +/- 35 (1 ng/ml), 912 +/- 74 (5 ng/ml), 1019 +/- 40 (25 ng/ml) and 1270 +/- 95 (50 ng/ml) cpm/microgram protein (mean +/- S.D.). In the presence of 10(-6) M oxymetazoline, 1, 5, 25 and 50 ng/ml EGF caused an increase of [3H]thymidine incorporation to 633 +/- 101, 1124 +/- 87, 1231 +/- 101, and 1561 +/- 89 cpm/microgram protein (mean +/- S.D.).(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation.

    PubMed

    Wu, Chin-Chung; Teng, Che-Ming

    2006-09-28

    Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis. PMID:16890935

  4. Total body irradiation of donors can alter the course of tolerance and induce acute rejection in a spontaneous tolerance rat liver transplantation model.

    PubMed

    Zhang, YeWei; Zhao, HeWei; Bo, Lin; Yang, YinXue; Lu, Xiang; Sun, JingFeng; Wen, JianFei; He, Xia; Yin, GuoWen

    2012-09-01

    Liver transplantation is an established therapy for end-stage liver diseases. Graft rejection occurs unless the recipient receives immunosuppression after transplantation. This study aimed to explore the mechanism of acute rejection of liver allografts in rats pre-treated with total body irradiation to eliminate passenger lymphocytes and to define the role of CD4(+)CD25(+) regulatory T cells in the induction of immunotolerance in the recipient. Male Lewis rats were used as donors and male DA rats were recipients. Rats were randomly assigned to the following four groups: control group, homogeneity liver transplantation group, idio-immunotolerance group and acute rejection group. After transplantation, the survival time of each group, serum alanine aminotransferase, total bilirubin levels, number of Foxp3(+)CD4(+)CD25(+) regulatory T cells, expression of glucocorticoid-induced tumor necrosis factor receptor on T cell subgroups, histopathology of the hepatic graft and spleen cytotoxic T lymphocyte lytic activity were measured. In the acute rejection group, where donors were preconditioned with total body irradiation before liver transplantation, all recipients died between day 17 and day 21. On day 14, serum alanine aminotransferase increased significantly to (459.2±76.9) U L(-1), total bilirubin increased to (124.1±33.7) μmol L(-1) (P<0.05) and the ratio of Foxp3(+)CD4(+)CD25(+) regulatory T cells decreased significantly to 1.50%±0.50% (P<0.05) compared with the other groups. Analysis of the T cell subpopulations in the acute rejection group varied from the other groups. Histological analysis showed typical changes of acute rejection in the acute rejection group only. Preconditioning of the donors with total body irradiation eliminated passenger lymphocytes of the liver graft, and thus affected the course of tolerance and induced acute rejection after liver transplantation.

  5. Schistosomes versus platelets.

    PubMed

    Da'dara, Akram A; Skelly, Patrick J

    2014-12-01

    Schistosomes are parasitic platyhelminths that currently infect >200million people and cause the chronic debilitating disease schistosomiasis. While these large intravascular parasites can disturb blood flow, they do not appear to activate platelets and provoke thrombus formation. Host-interactive tegumental molecules have been proposed to be important in this regard. For example, tegumental apyrase, SmATPDase1 can degrade the platelet-activating molecule ADP in the extracellular environment. The parasites themselves can produce prostaglandins (or may induce prostaglandin production by host cells) which could inhibit platelet aggregation. Additional tegumental proteins have been proposed to impede the coagulation cascade and to promote fibrinolysis. Platelets have been shown to be directly toxic to schistosomes. Platelets recovered from infected rats are able to kill larval parasites in culture and platelets obtained at later times post-infection are generally better at killing. Even platelets from uninfected rats can rapidly kill larval schistosomes if first exposed to a variety of activators (such as: serum from infected rats, the IgE fraction of that serum, C-reactive protein, cytokines (TNFα or TNFβ)). Passive transfer of stimulated platelets can protect rats against a challenge schistosome infection. Cytokines (TNFα, TNFβ, IFNγ or IL-6) have been shown to similarly promote normal human platelet killing of schistosomes in vitro. Platelet antimicrobial effector molecules (e.g. platelet microbicidal proteins) may mediate such killing. While platelets can be protective against schistosomes following infection of humans and mice, platelet numbers decline (but not so in the non-permissive rat host) and coagulopathy becomes more apparent as schistosome-induced pathology increases.

  6. Magnetic-field-induced orientational order in the isotropic phase of hard colloidal platelets

    SciTech Connect

    Beek, D. van der; Petukhov, A.V.; Vroege, G.J.; Lekkerkerker, H.N.W.; Davidson, P.; Ferre, J.; Jamet, J.P.; Wensink, H.H.; Bras, W.

    2006-04-15

    The magnetic-field-induced orientational order in the isotropic phase of colloidal gibbsite [Al(OH){sub 3}] platelets is studied by means of optical birefringence and small-angle x-ray scattering (SAXS) techniques. The suspensions display field-induced ordering at moderate field strengths (a few Tesla), which increases with increasing particle concentration. The gibbsite particles align their normals perpendicular to the magnetic field and hence possess a negative anisotropy of their diamagnetic susceptibility {delta}{chi}. The results can be described following a simple, Onsager-like approach. A simplified model is derived that allows one to obtain the orientational distribution function directly from the scattering data. However, it leads to an underestimate of the diamagnetic susceptibility anisotropy {delta}{chi}. This accounts for the difference between the {delta}{chi} values provided by the two experimental techniques (SAXS and magneto-optics). The order of magnitude {delta}{chi}{approx}10{sup -22} J/T{sup 2} lies in between that of goethite suspensions and that of suspensions of organic particles.

  7. Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis

    PubMed Central

    1994-01-01

    Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug. PMID:7516414

  8. Investigating the fluid mechanics behind red blood cell-induced lateral platelet motion

    NASA Astrophysics Data System (ADS)

    Crowl Erickson, Lindsay; Fogelson, Aaron

    2009-11-01

    Platelets play an essential role in blood clotting; they adhere to damaged tissue and release chemicals that activate other platelets. Yet in order to adhere, platelets must first come into contact with the injured vessel wall. Under arterial flow conditions, platelets have an enhanced concentration near blood vessel walls. This non-uniform cell distribution depends on the fluid dynamics of blood as a heterogeneous medium. We use a parallelized lattice Boltzmann-immersed boundary method to solve the flow dynamics of red cells and platelets in a periodic 2D vessel with no-slip boundary conditions. Red cells are treated as biconcave immersed boundary objects with isotropic Skalak membrane tension and an internal viscosity five times that of the surrounding plasma. Using this method we analyze the influence of shear rate, hematocrit, and red cell membrane properties on lateral platelet motion. We find that the effective diffusion of platelets is significantly lower near the vessel wall compared to the center of the vessel. Insight gained from this work could lead to significant improvements to current models for platelet adhesion where the presence of red blood cells is neglected due to computational intensity.

  9. Platelet Disorders

    MedlinePlus

    ... higher risk of blood clots. With other platelet disorders, the platelets do not work as they should. For example, in von Willebrand Disease, the platelets cannot stick together or cannot attach ...

  10. CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility

    PubMed Central

    Samuel, Stephen P; Santos-Martinez, Maria J; Medina, Carlos; Jain, Namrata; Radomski, Marek W; Prina-Mello, Adriele; Volkov, Yuri

    2015-01-01

    New nanomaterials intended for systemic administration have raised concerns regarding their biocompatibility and hemocompatibility. Quantum dots (QD) nanoparticles have been used for diagnostics, and recent work suggests their use for in vivo molecular and cellular imaging. However, the hemocompatibility of QDs and their constituent components has not been fully elucidated. In the present study, comprehensive investigation of QD–platelet interactions is presented. These interactions were shown using transmission electron microscopy. The effects of QDs on platelet function were investigated using light aggregometry, quartz crystal microbalance with dissipation, flow cytometry, and gelatin zymography. Platelet morphology was also analyzed by phase-contrast, immunofluorescence, atomic-force and transmission electron microscopy. We show that the QDs bind to platelet plasma membrane with the resultant upregulation of glycoprotein IIb/IIIa and P-selectin receptors, and release of matrix metalloproteinase-2. These findings unravel for the first time the mechanism of functional response of platelets to ultrasmall QDs in vitro. PMID:25897218

  11. CdTe quantum dots induce activation of human platelets: implications for nanoparticle hemocompatibility.

    PubMed

    Samuel, Stephen P; Santos-Martinez, Maria J; Medina, Carlos; Jain, Namrata; Radomski, Marek W; Prina-Mello, Adriele; Volkov, Yuri

    2015-01-01

    New nanomaterials intended for systemic administration have raised concerns regarding their biocompatibility and hemocompatibility. Quantum dots (QD) nanoparticles have been used for diagnostics, and recent work suggests their use for in vivo molecular and cellular imaging. However, the hemocompatibility of QDs and their constituent components has not been fully elucidated. In the present study, comprehensive investigation of QD-platelet interactions is presented. These interactions were shown using transmission electron microscopy. The effects of QDs on platelet function were investigated using light aggregometry, quartz crystal microbalance with dissipation, flow cytometry, and gelatin zymography. Platelet morphology was also analyzed by phase-contrast, immunofluorescence, atomic-force and transmission electron microscopy. We show that the QDs bind to platelet plasma membrane with the resultant upregulation of glycoprotein IIb/IIIa and P-selectin receptors, and release of matrix metalloproteinase-2. These findings unravel for the first time the mechanism of functional response of platelets to ultrasmall QDs in vitro. PMID:25897218

  12. Differential gene expression in Aspergillus fumigatus induced by human platelets in vitro.

    PubMed

    Perkhofer, Susanne; Zenzmaier, Christoph; Frealle, Emilie; Blatzer, Michael; Hackl, Hubert; Sartori, Bettina; Lass-Flörl, Cornelia

    2015-05-01

    Invasive aspergillosis is characterized by vascular invasion and thrombosis. In order to determine the antifungal activity of human platelets, hyphal elongation and metabolic activity of a clinical A. fumigatus isolate were measured. Genome-wide identification of differentially expressed genes in A. fumigatus was performed after exposure to platelets for 15, 30, 60 and 180 min. Data were analyzed by gene ontology annotation as well as functional categories (FunCat) and KEGG enrichment analyses. Platelets attenuated hyphal elongation and viability of A. fumigatus and in total 584 differentially expressed genes were identified, many of which were associated with regulation of biological processes, stress response, transport and metabolism. FunCat and KEGG enrichment analyses showed stress response and metabolic adaptation to be increased in response to platelets. Our findings demonstrate that A. fumigatus displayed a specific transcriptional response when exposed to platelets, thus reflecting their antifungal activities.

  13. Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

    SciTech Connect

    Meyers, K.M.; Boehme, M.; Inbar, O.

    1982-10-01

    Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.

  14. Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression.

    PubMed

    Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X; Walterscheid, Jeffrey P; Ullrich, Stephen E

    2004-03-15

    Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependent manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin. PMID:15020195

  15. Rejected applications

    PubMed Central

    2014-01-01

    Objective: To review membership application materials (especially rejected applications) to the American Academy of Neurology (AAN) during its formative years (1947–1953). Methods: Detailed study of materials in the AAN Historical Collection. Results: The author identified 73 rejected applications. Rejected applicants (71 male, 2 female) lived in 25 states. The largest number was for the Associate membership category (49). These were individuals “in related fields who have made and are making contributions to the field of neurology.” By contrast, few applicants to Active membership or Fellowship status were rejected. The largest numbers of rejectees were neuropsychiatrists (19), neurosurgeons (16), and psychiatrists (14). Conclusion: The AAN, established in the late 1940s, was a small and politically vulnerable organization. A defining feature of the fledgling society was its inclusiveness; its membership was less restrictive than that of the older American Neurological Association. At the same time, the society needed to preserve its core as a neurologic society rather than one of psychiatry or neurosurgery. Hence, the balance between inclusiveness and exclusive identity was a difficult one to maintain. The Associate membership category, more than any other, was at the heart of this issue of self-definition. Associate members were largely practitioners of psychiatry or neurosurgery. Their membership was a source of consternation and was to be carefully been held in check during these critical formative years. PMID:24944256

  16. Platelet preservation: agitation and containers.

    PubMed

    van der Meer, Pieter F; de Korte, Dirk

    2011-06-01

    For platelets to maintain their in vitro quality and in vivo effectiveness, they need to be stored at room temperature with gentle agitation in gas-permeable containers. The mode of agitation affects the quality of the platelets, and a gentle method of agitation, either a circular or a flat bed movement, provides the best results. Tumblers or elliptical agitators induce platelet activation and subsequent damage. As long as the platelets remain in suspension, the agitation speed is not important. Agitation of the platelet concentrates ensures that the platelets are continuously oxygenated, that sufficient oxygen can enter the storage container and that excess carbon dioxide can be expelled. During transportation of platelet concentrates, nowadays over long distances where they are held without controlled agitation, platelets may tolerate a certain period without agitation. However, evidence is accumulating that during the time without agitation, local hypoxia surrounding the platelets may induce irreversible harm to the platelets. Over the decades, more gas-permeable plastics have been used to manufacture platelet containers. The use of different plastics and their influence on the platelet quality both in vitro and in vivo is discussed. The improved gas-permeability has allowed the extension of platelet storage from 3 days in the early 1980s, to currently at least 7 days. In the light of new developments, particularly the introduction of pathogen reduction techniques, the use of platelet additive solutions and the availability of improved automated separators, further (renewed) research in this area is warranted.

  17. Effects of epinephrine on ADP-induced changes in platelet inositol phosphates

    SciTech Connect

    Vickers, J.D.; Keraly, C.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-03-01

    Epinephrine (EPI) does not aggregate rabbit platelets, but it does increase the labelling of inositol phosphate (IP) at 60s (21%, p < 0.05) in the presence of 20 mM Li/sup +/, in platelets prelabelled with (/sup 3/H) inositol. In contrast, 0.5 ..mu..M ADP which causes aggregation, increases the labelling of inositol bisphosphate (IP/sub 2/) by 30% (p < 0.01) at 10s and by 46% (p < 0.05) at 60s and IP by 26% (p < 0.05) at 60s. The combination of 0.5 ..mu..M ADP and 50 ..mu..M EPI causes more extensive aggregation and increases IP/sub 2/ by 154% (p < 0.01) and IP by 65% (p < 0.01) at 60s. The increase in IP/sub 2/ stimulated by ADP + EPI was greater than the increase caused by ADP (p < 0.05). The authors examined the effects of ..cap alpha..- and ..beta..-adrenergic receptor blockers on EPI + ADP-induced changes in the inositol phosphates. The ..beta..-adrenergic blocker Sotalol (50 ..mu..M), which had no effect by itself, enhanced the accumulation of IP/sub 2/ due to 0.2 ..mu..M ADP + 0.6 ..mu..M EPI by 70% (p < 0.01) at 60s, as well as aggregation. This is consistent with EPI inhibition mediated through stimulation of adenylate cyclase via the ..beta..-adrenergic receptor. The ..cap alpha..-adrenergic blocker phentolamine (50 ..mu..M), reduced aggregation stimulated by 0.5 ..mu..M ADP + 50 ..mu..M EPI, and reduced the accumulation of IP by 53% (p < 0.05) and IP/sub 2/ by 108% (0 < 0.05). These data are compatible with the hypothesis that the effect of EPI on ADP-induced aggregation involves IP/sub 2/ metabolism stimulated via the ..cap alpha..-adrenergic receptor.

  18. ADP-induced platelet aggregation after addition of tramadol in vitro in fed and fasted horses plasma.

    PubMed

    Casella, S; Giannetto, C; Giudice, E; Marafioti, S; Fazio, F; Assenza, A; Piccione, G

    2013-04-01

    Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 μM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting condition, ADP concentration and addition of tramadol. Findings showed statistical differences (P≤0.05) on studied parameters after addition of tramadol to different ADP concentrations in fed and fasted horses. The clinical relevance of these results is that tramadol provides many advantages as a therapeutic option; in fact, it is an inexpensive and a relatively new analgesic in equine veterinary medicine. Further investigations would be appropriate to compare the effects of different opioids but also using different concentrations of tramadol associated with other drugs in order to have substances which can regulate the functional activity of the platelets and to extend the knowledges on equine platelet aggregation. PMID:23031839

  19. Patterns of myocardial cell adhesion molecule expression in human endomyocardial biopsies after cardiac transplantation. Induced ICAM-1 and VCAM-1 related to implantation and rejection.

    PubMed Central

    Herskowitz, A.; Mayne, A. E.; Willoughby, S. B.; Kanter, K.; Ansari, A. A.

    1994-01-01

    Conflicting patterns of myocardial cell adhesion molecule expression associated with cardiac rejection have emerged from numerous studies of randomly selected cardiac biopsies. We designed a prospective, longitudinal study which reports both qualitative and quantitative levels of myocardial ICAM-1, VCAM-1, E-selectin, and P-selectin expression in sequential human cardiac allograft biopsies. Intense ICAM-1 and VCAM-1 staining was found in all biopsies during the first three weeks after transplant and coincided with elevated serum levels of troponin T, a sensitive marker of ischemic myocyte injury. Baseline ICAM-1 and VCAM-1 expression returned within three to four weeks, as did serum troponin T levels in all patients who did not develop rejection. All 29 rejection episodes encountered were associated with intense ICAM-1 staining, while 24 of the 29 (83%) had intense VCAM-1 staining. Increased ELAM-1 and CD62 staining was only rarely observed. Persistence of increased ICAM-1 and VCAM-1 staining after treated rejection episodes predicted a recurrent rejection episode within two months (75% positive and 100% negative predictive value). Objective quantitative measurements by radioimmunoassay (RIA) confirmed these patterns of induced ICAM-1 and VCAM-1 expression. Thus, longitudinal monitoring of serial biopsies for myocardial ICAM-1 and VCAM-1 expression could be useful in the early detection of rejection episodes and monitoring the efficacy of immunosuppressive therapy. Images Figure 2 PMID:7977640

  20. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets.

    PubMed

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2014-01-01

    Cyclic guanosine-3',5'-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  1. Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.

    PubMed Central

    Bussolino, F; Arese, M; Montrucchio, G; Barra, L; Primo, L; Benelli, R; Sanavio, F; Aglietta, M; Ghigo, D; Rola-Pleszczynski, M R

    1995-01-01

    Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS. Images PMID:7543496

  2. PLATELET COUNT IN WOMEN WITH PREGNANCY INDUCED HYPERTENSION IN UNIVERSITY HOSPITAL CENTER OF MOTHER AND CHILD HEALTHCARE “KOçO GLIOZHENI”, TIRANA, ALBANIA

    PubMed Central

    Damani, Zamir

    2016-01-01

    Introduction: One of the most common and potential life threatening complications of pregnancy is pregnancy induced hypertension. This cross-sectional study aimed to investigate the relationship between platelet count and pregnancy induced hypertension. Material and methods: Twenty (20) patients (subjects) and twenty (20) healthy pregnant women (control) visiting the Obstetrics and Gynecology Hospital University of “Koço Gliozheni” Tirana Albania were registered in the study and followed during their pregnancy. Both, subjects and control participants were subject to platelet count manually performed using standard methods on. Results: The mean platelet count of the control group (38448±235500) was significantly higher than that of the subject group (217050±50780.7) (p<0.03). In the first and second trimester was more prevalent low platelet counting with the mean platelet count (107 ±57.3) and (101 ±63.4), respectively. The mean age at marriage in subjects with PIH was found to be with low platelet count. Regular monitoring of platelet counts in women with Pregnancy Induced Hypertension must be subject of the management protocols.

  3. PLATELET COUNT IN WOMEN WITH PREGNANCY INDUCED HYPERTENSION IN UNIVERSITY HOSPITAL CENTER OF MOTHER AND CHILD HEALTHCARE “KOçO GLIOZHENI”, TIRANA, ALBANIA

    PubMed Central

    Damani, Zamir

    2016-01-01

    Introduction: One of the most common and potential life threatening complications of pregnancy is pregnancy induced hypertension. This cross-sectional study aimed to investigate the relationship between platelet count and pregnancy induced hypertension. Material and methods: Twenty (20) patients (subjects) and twenty (20) healthy pregnant women (control) visiting the Obstetrics and Gynecology Hospital University of “Koço Gliozheni” Tirana Albania were registered in the study and followed during their pregnancy. Both, subjects and control participants were subject to platelet count manually performed using standard methods on. Results: The mean platelet count of the control group (38448±235500) was significantly higher than that of the subject group (217050±50780.7) (p<0.03). In the first and second trimester was more prevalent low platelet counting with the mean platelet count (107 ±57.3) and (101 ±63.4), respectively. The mean age at marriage in subjects with PIH was found to be with low platelet count. Regular monitoring of platelet counts in women with Pregnancy Induced Hypertension must be subject of the management protocols. PMID:27698599

  4. Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

    PubMed Central

    Jiang, Peng; Loyau, Stéphane; Tchitchinadze, Maria; Ropers, Jacques; Jondeau, Guillaume; Jandrot-Perrus, Martine

    2015-01-01

    Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL-1 and 10 μg.mL-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy. Trial Registration ClinicalTrials.gov NCT00763893 PMID:26052700

  5. Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation

    SciTech Connect

    Bae, Ok-Nam; Lim, Kyung-Min; Chung, Jin-Ho

    2009-09-01

    Trivalent methylated metabolites of arsenic, monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}), have been found highly reactive and toxic in various cells and in vivo animal models, suggesting their roles in the arsenic-associated toxicity. However, their effects on cardiovascular system including blood cells, one of the most important targets for arsenic toxicity, remain poorly understood. Here we found that MMA{sup III} and DMA{sup III} could induce procoagulant activity and apoptosis in platelets, which play key roles in the development of various cardiovascular diseases (CVDs) through excessive thrombus formation. In freshly isolated human platelets, treatment of MMA{sup III} resulted in phosphatidylserine (PS) exposure, a hallmark of procoagulant activation, accompanied by distinctive apoptotic features including mitochondrial membrane potential disruption, cytochrome c release, and caspase-3 activation. These procoagulant activation and apoptotic features were found to be mediated by the depletion of protein thiol and intracellular ATP, and flippase inhibition by MMA{sup III}, while the intracellular calcium increase or reactive oxygen species generation was not involved. Importantly, increased platelet procoagulant activity by MMA{sup III} resulted in enhanced blood coagulation and excessive thrombus formation in a rat in vivo venous thrombosis model. DMA{sup III} also induced PS-exposure with apoptotic features mediated by protein thiol depletion, which resulted in enhanced thrombin generation. In summary, we believe that this study provides an important evidence for the role of trivalent methylated arsenic metabolites in arsenic-associated CVDs, giving a novel insight into the role of platelet apoptosis in toxicant-induced cardiovascular toxicity.

  6. Agonists-induced platelet activation varies considerably in healthy male individuals: studies by flow cytometry.

    PubMed

    Panzer, Simon; Höcker, Lisa; Koren, Daniela

    2006-02-01

    Flow cytometric evaluation of platelet function extends our understanding of platelets' role in various clinical conditions associated with either bleeding disorders, thrombosis, or monitoring of antiplatelet therapy. The use of suboptimal concentrations of various agonists may allow assessing the "activatability" of platelets. We determined platelet responsiveness to thrombin-receptor-activating peptide-6, arachidonic acid, adenosine 5c-diphosphate (ADP), epinephrine, collagen, and ristocetin at suboptimal concentrations by determination of P-selectin expression and binding of PAC-1 in 26 healthy male individuals. The response varied considerably from one individual to the next. However, within individuals, responses to all agonists except collagen correlated strongly (p<0.05), suggesting a global variability of platelet responses. Moreover, P-selectin expression and PAC-1 binding were strongly correlated (p<0.05). Interestingly, with epinephrine, PAC-1 positive events outnumbered P-selectin positive events, while this was not seen with the other agonists. Thus, epinephrine may specifically affect the conformational switch mechanism and receptor clustering. Our data indicate that the in vitro response to suboptimal concentrations of agonists varies, but individuals with selective platelet defects may still be identified based on data obtained with the various agonists. PMID:16283308

  7. Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbβ3, and platelet factor 4

    PubMed Central

    Krauel, Krystin; Tilley, Dorothea O.; Weber, Claudia; Cox, Dermot; Greinacher, Andreas; Kerrigan, Steven W.; Watson, Steve P.

    2014-01-01

    Bacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbβ3 engagement. Moreover, feedback agonists adenosine 5′-diphosphate and thromboxane A2 are mandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndrome α-granule–deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway. PMID:24642751

  8. Dual effect of nitrate therapy for cyclosporine-induced hypertension on vascular and platelet morphofunctional markers; an animal model.

    PubMed

    Reis, F; Teixeira de Lemos, E; Almeida, L; Parada, B; Garrido, A P; Rocha-Pereira, P; Santos-Silva, A; Santos-Dias, J; Dinis, A; Figueiredo, A; Costa-Almeida, C; Mota, A; Teixeira, F

    2007-10-01

    The present study sought to evaluate the prevention and reversion effects of isosorbide-5-mononitrate (Is-5-Mn) on the development of hypertension (HT) and on the underlying vascular and platelet morphofunctional disturbances, using an animal model of cyclosporine (CsA)-induced HT. The following rat groups (n = 8) were tested: (1) a control group (orange juice, for 7 weeks); (2) the CsA group (5 mg/kg/d for 7 weeks); (3) the Is-5-Mn group (150 mg/kg/d, twice a day for 7 weeks); (4) the prevention group (Is-5-Mn + CsA) treated for 2 weeks with Is-5-Mn only and thereafter with both drugs for 7 weeks; (5) the curative group (CsA + Is-5-Mn) beginning 7 weeks after CsA and following thereafter with both drugs for 5 weeks. Blood pressure, lipid profile, vascular lesion, platelet aggregation and morphology, and platelet thromboxane A(2)/vascular prostacyclin equilibrium were evaluated. Is-5-Mn + CsA therapy prevented (systolic blood pressure [SBP]: 114.3 +/- 1.9 mm Hg, P < .001; diastolic blood pressure [DBP]: 97.0 +/- 3.3 mm Hg, P < .001) the CsA-induced HT (SBP: 146.2 +/- 4.5 mm Hg, P < .001; DBP: 124.9 +/- 4.5 mm Hg, P < .001 vs control: SBP: 111.6 +/- 0.7 mm Hg; DBP: 94.6 +/- 1.0 mm Hg), as well as the vascular lesion and the platelet morphofunctional disturbances. The curative group did not show attenuated CsA-induced BP increase; it showed further enhancement of the HT effect (SBP: 159.7 +/- 5.5 mm Hg, P < .05; DBP: 132.8 +/- 2.8 mm Hg), as well as worsened vascular lesions and platelet function, namely a disruption in the TXA(2)/PGI(2) equilibrium. Our data suggested that Is-5-Mn therapy may be a valid choice to prevent the morphofunctional changes associated with CsA-induced HT, when used as a preventive therapy. A careful evaluation of the impact of nitrate therapy should be considered, particularly the negative effect on cardiovascular hemodynamics, when considering its use after previous CsA disturbances have been established.

  9. The inhibitory mechanism of crude saponin fraction from Korean Red Ginseng in collagen-induced platelet aggregation

    PubMed Central

    Jeon, Bo Ra; Kim, Su Jung; Hong, Seung Bok; Park, Hwa-Jin; Cho, Jae Youl; Rhee, Man Hee

    2015-01-01

    Background Korean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng’s therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. Methods The platelet aggregation was induced by collagen, the ligand of integrin αIIβI and glycoprotein VI. The crude saponin’s effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin αIIbβIII was examined by fluorocytometry. Results CSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed [Ca2+]i mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin αIIbβ3. Conclusion Our present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function. PMID:26199561

  10. Platelet-activating factor induces collagenase expression in corneal epithelial cells.

    PubMed Central

    Bazan, H E; Tao, Y; Bazan, N G

    1993-01-01

    Platelet-activating factor (PAF), a potent lipid mediator involved in inflammatory and immune responses, accumulates rapidly in response to injury in a variety of tissues, including the corneal epithelium. However, the precise role of this compound in the cascade of events following insult has not been defined. Here we examined the effect of PAF on gene expression in the epithelial cells of rabbit corneas in organ culture. We found that incubation with 100 nM methylcarbamoyl PAF, a nonhydrolyzable analog of PAF, produced rapid transient 2.8- and 3.5-fold increases in the expression of c-fos and c-jun, respectively, at 1 hr, followed by increased expression of the collagenase type I gene beginning at 3 hr and peaking at 14-fold by 8 hr. Addition of the protein-synthesis-inhibitor cycloheximide superinduced c-fos and c-jun, strongly potentiating the PAF effect, but inhibited the induction of collagenase type I expression, suggesting the existence of a transcriptional factor linking the two events. BN-50730, a selective antagonist of intracellular PAF-binding sites, blocked the expression of the immediate-early genes as well as the increase in collagenase type I mRNA. Our results suggest that one of the functions of PAF may be to enhance the breakdown of the extracellular matrix as a part of the remodeling process during corneal wound healing after injury. Pathologically, a PAF-induced overproduction of collagenase may be a factor in the development of corneal ulcers, as well as other pathophysiological conditions such as cartilage destruction in arthritis. If so, inhibitors of this signal-transduction pathway may be useful as tools for further investigation and, eventually, as therapeutic agents to treat such disorders. Images Fig. 1 Fig. 2 PMID:8378347

  11. Effect of Vitamin C Supplementation on Platelet Aggregation and Serum Electrolytes Levels in Streptozotocin-Induced Diabetes Mellitus in Rats.

    PubMed

    Owu, Daniel U; Nwokocha, Chukwuemeka R; Ikpi, Daniel E; Ogar, Emmanuel I

    2016-01-01

    Diabetes mellitus (DM) is a disease condition characterised by hyperglycemia; free radical and abnormalhaematological indices. Vitamin C can reduce free radical generation and ameliorate adverse conditions of diabetes mellitus.The aim of the present study is to investigate the effect of vitamin C on platelet aggregation and electrolyte levels in Type 1DM. Male Wistar rats were divided into four groups namely control, DM, DM +Vitamin C and Vitamin C groups. Rats weremade diabetic with a single dose of streptozotocin (65 mg/kg) intraperitoneally. Vitamin C was administered orally todiabetic and normal rats at 200 mg/kg body weight for 28 days. Blood samples were analyzed for hematological parameters,platelet aggregation, and serum electrolyte levels. Blood glucose in DM+ Vitamin C group (9.9 ± 1.8 mmol/L) wassignificantly reduced (p<0.01) compared to DM group (32.2 ± 2.1 mmol/L) and significantly higher (p<0.05) than control(4.4 ± 0.8 mmol/L). Haemoglobin (Hb) concentration in DM group (12 ± 0.1 g/dL) was significantly reduced (p<0.01) whencompared with control groups (14 ± 0.24 g/dL) and significantly increased (p<0.05) in the DM+vitamin C group (13.5 ± 0.5g/dL) compared with the diabetic group. The mean corpuscular volume values in DM (68.66 ± 0.5 fL) and DM+vitamin Cgroups (68.11 ± 0.4 fL) were significantly higher (p<0.01) than the control (59.49 ± 0.5fL). Platelet count in DM group (523± 8.5 x109/L) was significantly raised (p<0.01) when compared to control (356 ± 6.2 x109/L) and significantly reduced(p<0.01) in DM+ vitamin C-treated group (385 ± 7.8 x109/L) compared with DM group. Platelet aggregation and serumsodium/potassium ratios was significantly reduced (p<0.01) in DM+vitamin C compared with DM group. These resultssuggest that oral vitamin C administration increases haemoglobin, reduced plasma glucose level, platelet count, serumsodium/potassium ion ratio and inhibits platelet aggregation in streptozotocin-induced DM in rats. PMID:27574765

  12. Platelets mediate acetaminophen hepatotoxicity.

    PubMed

    Lam, Fong W; Rumbaut, Rolando E

    2015-10-01

    In this issue of Blood, Miyakawa et al show that platelets and protease-activated receptor (PAR)-4 contribute to acetaminophen (APAP)-induced liver damage. Using various strategies in a mouse model of APAP overdose, the authors demonstrate that platelets participate in the progression of liver damage, and that the direct thrombin inhibitor lepirudin and PAR-4 deficiency attenuate hepatotoxicity. These findings have the potential to help identify future therapeutic targets for APAP-induced hepatotoxicity. PMID:26450954

  13. In vitro generation of megakaryocytes and platelets from human embryonic stem cells and induced pluripotent stem cells.

    PubMed

    Takayama, Naoya; Eto, Koji

    2012-01-01

    Human embryonic stem cells (hESCs) represent a potential source of blood cells for transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. Moreover, human-induced pluripotent stem cells (hiPSCs), recently established by defined reprogramming factors expressed in somatic cells, represent a further source for the generation of hematopoietic cells. When undifferentiated hESCs or hiPSCs are cultured on either mesenchymal C3H10T1/2 cells or OP-9 stromal cells, they can be differentiated into a hematopoietic niche that concentrates hematopoietic progenitors, which we named "embryonic stem cell-derived sacs" (ES-sacs). We have optimized the in vitro culture condition for obtaining mature megakaryocytes derived from the hematopoietic progenitors within ES-sacs, which are then able to release platelets. These in vitro-generated platelets display integrin activation capability, indicating normal hemostatic function. This novel protocol thus provides a means of generating platelets from hESCs as well as hiPSCs, for the study of normal human thrombopoiesis and also thrombopoiesis in disease conditions using patient-specific hiPSCs.

  14. Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

    PubMed Central

    Balleisen, L; Nowack, H; Gay, S; Timpl, R

    1979-01-01

    Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen. Images PLATE 1 PMID:395952

  15. Potassium 2-(1-hydroxypentyl)-benzoate inhibits ADP-induced rat platelet aggregation through P2Y1-PLC signaling pathways.

    PubMed

    Yang, Hongyan; Xu, Shaofeng; Li, Jiang; Wang, Ling; Wang, Xiaoliang

    2015-09-01

    Potassium 2-(1-hydroxypenty1)-benzoate (dl-PHPB) is a new drug candidate for treatment of ischemic stroke with antiplatelet effect. In this study, we investigated the mechanisms of dl-PHPB in inhibiting platelet aggregation. The ADP-activated P2Y1-Gq-PLC and P2Y12-Gi-AC pathways were observed, respectively. Intravenous injection of dl-PHPB (1.3, 3.9, 12.9 mg/kg) significantly inhibited ADP-, collagen-, and arachidonic acid-induced rat platelet aggregation in a dose-dependent manner, and dl-PHPB had a relatively more potent inhibitory effect on ADP-induced rat platelet aggregation than other agonists. Dl-PHPB also showed a decreased expression of CD62P (a marker for platelet activation) mediated by ADP. Both dl-PHPB and ticlopidine (P2Y12 receptor antagonist) decreased cytoplasmic Ca(2+) concentration. But, dl-PHPB did not reverse the inhibition of PGE1-induced platelet cAMP formation by ADP, which was different from ticlopidine. Further, dl-PHPB instead of ticlopidine showed increasing phospholipase C-β phosphorylation (ser(1105)). The m-3M3FBS, a phospholipase C activator, attenuated the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation and enhanced IP1 accumulation in rat platelets. Dl-PHPB decreased IP1 accumulation induced by ADP but had no effect on IP1 level enhanced by m-3M3FBS. Our results suggest that dl-PHPB has a potent antiplatelet effect, which is mainly through blockade of P2Y1 receptor-PLC-IP3 pathway and decreasing cytoplasmic calcium.

  16. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  17. Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

    PubMed

    Huang, Jiqing; Kast, Juergen

    2015-08-01

    Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification. PMID:26159767

  18. Role of platelet adhesion in homeostasis and immunopathology.

    PubMed Central

    Männel, D N; Grau, G E

    1997-01-01

    Various molecules expressed on the surface of platelets have been shown to mediate the protective or deleterious role of these cells in immuno-inflammatory mechanisms. Increasing evidence points to the involvement of the cell adhesion molecules, gpIIb-IIIa, P-selectin, CD31, LFA-1, and CD36 in the interaction between platelets and endothelial cells as well as other cell types. The possible role of these molecules in the ability of platelets to support endothelium and to protect against tumour necrosis factor mediated cytolysis or parasitic invasion are reviewed. The involvement of platelets as effectors of tissue damage in cerebral malaria, lipopolysaccharide induced pathology, and pulmonary fibrosis is also discussed. This has then been extended to include the intercellular mechanisms underpinning their pathogenic role in metastasis, transplant rejection, stroke, brain hypoxia, and related conditions. A better understanding of the complex regulation and hierarchical organisation of these various platelet adhesion molecules may prove useful in the development of new approaches to the treatment of such diseases. Images PMID:9350300

  19. Taurine and platelet aggregation

    SciTech Connect

    Nauss-Karol, C.; VanderWende, C.; Gaut, Z.N.

    1986-03-01

    Taurine is a putative neurotransmitter or neuromodulator. The endogenous taurine concentration in human platelets, determined by amino acid analysis, is 15 ..mu..M/g. In spite of this high level, taurine is actively accumulated. Uptake is saturable, Na/sup +/ and temperature dependent, and suppressed by metabolic inhibitors, structural analogues, and several classes of centrally active substances. High, medium and low affinity transport processes have been characterized, and the platelet may represent a model system for taurine transport in the CNS. When platelets were incubated with /sup 14/C-taurine for 30 minutes, then resuspended in fresh medium and reincubated for one hour, essentially all of the taurine was retained within the cells. Taurine, at concentrations ranging from 10-1000 ..mu..M, had no effect on platelet aggregation induced by ADP or epinephrine. However, taurine may have a role in platelet aggregation since 35-39% of the taurine taken up by human platelets appears to be secreted during the release reaction induced by low concentrations of either epinephrine or ADP, respectively. This release phenomenon would imply that part of the taurine taken up is stored directly in the dense bodies of the platelet.

  20. Nature of hyperacute (accelerated second set) rejection in dog renal allografts and effects of heparin on rejection process.

    PubMed

    Amery, A H; Pegrum, G D; Risdon, R A; Williams, G

    1973-02-24

    Renal allografts were exchanged between unrelated mongrel dogs after previous sensitization with skin and kidney grafts from the same donors. Rapid rejection of the renal allografts was associated with the accumulation of platelets and leucocytes in the peritubular and glomerular capillaries but fibrin deposition was not demonstrated.Heparin infusion delayed but did not prevent the rejection process.

  1. Involvement of Rac in thromboxane A2‑induced human platelet activation: regulation of sCD40 ligand release and PDGF‑AB secretion.

    PubMed

    Kageyama, Yasunari; Doi, Tomoaki; Matsushima-Nishiwaki, Rie; Iida, Yuko; Akamatsu, Shigeru; Kondo, Akira; Kuroyanagi, Gen; Yamamoto, Naohiro; Mizutani, Jun; Otsuka, Takanobu; Tokuda, Haruhiko; Iida, Hiroki; Kozawa, Osamu; Ogura, Shinji

    2014-07-01

    We have previously shown that glycoprotein Ib/IX/V activation stimulates the release of the soluble CD40 ligand (sCD40L) via the generation of thromboxane A2 from human platelets. In the present study, the role of Rac, which is a member of the Rho family, was investigated in the thromboxane A2‑stimulated release of platelet‑derived growth factor (PDGF)‑AB and sCD40L in human platelets. U46619, a thromboxane receptor agonist, stimulated the activation of Rac time‑dependently in human platelets, and NSC23766, a selective inhibitor of the Rac‑guanine nucleotide exchange factor interaction, reduced the U46619‑induced platelet aggregation. NSC23766 markedly suppressed the U46619‑induced p38 mitogen-activated protein (MAP) kinase phosphorylation. The thromboxane A2‑induced release of PDGF‑AB and sCD40L was significantly suppressed by NSC23766 in a dose‑dependent manner. In addition, NSC23766 reduced the sCD40L release stimulated by ristocetin, a glycoprotein Ib/IX/V activator. These results indicate that Rac regulates the thromboxane A2‑induced stimulation of PDGF‑AB secretion and sCD40L release via the p38 MAP kinase in human platelets.

  2. Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor

    PubMed Central

    Chennupati, Ramesh; Nüsing, Rolf M.; Offermanns, Stefan

    2016-01-01

    19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists. PMID:27662627

  3. Quinine-dependent, platelet-reactive monoclonals mimic antibodies found in patients with quinine-induced immune thrombocytopenia

    PubMed Central

    Birenbaum, Jessica; Rasmussen, Mark; Poncz, Mortimer; Aster, Richard H.

    2009-01-01

    Drug-induced immune thrombocytopenia (DITP) is caused by drug-dependent antibodies (DDAbs) that are nonreactive in themselves but bind tightly to specific platelet membrane glycoproteins (GP) when soluble drug is present at pharmacologic concentrations. This reaction takes place without covalent linkage of drug to the target, indicating that drug does not function as a classical hapten to promote antibody binding. Studies to define other mechanism(s) responsible for this interaction have been frustrated by the polyclonal nature of human DDAbs and limited quantities of antibody usually available. We produced 2 monoclonal antibodies (mAbs), 314.1 and 314.3, from a mouse immunized with purified human GPIIb/IIIa and quinine that recognize the N terminus of the GPIIb β propeller domain only when soluble quinine is present. Both monoclonals closely mimic the behavior of antibodies from patients with quinine-induced immune thrombo-cytopenia in their reactions at various concentrations of quinine and quinine congeners. Sequencing studies showed that the 2 mAbs are closely related structurally and that mAb 314.3 probably evolved from mAb 314.1 in the course of the immune response. These monoclonal reagents are the first of their kind and should facilitate studies to define the molecular basis for drug-dependent antibody binding and platelet destruction in DITP. PMID:18948570

  4. Fluoxetine-induced alterations in human platelet serotonin transporter expression: serotonin transporter polymorphism effects

    PubMed Central

    Little, Karley Y.; Zhang, Lian; Cook, Edwin

    2006-01-01

    Objective Long-term antidepressant drug exposure may regulate its target molecule — the serotonin transporter (SERT). This effect could be related to an individual's genotype for an SERT promoter polymorphism (human serotonin transporter coding [5-HTTLPR]). We aimed to determine the effects of fluoxetine exposure on human platelet SERT levels. Method We harvested platelet samples from 21 healthy control subjects. The platelets were maintained alive ex vivo for 24 hours while being treated with 0.1 μM fluoxetine or vehicle. The effects on SERT immunoreactivity (IR) were then compared. Each individual's SERT promoter genotype was also determined to evaluate whether fluoxetine effects on SERT were related to genotype. Results Fluoxetine exposure replicably altered SERT IR within individuals. Both the magnitude and the direction of effect were related to a person's SERT genotype. People who were homozygous for the short gene (SS) displayed decreased SERT IR, whereas those who were homozygous for the long gene (LL) demonstrated increased SERT IR. A mechanistic experiment suggested that some individuals with the LL genotype might experience increased conversion of complexed SERT to primary SERT during treatment. Conclusions These preliminary results suggest that antidepressant effects after longer-term use may include changes in SERT expression levels and that the type and degree of effect may be related to the 5-HTTLPR polymorphism. PMID:16951736

  5. Lipopolysaccharide induces nitric oxide synthase expression and platelet-activating factor increases nitric oxide production in human fetal membranes in culture

    PubMed Central

    Seyffarth, Gunter; Nelson, Paul N; Dunmore, Simon J; Rodrigo, Nalinda; Murphy, Damian J; Carson, Ray J

    2004-01-01

    Background Platelet-activating factor and nitric oxide may be involved in the initiation of human labour as inflammatory mediators. The aim of this study was to test whether platelet-activating factor and lipopolysaccharide were able to induce nitric oxide synthase expression and stimulate the production of nitric oxide in human fetal membrane explants in culture. Methods Fetal membranes were collected from Caesarean sections at term. RNA was extracted from membranes and subjected to a qualitative RT-PCR to assess the baseline expression of iNOS. Discs of fetal membranes were cultured for 24 hours in the presence of platelet-activating factor at a dose range of 0.1 nanomolar – 1 micomolar or 1 microgram/ml lipopolysaccharide. Nitric oxide production was measured via nitrite ions in the culture medium and mRNA for iNOS was detected by RT-PCR. Results Culturing the membrane discs in medium containing serum induced nitric oxide synthase expression and platelet-activating factor significantly stimulated the production of nitric oxide under these conditions. When cultured without serum inducible nitric oxide synthase expression was induced by lipopolysaccharide, but not by platelet-activating factor. Conclusion Platelet-activating factor may have a role in the initiation of labour, at term or preterm, via the increased local production of nitric oxide as an inflammatory mediator. In this model of intrauterine infection, lipopolysaccharide was found to induce iNOS expression by fetal membranes, and this mechanism could be involved in preterm labour. PMID:15191613

  6. Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak21

    PubMed Central

    Han, Seung Hyun; Kim, Je Hak; Seo, Ho Seong; Martin, Michael H.; Chung, Gook-Hyun; Michalek, Suzanne M.; Nahm, Moon H.

    2006-01-01

    NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-α and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-α. Jak2 tyrosine kinase blocked LTA-induced production of NO but not TNF-α. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-β expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-α production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-β, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production. PMID:16365452

  7. Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

    PubMed Central

    Montrucchio, G.; Lupia, E.; de Martino, A.; Battaglia, E.; Arese, M.; Tizzani, A.; Bussolino, F.; Camussi, G.

    1997-01-01

    We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent. Images Figure 3 Figure 4 PMID:9250168

  8. Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry

    PubMed Central

    Przygodzki, Tomasz; Talar, Marcin; Blazejczyk, Agnieszka; Kalchenko, Vyacheslav; Watala, Cezary

    2016-01-01

    Introduction The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry. Materials and Methods Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection. Results The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung. Conclusions Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates. PMID:26751810

  9. Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

    PubMed Central

    Kahn, N N; Bauman, W A; Sinha, A K

    1996-01-01

    Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI. PMID:8552614

  10. Hydrogen peroxide generation induces pp60src activation in human platelets: evidence for the involvement of this pathway in store-mediated calcium entry.

    PubMed

    Rosado, Juan A; Redondo, Pedro C; Salido, Ginés M; Gómez-Arteta, Emilio; Sage, Stewart O; Pariente, Jose A

    2004-01-16

    Reactive oxygen species, such as H2O2, have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60(src) by H2O2, a mechanism required for the activation of store-mediated Ca2+ entry (SMCE) in human platelets. Treatment of platelets with H2O2 resulted in a time- and concentration-dependent activation of pp60(src). Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H2O2-induced pp60(src) activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60(src) by H2O2 is independent of increases in [Ca2+](i). Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H2O2-induced pp60(src) activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H2O2 production, a mechanism independent of elevations in [Ca2+](i). Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60(src). In addition, catalase as well as the pp60(src) inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP(3)R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60(src) by H2O2, the PKC inhibitors GF 109202X and Ro-31-8220 were found to reduced SMCE in platelets. This study suggests that platelet activation with TG plus Iono or thrombin is associated with H2O2 production, which acts as a second messenger by stimulating pp60(src) by a PKC-dependent pathway and is involved in the activation of SMCE in these cells.

  11. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  12. Favorable Vocal Fold Wound Healing Induced by Platelet-Rich Plasma Injection

    PubMed Central

    Woo, Seung Hoon; Jeong, Han-Sin; Kim, Jin Pyeong; Koh, Eun-Ha; Lee, Seon Uk; Jin, Sung Min; Kim, Dong Hoon; Sohn, Jin Hee

    2014-01-01

    Objectives To introduce a new injection material for vocal fold diseases, which could be readily translated to clinical practice, we investigated the effectiveness of platelet-rich plasma (PRP) injection on the injured vocal fold in terms of histological recovery. Methods Blood samples were drawn from New Zealand White rabbits and PRP was isolated through centrifugation and separation of the samples. Using a CO2 laser, we made a linear wound in the 24 vocal fold sides of 12 rabbits and injected each wound with PRP on one vocal fold side and normal saline (NS) on the other. Morphologic analyses were conducted at 2, 4, and 12 weeks after injection, and inflammatory response, collagen deposit, and changes in growth factors were assessed using H&E and masson trichrome (MT) staining and western blot assay. Results PRP was prepared in approximately 40 minutes. The mean platelet concentration was 1,315,000 platelets/mm3. In morphological analyses, decreased granulation was observed in the PRP-injected vocal folds (P<0.05). However, the irregular surface and atrophic change were not difference. Histological findings revealed significant inflammation and collagen deposition in NS-injected vocal folds, whereas the PRP-injected vocal folds exhibited less (P<0.05). However, the inflammatory reaction and fibrosis were not difference. In western blot assay, increased amounts of growth factors were observed in PRP-injected vocal folds. Conclusion Injection of injured rabbit vocal folds with PRP led to improved wound healing and fewer signs of scarring as demonstrated by decreased inflammation and collagen deposition. The increased vocal fold regeneration may be due to the growth factors associated with PRP. PMID:24587881

  13. Platelet proteomics.

    PubMed

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  14. Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

    PubMed Central

    Concha, Claudia; Carretta, María Daniella; Alarcón, Pablo; Conejeros, Ivan; Gallardo, Diego; Hidalgo, Alejandra Isabel; Tadich, Nestor; Cáceres, Dante Daniel; Hidalgo, María Angélica

    2014-01-01

    Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response. PMID:25013355

  15. A Histologically Distinctive Interstitial Pneumonia Induced by Overexpression of the Interleukin 6, Transforming Growth Factor β1, or Platelet-Derived Growth Factor B Gene

    NASA Astrophysics Data System (ADS)

    Yoshida, Mitsuhiro; Sakuma, Junko; Hayashi, Seiji; Abe, Kin'ya; Saito, Izumu; Harada, Shizuko; Sakatani, Mitsunoir; Yamamoto, Satoru; Matsumoto, Norinao; Kaneda, Yasufumi; Kishmoto, Tadamitsu

    1995-10-01

    Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor β1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor β1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

  16. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

    PubMed

    Dieudé, Mélanie; Bell, Christina; Turgeon, Julie; Beillevaire, Deborah; Pomerleau, Luc; Yang, Bing; Hamelin, Katia; Qi, Shijie; Pallet, Nicolas; Béland, Chanel; Dhahri, Wahiba; Cailhier, Jean-François; Rousseau, Matthieu; Duchez, Anne-Claire; Lévesque, Tania; Lau, Arthur; Rondeau, Christiane; Gingras, Diane; Muruve, Danie; Rivard, Alain; Cardinal, Héloise; Perreault, Claude; Desjardins, Michel; Boilard, Éric; Thibault, Pierre; Hébert, Marie-Josée

    2015-12-16

    Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. PMID:26676607

  17. Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

    PubMed Central

    Tada, Tomoki; Aki, Kensaku; Oboshi, Wataru; Kawazoe, Kazuyoshi; Yasui, Toshiyuki; Hosoi, Eiji

    2016-01-01

    Background The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. Objective The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. Materials and methods Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. Results There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. Conclusion It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions

  18. Platelets and Infection – An Emerging Role of Platelets in Viral Infection

    PubMed Central

    Assinger, Alice

    2014-01-01

    Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen–antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses. PMID:25566260

  19. Platelets and infection - an emerging role of platelets in viral infection.

    PubMed

    Assinger, Alice

    2014-01-01

    Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis. While platelet activation and elevated platelet counts (thrombocytosis) are associated with increased risk of thrombotic complications, low platelet counts (thrombocytopenia) and several platelet function disorders increase the risk of bleeding. Over the last years, more and more evidence has emerged that platelets and their activation state can also modulate innate and adaptive immune responses and low platelet counts have been identified as a surrogate marker for poor prognosis in septic patients. Viral infections often coincide with platelet activation. Host inflammatory responses result in the release of platelet activating mediators and a pro-oxidative and pro-coagulant environment, which favors platelet activation. However, viruses can also directly interact with platelets and megakaryocytes and modulate their function. Furthermore, platelets can be activated by viral antigen-antibody complexes and in response to some viruses B-lymphocytes also generate anti-platelet antibodies. All these processes contributing to platelet activation result in increased platelet consumption and removal and often lead to thrombocytopenia, which is frequently observed during viral infection. However, virus-induced platelet activation does not only modulate platelet count but also shape immune responses. Platelets and their released products have been reported to directly and indirectly suppress infection and to support virus persistence in response to certain viruses, making platelets a double-edged sword during viral infections. This review aims to summarize the current knowledge on platelet interaction with different types of viruses, the viral impact on platelet activation, and platelet-mediated modulations of innate and adaptive immune responses.

  20. Effect of in-water oxygen prebreathing at different depths on decompression-induced bubble formation and platelet activation.

    PubMed

    Bosco, Gerardo; Yang, Zhong-jin; Di Tano, Guglielmo; Camporesi, Enrico M; Faralli, Fabio; Savini, Fabio; Landolfi, Angelo; Doria, Christian; Fanò, Giorgio

    2010-05-01

    Effect of in-water oxygen prebreathing at different depths on decompression-induced bubble formation and platelet activation in scuba divers was evaluated. Six volunteers participated in four diving protocols, with 2 wk of recovery between dives. On dive 1, before diving, all divers breathed normally for 20 min at the surface of the sea (Air). On dive 2, before diving, all divers breathed 100% oxygen for 20 min at the surface of the sea [normobaric oxygenation (NBO)]. On dive 3, before diving, all divers breathed 100% O2 for 20 min at 6 m of seawater [msw; hyperbaric oxygenation (HBO) 1.6 atmospheres absolute (ATA)]. On dive 4, before diving, all divers breathed 100% O2 for 20 min at 12 msw (HBO 2.2 ATA). Then they dove to 30 msw (4 ATA) for 20 min breathing air from scuba. After each dive, blood samples were collected as soon as the divers surfaced. Bubbles were measured at 20 and 50 min after decompression and converted to bubble count estimate (BCE) and numeric bubble grade (NBG). BCE and NBG were significantly lower in NBO than in Air [0.142+/-0.034 vs. 0.191+/-0.066 (P<0.05) and 1.61+/-0.25 vs. 1.89+/-0.31 (P<0.05), respectively] at 20 min, but not at 50 min. HBO at 1.6 ATA and 2.2 ATA has a similar significant effect of reducing BCE and NBG. BCE was 0.067+/-0.026 and 0.040+/-0.018 at 20 min and 0.030+/-0.022 and 0.020+/-0.020 at 50 min. NBG was 1.11+/-0.17 and 0.92+/-0.16 at 20 min and 0.83+/-0.18 and 0.75+/-0.16 at 50 min. Prebreathing NBO and HBO significantly alleviated decompression-induced platelet activation. Activation of CD62p was 3.0+/-0.4, 13.5+/-1.3, 10.7+/-0.9, 4.5+/-0.7, and 7.6+/-0.8% for baseline, Air, NBO, HBO at 1.6 ATA, and HBO at 2.2 ATA, respectively. The data show that prebreathing oxygen, more effective with HBO than NBO, decreases air bubbles and platelet activation and, therefore, may be beneficial in reducing the development of decompression sickness.

  1. Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity.

    PubMed

    Brandt, S; Krauel, K; Gottschalk, K E; Renné, T; Helm, C A; Greinacher, A; Block, S

    2014-07-01

    Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32-38, which form together with C-terminal AA (66-70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express "HIT antigens". The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

  2. The matricellular protein Cyr61 is a key mediator of platelet-derived growth factor-induced cell migration.

    PubMed

    Zhang, Fuqiang; Hao, Feng; An, Dong; Zeng, Linlin; Wang, Yi; Xu, Xuemin; Cui, Mei-Zhen

    2015-03-27

    Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

  3. Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx.

    PubMed

    Park, Jae-Hyung; Kim, Jeong-Nam; Jang, Byeong-Churl; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

    2016-03-01

    Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of D-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca(2+) ([Ca(2+)]i) increase through store-operated Ca(2+) channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced pro-inflammatory markers through NFκB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca(2+) influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFκB/COX-2 inhibition in PAF-stimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation. PMID:26745504

  4. Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

    PubMed Central

    Du, Lily M.; Nurden, Paquita; Nurden, Alan T.; Nichols, Timothy C.; Bellinger, Dwight A.; Jensen, Eric S.; Haberichter, Sandra L.; Merricks, Elizabeth; Raymer, Robin A.; Fang, Juan; Koukouritaki, Sevasti B.; Jacobi, Paula M.; Hawkins, Troy B.; Cornetta, Kenneth; Shi, Qizhen; Wilcox, David A.

    2013-01-01

    It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A. PMID:24253479

  5. Resveratrol inhibits collagen-induced platelet stimulation through suppressing NADPH oxidase and oxidative inactivation of SH2 domain-containing protein tyrosine phosphatase-2.

    PubMed

    Jang, Ji Yong; Min, Ji Hyun; Wang, Su Bin; Chae, Yun Hee; Baek, Jin Young; Kim, Myunghee; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-12-01

    Reactive oxygen species (ROS) produced upon collagen stimulation are implicated in propagating various platelet-activating pathways. Among ROS-producing enzymes, NADPH oxidase (NOX) is largely responsible for collagen receptor-dependent ROS production. Therefore, NOX has been proposed as a novel target for the development of antiplatelet agent. We here investigate whether resveratrol inhibits collagen-induced NOX activation and further examine the effects of resveratrol on ROS-dependent signaling pathways in collagen-stimulated platelets. Collagen-induced superoxide anion production in platelets was inhibited by resveratrol. Resveratrol suppressed collagen-induced phosphorylation of p47(phox), a major regulatory subunit of NOX. Correlated with the inhibitory effects on NOX, resveratrol protected SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) from ROS-mediated inactivation and subsequently attenuated the specific tyrosine phosphorylation of key components (spleen tyrosine kinase, Vav1, Bruton's tyrosine kinase, and phospholipase Cγ2) for collagen receptor signaling cascades. Resveratrol also inhibited downstream responses such as cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Furthermore, resveratrol inhibited platelet aggregation and adhesion in response to collagen. The antiplatelet effects of resveratrol through the inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2 suggest that resveratrol is a potential compound for prevention and treatment of thrombovascular diseases.

  6. Heterogenous graft rejection pathways in class I major histocompatibility complex-disparate combinations and their differential susceptibility to immunomodulation induced by intravenous presensitization with relevant alloantigens

    PubMed Central

    1991-01-01

    The present study investigates the heterogeneity of graft rejection pathways in class I major histocompatibility complex (MHC)-disparate combinations and the susceptibility of each pathway to immunomodulation induced by intravenous presensitization with alloantigens. Depletion of CD8+ T cells was induced by repeated administration of anti-CD8 monoclonal antibody. CD8+ T cell-depleted mice failed to generate anti- allo class I MHC cytotoxic T cell (CTL) responses but exhibited anti- allo class I MHC T cell responses, such as mixed lymphocyte reaction (MLR)/IL-2 production, that were induced by CD4+ T cells. In contrast, donor-specific intravenous presensitization (DSP), as a model of donor- specific transfusion, induced almost complete elimination of CD4+ and CD8+ T cell-mediated MLR/IL-2 production, whereas this regimen did not affect the generation of CTL responses induced by DSP-resistant elements (CD8+ CTL precursors and CD4+ CTL helpers). Prolongation of skin graft survival was not induced by either of the above two regimens alone, but by the combination of these. Prolonged graft survival was obtained irrespective of whether the administration of anti-CD8 antibody capable of eliminating CTL was started before or after DSP. The combination of DSP with injection of anti-CD4 antibody also effectively prolonged graft survival. However, this was the case only when the injection of antibody was started before DSP, because such antibody administration was capable of inhibiting the generation of CTL responses by eliminating DSP-resistant CD4+ CTL helpers. These results indicate that (a) the graft rejection in class I-disparate combinations is induced by CD8+ CTL-involved and -independent pathways that are resistant and susceptible to DSP, respectively; (b) DSP contributes to, but is not sufficient for, the prolongation of graft survival; and (c) the suppression of graft rejection requires an additional treatment for reducing DSP-resistant CTL responses. The results are

  7. Dengue platelets meet Sir Arthur Conan Doyle.

    PubMed

    Bray, Paul F

    2013-11-14

    In this issue of Blood, Hottz et al provide compelling evidence that dengue virus (DV) induces (1) platelet synthesis of interleukin-1b (IL-1b); (2) platelet-derived IL-1b–containing microvesicles (MVs) that increase vascular permeability; and (3) DV-triggered inflammasome activation in platelets.

  8. Acetal phosphatidic acids: novel platelet aggregating agents.

    PubMed

    Brammer, J P; Maguire, M H; Walaszek, E J; Wiley, R A

    1983-05-01

    1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids

  9. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes: III. Shear and extrinsic fibrinogen-dependent effects.

    PubMed

    Goldsmith, H L; Frojmovic, M M; Braovac, S; McIntosh, F; Wong, T

    1994-01-01

    The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23 degrees C was studied using a previously described double infusion technique and resistive particle counter size analysis. Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 x 10(5) microliters-1; (17)] with [fibrinogen] from 0 to 1.2 microM, the rate and extent of aggregation with 0.7 microM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, G, = 41.9, 335 and 1,335 s-1. As measured by the decrease in singlet concentration, aggregation at 1.2 microM fibrinogen increased with increasing G up to 1,335 s-1, in contrast to that previously reported in citrated plasma, in which aggregation reached a maximum at G = 335 s-1. Without added fibrinogen, there was no aggregation at G = 41.9 s-1; at G = 335 s-1, there was significant aggregation but with an initial lag time, aggregation increasing further at G = 1,335 s-1. Without added fibrinogen, aggregation was abolished at all G upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab')2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37 degrees C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab')2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of

  10. Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

    PubMed Central

    Puebla-Osorio, N; Damiani, E; Bover, L; Ullrich, S E

    2015-01-01

    Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment. PMID:25950475

  11. The effect of platelet activating factor antagonist on ozone-induced airway inflammation and bronchial hyperresponsiveness in guinea pigs

    SciTech Connect

    Tan, W.C.; Bethel, R.A. )

    1992-10-01

    We investigated the role of platelet-activating factor (PAF) in ozone-induced airway responses by examining the effects of L659,989, a potent PAF antagonist, on bronchial hyperresponsiveness and airway inflammation. Twenty-four male guinea pigs were studied in four equal groups. Total lung resistance (RL) in intubated and spontaneously breathing animals was measured in a constant-volume body plethysmograph. Dose-response curves to methacholine were determined in all animals at the start of the experiment. These were repeated on a separate day after the following types of treatments: air exposure in Group 1, intraperitoneally administered alcohol and air exposure in Group 2; intraperitoneally administered alcohol and ozone exposure in Group 3, and intraperitoneally administered L659,989 (a specific PAF antagonist), 5 mg/kg dissolved in alcohol, and ozone exposure in Group 4. Bronchoalveolar lavage (BAL) was performed after the second methacholine challenge, and the bronchial mucosa was also examined for inflammatory cells. Exposure to 3 ppm ozone for 2 h resulted in a three-doubling concentration increase in bronchial responsiveness, which was not significantly inhibited by prior treatment with L659,989. Ozone induced a 1.8-fold increase in BAL total cell count, increased eosinophilic influx into the airways, and increased eosinophilic infiltration in the bronchial mucosa, which were all not inhibited by L659,989 pretreatment. The results suggest that PAF may not have an essential role in ozone-induced airway hyperresponsiveness and nonallergic airway inflammation.

  12. Valproic acid and all trans retinoic acid differentially induce megakaryopoiesis and platelet-like particle formation from the megakaryoblastic cell line MEG-01.

    PubMed

    Schweinfurth, N; Hohmann, S; Deuschle, M; Lederbogen, F; Schloss, P

    2010-01-01

    Both, the activity of transcription factors as well as epigenetic alterations in defined DNA regions regulate cellular differentiation processes. Hence, neuronal differentiation from neural progenitor cells is promoted by the transcription factor all trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid (VPA). VPA has also been shown to be involved in differentiation of tumor cells and to greatly improve the reprogramming of human somatic cells to induced pluripotent stem cells. Here we have investigated the impact of ATRA and VPA on the differentiation of megakaryoctes and platelets from the megakaryocyte progenitor cell line MEG-01. Our results show that treatment with ATRA (10⁻¹¹ M) and VPA (2 × 10⁻³ M) induces megakaryopoiesis of MEG-01 cells as estimated by polyploidy, formation of characteristic proplatelets and elevated expression of the megakaryocytic markers CD41 and CD61. The resulting megakaryocytes stayed viable for more than 3 weeks and shed platelet-like particles positive for CD41, CD61 and CD42b into the supernatant. Platelet-like particles responded to thrombin receptor activating peptide (TRAP-6) with increased externalization of P-selectin. Thus, ATRA and VPA proved to be efficient agents for the gentle induction of megakaryopoiesis and thrombopoiesis of MEG-01 cells providing the possibility to study molecular events underlying megakaryopoiesis and human platelet production over longer time periods. PMID:20942599

  13. Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction.

    PubMed

    Akand, M; Gencer, E; Yaman, Ö; Erişgen, G; Tekin, D; Özdiler, E

    2015-12-01

    To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.

  14. Membrane Permeabilization by Thrombin-Induced Platelet Microbicidal Protein 1 Is Modulated by Transmembrane Voltage Polarity and Magnitude

    PubMed Central

    Koo, Su-Pin; Bayer, Arnold S.; Kagan, Bruce L.; Yeaman, Michael R.

    1999-01-01

    Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758–3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178–187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at −90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at −90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct

  15. The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein.

    PubMed Central

    Koo, S P; Yeaman, M R; Nast, C C; Bayer, A S

    1997-01-01

    Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner. PMID:9353067

  16. Platelet Count

    MedlinePlus

    ... rash Small purplish spots on the skin called purpura, caused by bleeding under the skin Testing may ... Idiopathic thrombocytopenia (ITP), also known as immune thrombocytopenic purpura, is the result of antibody production against platelets. ...

  17. The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts).

    PubMed

    Maurice, Pascal; Waeckel, Ludovic; Pires, Viviane; Sonnet, Pascal; Lemesle, Monique; Arbeille, Brigitte; Vassy, Jany; Rochette, Jacques; Legrand, Chantal; Fauvel-Lafève, Françoise

    2006-04-01

    Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and alpha2beta1 integrin) for efficient platelet activation. PMID:16205938

  18. Platelet-derived growth factor inhibits bone regeneration induced by osteogenin, a bone morphogenetic protein, in rat craniotomy defects.

    PubMed Central

    Marden, L J; Fan, R S; Pierce, G F; Reddi, A H; Hollinger, J O

    1993-01-01

    Platelet-derived growth factor (PDGF) is a potent moderator of soft tissue repair through induction of the inflammatory phase of repair and subsequent enhanced collagen deposition. We examined the effect of recombinant BB homodimer PDGF (rPDGF-BB) applied to rat craniotomy defects, treated with and without bovine osteogenin (OG), to see if bone regeneration would be stimulated. Implants containing 0, 20, 60, or 200 micrograms rPDGF-BB, reconstituted with insoluble rat collagenous bone matrix containing 0, 30, or 150 micrograms OG, were placed into 8-mm craniotomies. After 11 d, 21 of the 144 rats presented subcutaneous masses superior to the defect sites. The masses, comprised of serosanguinous fluid encapsulated by fibrous connective tissue, were larger and occurred more frequently in rats treated with 200 micrograms rPDGF-BB, and were absent in rats not treated with rPDGF-BB. The masses underwent resorption within 28 d after surgery. OG (2-256 micrograms) caused a dose-dependent increase in radiopacity and a marked regeneration of calcified tissue in a dose-dependent fashion within defect sites. However, OG-induced bone regeneration was inhibited 17-53% in the presence of rPDGF-BB. These results suggest that rPDGF-BB inhibited OG-induced bone regeneration and stimulated a soft tissue repair wound phenotype and response. Images PMID:8254045

  19. Protein kinase A regulates 3-phosphatidylinositide dynamics during platelet-derived growth factor-induced membrane ruffling and chemotaxis.

    PubMed

    Deming, Paula B; Campbell, Shirley L; Baldor, Linda C; Howe, Alan K

    2008-12-12

    Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s) by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP(3)-rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP(3) following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP(3) dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP(3) marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP(3) and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP(3)/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events.

  20. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.

    PubMed

    Zhou, Weilin; Ibe, Basil O; Raj, J Usha

    2007-06-01

    We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC. PMID:17322418

  1. Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.

    PubMed

    Travers, J B; Harrison, K A; Johnson, C A; Clay, K L; Morelli, J G

    1996-07-01

    Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.

  2. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  3. Congenital platelet function defects

    MedlinePlus

    Platelet storage pool disorder; Glanzmann's thrombasthenia; Bernard-Soulier syndrome; Platelet function defects - congenital ... disorder may also cause severe bleeding. Platelet storage pool disorder (also called platelet secretion disorder) occurs when ...

  4. [STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

    PubMed

    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Litvinov, R I

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states. PMID:27228656

  5. The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

    PubMed

    Hviid, Claus Vinter B; Samulin Erdem, Johanna; Drechsler, Susanne; Weixelbaumer, Katrin; Ahmed, M Shakil; Attramadal, Håvard; Redl, Heinz; Bahrami, Soheyl; Osuchowski, Marcin F; Aasen, Ansgar O

    2014-03-01

    Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis. PMID:24430538

  6. Redistribution of P-selectin ligands on neutrophil cell membranes and the formation of platelet-neutrophil complex induced by hemodialysis membranes.

    PubMed

    Itoh, Saotomo; Takeshita, Kana; Susuki, Chie; Shige-Eda, Kazunori; Tsuji, Tsutomu

    2008-07-01

    The formation of platelet-neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet-neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet-neutrophil microaggregate formation in developing hemodialysis-associated complications.

  7. The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

    PubMed

    Hviid, Claus Vinter B; Samulin Erdem, Johanna; Drechsler, Susanne; Weixelbaumer, Katrin; Ahmed, M Shakil; Attramadal, Håvard; Redl, Heinz; Bahrami, Soheyl; Osuchowski, Marcin F; Aasen, Ansgar O

    2014-03-01

    Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.

  8. Splenic release of platelets contributes to increased circulating platelet size and inflammation after myocardial infarction.

    PubMed

    Gao, Xiao-Ming; Moore, Xiao-Lei; Liu, Yang; Wang, Xin-Yu; Han, Li-Ping; Su, Yidan; Tsai, Alan; Xu, Qi; Zhang, Ming; Lambert, Gavin W; Kiriazis, Helen; Gao, Wei; Dart, Anthony M; Du, Xiao-Jun

    2016-07-01

    Acute myocardial infarction (AMI) is characterized by a rapid increase in circulating platelet size but the mechanism for this is unclear. Large platelets are hyperactive and associated with adverse clinical outcomes. We determined mean platelet volume (MPV) and platelet-monocyte conjugation (PMC) using blood samples from patients, and blood and the spleen from mice with AMI. We further measured changes in platelet size, PMC, cardiac and splenic contents of platelets and leucocyte infiltration into the mouse heart. In AMI patients, circulating MPV and PMC increased at 1-3 h post-MI and MPV returned to reference levels within 24 h after admission. In mice with MI, increases in platelet size and PMC became evident within 12 h and were sustained up to 72 h. Splenic platelets are bigger than circulating platelets in normal or infarct mice. At 24 h post-MI, splenic platelet storage was halved whereas cardiac platelets increased by 4-fold. Splenectomy attenuated all changes observed in the blood, reduced leucocyte and platelet accumulation in the infarct myocardium, limited infarct size and alleviated cardiac dilatation and dysfunction. AMI-induced elevated circulating levels of adenosine diphosphate and catecholamines in both human and the mouse, which may trigger splenic platelet release. Pharmacological inhibition of angiotensin-converting enzyme, β1-adrenergic receptor or platelet P2Y12 receptor reduced platelet abundance in the murine infarct myocardium albeit having diverse effects on platelet size and PMC. In conclusion, AMI evokes release of splenic platelets, which contributes to the increase in platelet size and PMC and facilitates myocardial accumulation of platelets and leucocytes, thereby promoting post-infarct inflammation. PMID:27129192

  9. Effect of troxerutin on laser-induced thrombus formation in rat mesenteric vessels, coagulation parameters and platelet function.

    PubMed

    Krupiński, K; Giedrojć, J; Bielawiec, M

    1996-01-01

    The antithrombotic effect of Troxerutin have been studied in an experimental model of thrombosis in which rat mesenteric vessels (arterioles and venules) 25-30 microns in diameter were injured by well defined argon laser lesions. Furthermore in vitro effect of this agent on coagulation parameters (IIa, Xa inhibition, TT, heptest), and platelet function (platelet adhesion to the siliconised glass and extracellular matrix, platelet spreading) has been investigated 2 h after oral drug administration. Troxerutin at a dose of 10 mg/kg markedly inhibited thrombus formation in venules. Higher dose (50 mg/kg) was needed to obtain the same antithrombotic effect when arterioles were studied. After application of a single dose of Troxerutin (100 mg/kg) antithrombotic effect lasted for 6 h to 7.5 h when venules were studied, and for 4.5 h to 6 h when arterioles were investigated. In in vitro study we did not observe any effect of Troxerutin on coagulation parameters. In concentrations of 100 micrograms/ml in platelet rich plasma Troxerutin significantly inhibited platelet adhesion to the extracellular matrix and siliconised glass as well as platelet spreading. It is likely that this drug possesses antithrombotic effect evaluated by inhibition of platelet function and protection of endothelial cells.

  10. Piperine inhibits platelet-derived growth factor-BB-induced proliferation and migration in vascular smooth muscle cells.

    PubMed

    Lee, Kang Pa; Lee, Kwan; Park, Won-Hwan; Kim, Hyuck; Hong, Heeok

    2015-02-01

    The proliferation and migration of vascular smooth muscle cells (VSMCs) in blood vessels are important in the pathogenesis of vascular disorders such as atherosclerosis and restenosis. Piperine, a major component of black pepper, has antioxidant, anticancer, and anti-inflammatory activity. However, the antiatherosclerotic effects of piperine have not been investigated. In this study, the effects of piperine on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of VSMCs were investigated. The antiproliferative effects of piperine were determined using MTT assays, cell counting, real-time polymerase chain reaction, and western blots. Our results showed that piperine significantly attenuated the proliferation of VSMCs by increasing the expression of p27(kip1), regulating the mRNA expression of cell cycle enzymes (cyclin D, cyclin E, and PCNA), and decreasing the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in a noncytotoxic concentration-dependent manner (30-100 μM). Moreover, we examined the effects of piperine on the migration of PDGF-BB-stimulated VSMCs, as determined by the Boyden chamber assay, H2DCFDA staining, and western blots. Our results showed that 100 μM piperine decreased cell migration, the production of reactive oxygen species (ROS), and phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Taken together, our results suggest that piperine inhibits PDGF-BB-induced proliferation and the migration of VSMCs by inducing cell cycle arrest and suppressing MAPK phosphorylation and ROS. These findings suggest that piperine may be beneficial for the treatment of vascular-related disorders and diseases.

  11. Piperine inhibits platelet-derived growth factor-BB-induced proliferation and migration in vascular smooth muscle cells.

    PubMed

    Lee, Kang Pa; Lee, Kwan; Park, Won-Hwan; Kim, Hyuck; Hong, Heeok

    2015-02-01

    The proliferation and migration of vascular smooth muscle cells (VSMCs) in blood vessels are important in the pathogenesis of vascular disorders such as atherosclerosis and restenosis. Piperine, a major component of black pepper, has antioxidant, anticancer, and anti-inflammatory activity. However, the antiatherosclerotic effects of piperine have not been investigated. In this study, the effects of piperine on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of VSMCs were investigated. The antiproliferative effects of piperine were determined using MTT assays, cell counting, real-time polymerase chain reaction, and western blots. Our results showed that piperine significantly attenuated the proliferation of VSMCs by increasing the expression of p27(kip1), regulating the mRNA expression of cell cycle enzymes (cyclin D, cyclin E, and PCNA), and decreasing the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in a noncytotoxic concentration-dependent manner (30-100 μM). Moreover, we examined the effects of piperine on the migration of PDGF-BB-stimulated VSMCs, as determined by the Boyden chamber assay, H2DCFDA staining, and western blots. Our results showed that 100 μM piperine decreased cell migration, the production of reactive oxygen species (ROS), and phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Taken together, our results suggest that piperine inhibits PDGF-BB-induced proliferation and the migration of VSMCs by inducing cell cycle arrest and suppressing MAPK phosphorylation and ROS. These findings suggest that piperine may be beneficial for the treatment of vascular-related disorders and diseases. PMID:25384161

  12. Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

    PubMed Central

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  13. Ex vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect.

    PubMed

    Murayama, M

    1984-03-01

    Decompression of human platelet-rich plasma (PRP) in siliconized glass or plastic to 380 mm Hg for 3 hours at 38 degrees C produced platelet aggregation independent of pO2. Aggregation also took place when PRP was compressed to 8,000 PSI and then decompressed slowly to one atmosphere (14.7 PSI) without gas bubble formation. Platelets also aggregated when plasma was decompressed hydrodynamically (Bernoulli effect) at room temperature. It was also found that the drugs piracetam (2-oxypyrolidine acetamide) and pentoxifylline (1-(5-oxohexyl)-theobromine) at 0.5 and 1.0 mM prevent thrombocyte aggregation. Implications for mountain sickness are discussed.

  14. Platelet-activating factor modulates fat storage in the liver induced by a high-refined carbohydrate-containing diet.

    PubMed

    de Oliveira, Marina Chaves; Menezes-Garcia, Zélia; Arifa, Raquel Duque do Nascimento; de Paula, Talles Prosperi; Andrade, João Marcus Oliveira; Santos, Sérgio Henrique Sousa; de Menezes, Gustavo Batista; de Souza, Danielle da Glória; Teixeira, Mauro Martins; Ferreira, Adaliene Versiani Matos

    2015-09-01

    Hepatic diseases are comorbidities caused by obesity and are influenced by diet composition. The aim of this study was to evaluate the kinetics of metabolic and inflammatory liver dysfunction induced by a high-refined carbohydrate-containing (HC) diet and to determine how platelet-activating factor (PAF) modulates the liver lipid content of mice. BALB/c mice were fed a chow or HC diet for the following experimental periods: 1 and 3 days, 1, 2, 4, 6, 8, 10 and 12 weeks. Wild-type (WT) and PAF receptor-deficient (PAFR(-/-)) mice were fed the same diets for 8 weeks. Mice fed with HC diet showed higher triglycerides and cholesterol levels, fibrosis and inflammation in the liver. The number of neutrophils migrating into the liver was also increased in mice fed with HC diet. However, transaminase levels did not change. PAFR(-/-) mice fed with HC diet showed more steatosis, oxidative stress and higher transaminases levels associated with lower inflammation than WT mice. The consumption of HC diet altered the metabolic and inflammatory response in the liver and was worse in PAFR(-/-) mice. We suggest that PAF regulates liver lipid content and dyslipidemia, protecting the mice from lipotoxicity and liver damage.

  15. Platelet-Rich Plasma in Treatment of Zoledronic Acid-Induced Bisphosphonate-related Osteonecrosis of the Jaws

    PubMed Central

    Sarkarat, Farzin; Kalantar Motamedi, Mohammad Hosein; Jahanbani, Jahanfar; Sepehri, Dena; Kahali, Roozbeh; Nematollahi, Zahra

    2014-01-01

    Background: Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a well-known challenging entity warranting management. Platelet-Rich Plasma (PRP) plays an important role in bone biology by enhancing bone repair and regeneration. Objectives: The aim of this animal study was to evaluate the effects of PRP on zoledronic acid-induced BRONJ. Materials and Methods: Seven rats were given 0.04 mg Zoledronic acid intravenously once a week for five weeks. Two weeks later, the animals underwent extraction of their first lower molars, bilaterally. After clinical confirmation of the osteonecrosis, PRP was injected randomly into one of the extraction sockets of each rat. Three weeks later, all rats were sacrificed in order to obtain histological sections. The analysis of epithelialization was performed by McNamar’s test, and the analysis of osteogenesis and angiogenesis was performed by the Wilcoxon Sign Rank test. P value was set at 0.05. Results: We found no significant differences between the two groups regarding the amount of epithelialization, angiogenesis or sequestrum formation (P > 0.05), but a significant difference was seen between the two groups regarding the amount of existing vital bone (P < 0.05). Conclusions: Our study demonstrates positive results (preservation or regeneration of bone) using PRP in treatment of BRONJ. Although PRP may enhance osseous regeneration, long-term follow-ups are required to confirm its benefits. PMID:25032151

  16. Autologous leukocyte-reduced platelet-rich plasma therapy for Achilles tendinopathy induced by collagenase in a rabbit model

    PubMed Central

    González, Juan C.; López, Catalina; Álvarez, María E.; Pérez, Jorge E.; Carmona, Jorge U.

    2016-01-01

    Leukocyte-reduced platelet-rich plasma (LR-PRP) is a therapy for tendinopathy of the Achilles tendon (TAT); however, there is scarce information regarding LR-PRP effects in rabbit models of TAT. We compared, at 4 and 12 weeks (w), the LR-PRP and placebo (PBS) effects on ultrasonography, histology and relative gene expression of collagen types I (COL1A1) and III (COL3A1) and vascular endothelial growth factor (VEGF) in 24 rabbits with TAT induced by collagenase. The rabbits (treated with both treatments) were euthanatised after either 4 or 12 w. A healthy group (HG (n = 6)) was included. At 4 and 12 w, the LR-PRP group had a no statistically different histology score to the HG. At w 4, the COL1A1 expression was significantly higher in the LR-PRP group when compared to HG, and the expression of COL3A1from both LR-PRP and PBS-treated tendons was significantly higher when compared to the HG. At w 12, the expression of COL3A1 remained significantly higher in the PBS group in comparison to the LR-PRP group and the HG. At w 4, the LR-PRP group presented a significantly higher expression of VEGF when compared to the PBS group and the HG. In conclusion, LR-PRP treatment showed regenerative properties in rabbits with TAT. PMID:26781753

  17. Autologous leukocyte-reduced platelet-rich plasma therapy for Achilles tendinopathy induced by collagenase in a rabbit model.

    PubMed

    González, Juan C; López, Catalina; Álvarez, María E; Pérez, Jorge E; Carmona, Jorge U

    2016-01-01

    Leukocyte-reduced platelet-rich plasma (LR-PRP) is a therapy for tendinopathy of the Achilles tendon (TAT); however, there is scarce information regarding LR-PRP effects in rabbit models of TAT. We compared, at 4 and 12 weeks (w), the LR-PRP and placebo (PBS) effects on ultrasonography, histology and relative gene expression of collagen types I (COL1A1) and III (COL3A1) and vascular endothelial growth factor (VEGF) in 24 rabbits with TAT induced by collagenase. The rabbits (treated with both treatments) were euthanatised after either 4 or 12 w. A healthy group (HG (n = 6)) was included. At 4 and 12 w, the LR-PRP group had a no statistically different histology score to the HG. At w 4, the COL1A1 expression was significantly higher in the LR-PRP group when compared to HG, and the expression of COL3A1 from both LR-PRP and PBS-treated tendons was significantly higher when compared to the HG. At w 12, the expression of COL3A1 remained significantly higher in the PBS group in comparison to the LR-PRP group and the HG. At w 4, the LR-PRP group presented a significantly higher expression of VEGF when compared to the PBS group and the HG. In conclusion, LR-PRP treatment showed regenerative properties in rabbits with TAT. PMID:26781753

  18. Anti-platelet activity of water dispersible curcuminoids in rat platelets.

    PubMed

    Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

    2015-03-01

    Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders.

  19. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

    PubMed Central

    Visentin, G P; Ford, S E; Scott, J P; Aster, R H

    1994-01-01

    Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin. PMID:8282825

  20. A myocardial ischemia- and reperfusion-induced injury is mediated by reactive oxygen species released from blood platelets.

    PubMed

    Seligmann, Christian; Prechtl, Gerald; Kusus-Seligmann, Magda; Daniel, Werner G

    2013-01-01

    In recent experimental studies, blood platelets have been found to exhibit some cardiodepressive effects in ischemic and reperfused guinea pig hearts independent of thrombus formation. These effects seemed to be mediated by reactive oxygen species (ROS). However, the source of these ROS - platelets or heart - remained still unknown. Isolated, buffer-perfused and pressure-volume work performing guinea pig hearts were exposed to a low-flow ischemia (1 ml/min) of 30 min duration and reperfused at a constant flow of 5 ml/min. Human thrombocytes were administered as 1 min bolus (20 000 thrombocytes/µl perfusion buffer) in the 15th min of ischemia or in the 1st or 5th min of reperfusion in the presence of thrombin (0.3 U/ml perfusion buffer). Recovery of external heart work (REHW) was expressed as ratio between postischemic and preischemic EHW in percent. Intracoronary platelet retention (RET) was quantified as percent of platelets applied. In a second set of experiments, thrombocytes were incubated with 10 µM of the irreversible NADPH oxidase blocker diphenyliodonium chloride and washed twice, thereafter, and administered according to the same protocol as described above. Hearts exposed to ischemia and reperfusion in the presence of thrombin but without application of platelets served as controls. Controls without application of platelets did not reveal a severe compromisation of myocardial function (REHW 85.5 ± 1%). However, addition of platelets during ischemia or in the 1st or 5th min of reperfusion led to a significant reduction of REHW as compared with controls (REHW 62.4 ± 6, 53.9 ± 3, 40.5 ± 3, respectively). Application of platelets pretreated with diphenyliodonium chloride did not reveal any cardiodepressive effects being significantly different from controls without platelet application. Moreover, treatment of platelets with diphenyliodonium chloride did not significantly decrease intracoronary platelet retention. In conclusion, these results demonstrate

  1. [Platelet hyperreactivity and antiaggregatory properties of nootropic drugs under conditions of alloxan-induced diabetes in rats].

    PubMed

    Zhiliuk, V I; Levykh, A É; Mamchur, V I

    2012-01-01

    The effects of nootropic drugs (noopept, pentoxifylline, piracetam, pramiracetam, Ginkgo biloba extract, entrop, cerebrocurin and citicoline) on platelet aggregation in rats with experimental diabetes have been studied. It is established that all these drugs exhibit an inhibitory action of various degrees against platelet hyperreactivity under conditions of chronic hyperglycemia. The maximum universality of the antiaggregatory action is characteristic of pramiracetam, entrop and Ginkgo biloba extract. PMID:22702111

  2. Impact of Dabigatran versus Phenprocoumon on ADP Induced Platelet Aggregation in Patients with Atrial Fibrillation with or without Concomitant Clopidogrel Therapy (the Dabi-ADP-1 and Dabi-ADP-2 Trials)

    PubMed Central

    Martischnig, Amadea M.; Mehilli, Julinda; Pollak, Janina; Petzold, Tobias; Fiedler, Anette K.; Mayer, Katharina; Schulz-Schüpke, Stefanie; Sibbing, Dirk; Massberg, Steffen; Kastrati, Adnan; Sarafoff, Nikolaus

    2015-01-01

    Background. A relevant number of patients receive triple therapy with clopidogrel, aspirin, and oral anticoagulation. Clopidogrel's efficacy on ADP induced platelet function may be influenced by concomitant antithrombotic therapies. Data regarding the effect of dabigatran on platelet function is limited to in vitro studies and healthy individuals. Methods. The “Dabi-ADP-1” and “Dabi-ADP-2” trials randomized patients with atrial fibrillation to either dabigatran or phenprocoumon for a 2-week period. In Dabi-ADP-1 (n = 70) patients with clopidogrel therapy were excluded and in Dabi-ADP-2 (n = 46) patients had to be treated concomitantly with clopidogrel. The primary endpoint was ADP-induced platelet aggregation between dabigatran and phenprocoumon at 14 days. Secondary endpoints were ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Results. There was no significant difference regarding the primary endpoint between both groups in either trial (Dabi-ADP-1: Dabigatran: 846 [650–983] AU × min versus phenprocoumon: 839 [666–1039] AU × min, P = 0.90 and Dabi-ADP-2: 326 [268–462] versus 350 [214–535], P = 0.70) or regarding the secondary endpoints, ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Conclusion. Dabigatran as compared to phenprocoumon has no impact on ADP-induced platelet aggregation in atrial fibrillation patients neither with nor without concomitant clopidogrel therapy. PMID:26229963

  3. Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression

    PubMed Central

    2016-01-01

    We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1. PMID:27588757

  4. Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression.

    PubMed

    Ando, Yusuke; Oku, Teruaki; Tsuji, Tsutomu

    2016-01-01

    We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1. PMID:27588757

  5. Anti-human protein S antibody induces tissue factor expression through a direct interaction with platelet phosphofructokinase

    PubMed Central

    Chen, Changyi; Liao, Dan; Wang, Jing; Liang, Zhengdong; Yao, Qizhi

    2013-01-01

    Introduction Autoantibodies including anti-human protein S antibody (anti-hPS Ab) and anti-human protein C antibody (anti-hPC Ab) can be detected in patients with autoimmune diseases with hypercoagulability. The objective of the present study was to determine the effects and molecular pathways of these autoantibodies on tissue factor (TF) expression in human coronary artery endothelial cells (HCAECs). Materials and Methods HCAECs were treated with anti-hPS Ab or anti-hPC Ab for 3 hours. TF expression was measured by real-time PCR and Western blot. TF-mediated procoagulant activity was determined by a commercial kit. MAPK phosphorylation was analyzed by Bio-Plex luminex immunoassay and Western blot. The potential proteins interacting with anti-hPS Ab were studied by immunoprecipitation, mass spectrometry and in vitro pull-down assay. Results Anti-hPS Ab, but not anti-hPC Ab, specifically induced TF expression and TF-mediated procoagulant activity in HCAECs in a concentration-dependent manner. This effect was confirmed in human umbilical endothelial cells (HUVECs). ERK1/2 phosphorylation was induced by anti-hPS Ab treatment, while inhibition of ERK1/2 by U0216 partially blocked anti-hPS Ab-induced TF upregulation (P<0.05). In addition, anti-hPS Ab specifically cross-interacted with platelet phosphofructokinase (PFKP) in HCAECs. Anti-hPS Ab was able to directly inhibit PFKP activities in HCAECs. Furthermore, silencing of PFKP by PFKP shRNA resulted in TF upregulation in HCAECs, while activation of PFKP by fructose-6-phosphate partially blocked the effect of anti-hPS Ab on TF upregulation (P<0.05). Conclusions Anti-hPS Ab induces TF expression through a direct interaction with PFKP and ERK1/2 activation in HCAECs. Anti-hPS Ab may directly contribute to vascular thrombosis in the patient with autoimmune disorders. PMID:24331211

  6. Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies.

    PubMed

    Krauel, Krystin; Hackbarth, Christine; Fürll, Birgitt; Greinacher, Andreas

    2012-02-01

    Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin. PMID:22049520

  7. Cancer procoagulant and blood platelet activation.

    PubMed

    Olas, B; Wachowicz, B; Mielicki, W P

    2001-08-28

    The effects of cancer procoagulant (CP), cysteine protease (EC 3.4.22.26), on the pig blood platelet secretory process and platelet aggregation have been studied. The response of platelets to CP was compared with the response of these cells to thrombin. The obtained results show that blood platelets treated with CP (0.5, 1, 2.5, and 5 microg/ml, 2-30 min, 37 degrees C) released adenine nucleotides (P < 0.05) and proteins (P < 0.05). The secretion of compounds from blood platelets after incubation with CP does not correlate with the release of platelet lactic dehydrogenase activity (marker of cell lysis) into the extracellular medium. In comparison with thrombin action, CP stimulates secretory process to a smaller extent than thrombin alone. In the presence of CP, the thrombin action is suppressed (P < 0.05). We noticed that CP does not induce platelet aggregation.

  8. Quinidine, but not eicosanoid antagonists or dexamethasone, protect the gut from platelet activating factor-induced vasoconstriction, edema and paralysis.

    PubMed

    Lautenschläger, Ingmar; Frerichs, Inéz; Dombrowsky, Heike; Sarau, Jürgen; Goldmann, Torsten; Zitta, Karina; Albrecht, Martin; Weiler, Norbert; Uhlig, Stefan

    2015-01-01

    Intestinal circulatory disturbances, atony, edema and swelling are of great clinical relevance, but the related mechanisms and possible therapeutic options are poorly characterized, in part because of the difficulties to comprehensively analyze these conditions. To overcome these limitations we have developed a model of the isolated perfused rat small intestine where all of these symptoms can be studied simultaneously. Here we used this model to study the role of eicosanoids, steroids and quinidine in platelet-activating factor (PAF)-induced intestinal disorders. A vascular bolus of PAF (0.5 nmol) triggered release of thromboxane and peptidoleukotrienes into the vascular bed (peak concentration 35 nM and 0.8 nM) and reproduced all symptoms of intestinal failure: mesenteric vasoconstriction, translocation of fluid and macromolecules from the vasculature to the lumen and lymphatics, intestinal edema formation, loss of intestinal peristalsis and decreased galactose uptake. All effects of PAF were abolished by the PAF-receptor antagonist ABT491 (2.5 μM). The COX and LOX inhibitors ASA and AA861 (500 μM, 10 μM) did not exhibit barrier-protective effects and the eicosanoid antagonists SQ29548 and MK571 (10 μM, each) only moderately attenuated the loss of vascular fluid, the redistribution to the lumen and the transfer of FITC dextran to the lumen. The steroid dexamethasone (10 μM) showed no barrier-protective properties and failed to prevent edema formation. Quinidine (100 μM) inhibited the increase in arterial pressure, stabilized all the intestinal barriers, and reduced lymph production and the transfer of FITC dextran to the lymph. While quinidine by itself reduced peristalsis, it also obviated paralysis, preserved intestinal functions and prevented edema formation. We conclude that quinidine exerts multiple protective effects against vasoconstriction, edema formation and paralysis in the intestine. The therapeutic use of quinidine for intestinal ailments

  9. Quinidine, but Not Eicosanoid Antagonists or Dexamethasone, Protect the Gut from Platelet Activating Factor-Induced Vasoconstriction, Edema and Paralysis

    PubMed Central

    Lautenschläger, Ingmar; Frerichs, Inéz; Dombrowsky, Heike; Sarau, Jürgen; Goldmann, Torsten; Zitta, Karina; Albrecht, Martin; Weiler, Norbert; Uhlig, Stefan

    2015-01-01

    Intestinal circulatory disturbances, atony, edema and swelling are of great clinical relevance, but the related mechanisms and possible therapeutic options are poorly characterized, in part because of the difficulties to comprehensively analyze these conditions. To overcome these limitations we have developed a model of the isolated perfused rat small intestine where all of these symptoms can be studied simultaneously. Here we used this model to study the role of eicosanoids, steroids and quinidine in platelet-activating factor (PAF)-induced intestinal disorders. A vascular bolus of PAF (0.5 nmol) triggered release of thromboxane and peptidoleukotrienes into the vascular bed (peak concentration 35 nM and 0.8 nM) and reproduced all symptoms of intestinal failure: mesenteric vasoconstriction, translocation of fluid and macromolecules from the vasculature to the lumen and lymphatics, intestinal edema formation, loss of intestinal peristalsis and decreased galactose uptake. All effects of PAF were abolished by the PAF-receptor antagonist ABT491 (2.5 μM). The COX and LOX inhibitors ASA and AA861 (500 μM, 10 μM) did not exhibit barrier-protective effects and the eicosanoid antagonists SQ29548 and MK571 (10 μM, each) only moderately attenuated the loss of vascular fluid, the redistribution to the lumen and the transfer of FITC dextran to the lumen. The steroid dexamethasone (10 μM) showed no barrier-protective properties and failed to prevent edema formation. Quinidine (100 μM) inhibited the increase in arterial pressure, stabilized all the intestinal barriers, and reduced lymph production and the transfer of FITC dextran to the lymph. While quinidine by itself reduced peristalsis, it also obviated paralysis, preserved intestinal functions and prevented edema formation. We conclude that quinidine exerts multiple protective effects against vasoconstriction, edema formation and paralysis in the intestine. The therapeutic use of quinidine for intestinal ailments

  10. Platelets in inflammation and infection.

    PubMed

    Jenne, Craig N; Kubes, Paul

    2015-01-01

    Although platelets are traditionally recognized for their central role in hemostasis, many lines of research clearly demonstrate these rather ubiquitous blood components are potent immune modulators and effectors. Platelets have been shown to directly recognize, sequester and kill pathogens, to activated and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behavior, enhancing their ability to phagocytose and kill pathogens and inducing unique effector functions, such as the production of Neutrophil Extracellular Traps (NETs). This multifaceted response to infection and inflammation is due, in part, to the huge array of soluble mediators and cell surface molecules expressed by platelets. From their earliest origins as primordial hemocytes in invertebrates to their current form as megakaryocyte-derived cytoplasts, platelets have evolved to be one of the key regulators of host intravascular immunity and inflammation. In this review, we present the diverse roles platelets play in immunity and inflammation associated with autoimmune diseases and infection. Additionally, we highlight recent advances in our understanding of platelet behavior made possible through the use of advanced imaging techniques that allow us to visualize platelets and their interactions, in real-time, within the intact blood vessels of a living host.

  11. Platelets: bridging hemostasis, inflammation, and immunity.

    PubMed

    Jenne, C N; Urrutia, R; Kubes, P

    2013-06-01

    Although the function of platelets in the maintenance of hemostasis has been studied in great detail, more recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Platelets by virtue of their large numbers and their ability to rapidly release a broad spectrum of immunomodulatory cytokines, chemokines, and other mediators act as circulating sentinels. Upon detection of a pathogen, platelets quickly activate and begin to drive the ensuing inflammatory response. Platelets have the ability to directly modulate the activity of neutrophils (phagocytosis, oxidative burst), endothelium (adhesion molecule and chemokine expression), and lymphocytes. Due to their diverse array of adhesion molecules and preformed chemokines, platelets are able to adhere to leukocytes and facilitate their recruitment to sites of tissue damage or infection. Furthermore, platelets directly participate in the capture and sequestration of pathogens within the vasculature. Platelet-neutrophil interactions are known to induce the release of neutrophil extracellular traps (NETs) in response to either bacterial or viral infection, and platelets have been shown to internalize pathogens, sequestering them in engulfment vacuoles. Finally, emerging data indicate that platelets also participate in the host immune response by directly killing infected cells. This review will highlight the central role platelets play in the initiation and modulation of the host inflammatory and immune responses.

  12. Integrated analysis of miRNA and mRNA gene expression microarrays: Influence on platelet reactivity, clopidogrel response and drug-induced toxicity.

    PubMed

    Freitas, Renata Caroline Costa de; Bortolin, Raul Hernandes; Lopes, Mariana Borges; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo; Silbiger, Vivian Nogueira; Luchessi, André Ducati

    2016-11-15

    Genetic and epigenetic variability may influence the efficacy and safety of antiplatelet therapies, including clopidogrel. Therefore, the miRNA-mRNA interactions and drug toxicity were investigated in silico using available microarray data. Expressions profiles of platelet miRNA (GSE59488) from acute coronary syndrome and mRNA in peripheral blood cells (GSE32226) from coronary artery disease patients were used to miRNA-target mRNA integrated analysis by Ingenuity Pathways Analysis 6 software (IPA). Results showed that ST13 mRNA is regulated by hsa-miR-107 (miR-103-3p); BTNL3 and CFD mRNAs are regulated by hsa-miR-4701-3p (miR-1262); SLC7A8 is regulated by hsa-miR-145-5p (miR-145-5p); and SENP5 mRNA is regulated by hsa-miR-15b-5p (miR-16-5p) and hsa-miR-26a-5p (miR-26a-5p). Drug toxicity IPA tool showed that these miRNAs/mRNAs are associated with clopidogrel-related liver and renal injury. In conclusion, these results demonstrate that differential expression of miRNAs in platelets and interactions with their target mRNAs are associated with variability in platelet reactivity, clopidogrel response and drug-induced toxicity. PMID:27543010

  13. Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3.

    PubMed

    Pampolina, Caroline; McNicol, Archibald

    2005-05-01

    The low-affinity IgG receptor, FcgammaRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcgammaRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017-78 caused the tyrosine phosphorylation of FcgammaRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCgamma2. These early events were dependent on Src family kinases but independent of either TxA(2) or the engagement of the alpha(IIb)beta(3) integrin. During the lag phase prior to platelet aggregation, FcgammaRIIA, Syk, LAT, and PLCgamma2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017-78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcgammaRIIA, Syk, LAT, and PLCgamma2 were rephosphorylated in an RGDS-sensitive, and therefore alpha(IIb)beta(3)-dependent, manner. Additionally, TxA(2) release, 5-hydroxytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited alpha(IIb)beta(3) -mediated re-phosphorylation. Therefore, S. sanguis -bound IgG cross links FcgammaRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of alpha(IIb)beta(3) leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA(2

  14. Emerging roles for platelets as immune and inflammatory cells.

    PubMed

    Morrell, Craig N; Aggrey, Angela A; Chapman, Lesley M; Modjeski, Kristina L

    2014-05-01

    Despite their small size and anucleate status, platelets have diverse roles in vascular biology. Not only are platelets the cellular mediator of thrombosis, but platelets are also immune cells that initiate and accelerate many vascular inflammatory conditions. Platelets are linked to the pathogenesis of inflammatory diseases such as atherosclerosis, malaria infection, transplant rejection, and rheumatoid arthritis. In some contexts, platelet immune functions are protective, whereas in others platelets contribute to adverse inflammatory outcomes. In this review, we will discuss platelet and platelet-derived mediator interactions with the innate and acquired arms of the immune system and platelet-vessel wall interactions that drive inflammatory disease. There have been many recent publications indicating both important protective and adverse roles for platelets in infectious disease. Because of this new accumulating data, and the fact that infectious disease continues to be a leading cause of death globally, we will also focus on new and emerging concepts related to platelet immune and inflammatory functions in the context of infectious disease.

  15. Factor VIII is a positive regulator of platelet function.

    PubMed

    Obergfell, A; Sturm, A; Speer, C P; Walter, U; Grossmann, R

    2006-11-01

    FVIII is an important cofactor in the tenase coagulation factor complex, lack of FVIII causes severe bleeding, whereas high FVIII levels seem to be associated with venous and arterial thromboembolism. Resting platelets do not bind FVIII, but activated platelets bind unactivated FVIII if vWF is not present. We investigated a possible influence of platelet bound FVIII on platelet function itself as it is unclear if there is a direct effect of FVIII on platelet function. The influence of FVIII on platelet function was investigated by flow cytometric analysis of P-selectin expression (CD62P) and PAC-1 binding before and after submaximal stimulation with TRAP-6 (5 microM final concentration), by confocal microscopy and by platelet aggregometry. For flow cytometry and confocal microscopy, washed platelets were incubated with human recombinant FVIII for 5 min at 37 degrees C. Analysis of platelet surface area was measured by computerized image analysis. Treatment with FVIII only caused no changes in P-selectin expression or PAC-1 binding, respectively. Stimulation of platelets with TRAP-6 increased the expression of P-selectin (445%) and PAC-1 binding (934%) as expected. These effects were further increased when platelets were stimulated with TRAP-6 and FVIII (P-selectin 499%, difference not significant; PAC-1 1626%, P < 0.05. Values were expressed in%, related to unstimulated, buffer treated platelets). Platelet spreading on fibrinogen was significantly increased when platelets were treated with FVIII and TRAP-6 compared to TRAP-6 alone (368 vs. 307 average pixel/platelet, P<0.05). In addition platelet aggregation was enhanced when platelets were stimulated with FVIII and TRAP-6 compared to TRAP-6 alone. FVIII can act as a positive regulator of platelet function in TRAP-co-stimulated platelets. We hypothesize that FVIII induced increase in platelet activation might contribute to venous and even arterial thrombus formation in patients with high FVIII levels. PMID:17074720

  16. Increasing platelet aggregability after venepuncture is platelet, not plasma derived.

    PubMed

    Terres, W; Becker, B F; Kratzer, M A; Gerlach, E

    1986-05-15

    The time course of ADP induced aggregation of human platelets was determined in aliquots of stored platelet rich plasma 3.5, 10, 30 and 100 minutes after venepuncture. The maximal rate of aggregation was found to increase throughout this entire period, even though pH (7.4), CO2 (7 volume per cent) and temperature (35 degrees C) of the samples were kept constant. The mean acceleration (+/- SEM) between 3.5 and 100 minutes was 41.7 +/- 6.9 per cent (n = 67) at an ADP-concentration of 1 mumol/l and 18.3 +/- 6.2 per cent (n = 23) at 2 mumol/l ADP. The effect did not result from changes of any platelet regulatory factors putatively present alone in the plasma. Acceleration of aggregability was only found when the platelets themselves underwent storage, but not when freshly prepared plasma was given to prestored platelets. The change in aggregability was not diminished after inhibition of platelet cyclooxygenase by oral administration of acetylsalicylic acid. PMID:3715816

  17. Sphingosine 1-phosphate induces platelet/endothelial cell adhesion molecule-1 phosphorylation in human endothelial cells through cSrc and Fyn.

    PubMed

    Huang, Yu-Ting; Chen, Shee-Uan; Chou, Chia-Hong; Lee, Hsinyu

    2008-08-01

    Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells. PMID:18502612

  18. The relationship between platelet endothelial cell adhesion molecule-1 and paraquat-induced lung injury in rabbits

    PubMed Central

    Shi, Jing; Hu, Chun-lin; Gao, Yu-feng; Liao, Xiao-xing; Xu, Hope

    2012-01-01

    BACKGROUND: Platelet endothelial cell adhesion molecule-1 (PECAM-1), also known as CD31, is mainly distributed in vascular endothelial cells. Studies have shown that PECAM-1 is a very significant indicator of angiogenesis, and has been used as an indicator for vascular endothelial cells. The present study aimed to explore the relationship between the expression of PECAM-1 and the degree of acute lung injury (ALI) and fibrosis in paraquat (PQ) induced lung injury in rabbits. METHODS: Thirty-six adult New Zealand rabbits were randomly divided into three groups (12 rabbits in each group) according to PQ dosage: 8 mg/kg (group A), 16 mg/kg (group B), and 32 mg/kg (group C). After PQ infusion, the rabbits were monitored for 7 days and then euthanized. The lungs were removed for histological evaluation. Masson staining was used to determine the degree of lung fibrosis (LF), and semi-quantitative immune-histochemistry analysis to determine the expression of PECAM-1. Pearson’s product-moment correlation analysis was performed to evaluate the relationship between the expression of PECAM-1 and the extent of lung injuries expressed by ALI score and degree of LF. RESULTS: Rabbits in the three groups showed apparent poisoning. The rabbits survived longer in group A than in groups B and C (6.47±0.99 days vs. 6.09±1.04 days vs. 4.77±2.04 days) (P<0.05). ALI score was lower in group A than in groups B and C (8.33±1.03 vs. 9.83±1.17 vs. 11.50±1.38) (P<0.05), and there was statistically significant difference between group B and group C (P=0.03). LF was slighter in group A than in groups B and C (31.09%±2.05 % vs. 34.37%±1.62 % vs. 36.54%±0.44%) (P<0.05), and there was statistically significant difference between group B and group C (P=0.026). The PEACAM-1 expression was higher in group A than in groups B and C (20.31%±0.70% vs. 19.34%±0.68% vs. 18.37%±0.46%) (P<0.05), and there was statistically significant difference between group B and group C (P=0.017). Pearson

  19. LC-MS/MS Analysis and Comparison of Oxidative Damages on Peptides Induced by Pathogen Reduction Technologies for Platelets

    NASA Astrophysics Data System (ADS)

    Prudent, Michel; Sonego, Giona; Abonnenc, Mélanie; Tissot, Jean-Daniel; Lion, Niels

    2014-04-01

    Pathogen reduction technologies (PRT) are photochemical processes that use a combination of photosensitizers and UV-light to inactivate pathogens in platelet concentrates (PCs), a blood-derived product used to prevent hemorrhage. However, different studies have questioned the impact of PRT on platelet function and transfusion efficacy, and several proteomic analyses revealed possible oxidative damages to proteins. The present work focused on the oxidative damages produced by the two main PRT on peptides. Model peptides containing residues prone to oxidation (tyrosine, histidine, tryptophane, and cysteine) were irradiated with a combination of amotosalen/UVA (Intercept process) or riboflavin/UVB (Mirasol-like process). Modifications were identified and quantified by liquid chromatography coupled to tandem mass spectrometry. Cysteine-containing peptides formed disulfide bridges (R-SS-R, -2 Da; favored following amotosalen/UVA), sulfenic and sulfonic acids (R-SOH, +16 Da, R-SO3H, +48 Da, favored following riboflavin/UVB) upon treatment and the other amino acids exhibited different oxidations revealed by mass shifts from +4 to +34 Da involving different mechanisms; no photoadducts were detected. These amino acids were not equally affected by the PRT and the combination riboflavin/UVB generated more oxidation than amotosalen/UVA. This work identifies the different types and sites of peptide oxidations under the photochemical treatments and demonstrates that the two PRT may behave differently. The potential impact on proteins and platelet functions may thus be PRT-dependent.

  20. Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbβ3 integrin

    PubMed Central

    Wang, Lu; Zhou, Junsong; Ahmad, Syed S.; Mutus, Bulent; Garbi, Natalio; Hämmerling, Günter; Liu, Junling

    2013-01-01

    The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbβ3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488−labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbβ3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus. PMID:24030382

  1. Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the αIIbβ3 integrin.

    PubMed

    Wang, Lu; Wu, Yi; Zhou, Junsong; Ahmad, Syed S; Mutus, Bulent; Garbi, Natalio; Hämmerling, Günter; Liu, Junling; Essex, David W

    2013-11-21

    The platelet protein disulfide isomerase called ERp57 mediates platelet aggregation, but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis, we generated a megakaryocyte/platelet-specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times with FeCl3-induced carotid artery injury. Using a mesenteric artery thrombosis model, we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Platelets lacking ERp57 have defective activation of the αIIbβ3 integrin and platelet aggregation. The defect in aggregation was corrected by the addition of exogenous ERp57, implicating surface ERp57 in platelet aggregation. Using mutants of ERp57, we demonstrate the second active site targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn(2+)-treated platelets lacking β3 was decreased substantially, suggesting a direct interaction of ERp57 with αIIbβ3. Surface expression of ERp57 protein and activity in human platelets increased with platelet activation, with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus. PMID:24030382

  2. Platelet-cytokine Complex Suppresses Tumour Growth by Exploiting Intratumoural Thrombin-dependent Platelet Aggregation

    PubMed Central

    Li, Yu-Tung; Nishikawa, Tomoyuki; Kaneda, Yasufumi

    2016-01-01

    Tumours constitute unique microenvironments where various blood cells and factors are exposed as a result of leaky vasculature. In the present study, we report that thrombin enrichment in B16F10 melanoma led to platelet aggregation, and this property was exploited to administer an anticancer cytokine, interferon-gamma induced protein 10 (IP10), through the formation of a platelet-IP10 complex. When intravenously infused, the complex reached platelet microaggregates in the tumour. The responses induced by the complex were solely immune-mediated, and tumour cytotoxicity was not observed. The complex suppressed the growth of mouse melanoma in vivo, while both platelets and the complex suppressed the accumulation of FoxP3+ regulatory T cells in the tumour. These results demonstrated that thrombin-dependent platelet aggregation in B16F10 tumours defines platelets as a vector to deliver anticancer cytokines and provide specific treatment benefits. PMID:27117228

  3. Interaction of polypeptide antibiotic gramicidin S with platelets.

    PubMed

    Hackl, Ellen V; Berest, Vladimir P; Gatash, Sergey V

    2012-12-01

    Gramicidin S (GS) is a cyclic decapeptide antibiotic active against both Gram-positive and Gram-negative bacteria as well as against several pathogenic fungi. However, clinical application of GS is limited because of GS hemolytic activity. The large number of GS analogues with potentially attenuated hemolytic activity has been developed over the last two decades. For all new GS derivatives, the antimicrobial test is accompanied with the hemolytic activity assay. At the same time, neither GS nor its analogues were tested against other blood cells. In the present work, the effects of GS on platelets and platelet aggregates have been studied. GS interaction with platelets is concentration dependent and leads either to platelet swelling or platelet shape change. Effect of GS on platelets is independent of platelet aggregation mechanism. GS induces disaggregation of platelet aggregates formed in the presence of aggregation agonists. The rate of the GS interaction with platelet membranes depends on membrane lipid mobility and significantly increases with temperature. The interaction of GS with the platelet membranes depends strongly on the state of the membrane lipids. Factors affecting the membrane lipids (temperature, lipid peroxidation and ionising irradiation) modify GS interaction with platelets. Our results show that GS is active not only against erythrocytes but also against other blood cells (platelets). The estimated numbers of GS molecules per 1 µm2 of a blood cell required to induce erythrocyte hemolysis and disaggregation of platelet aggregates are comparable. This must be considered when developing new antimicrobial GS analogues with improved hemolytic properties.

  4. Inherited platelet disorders.

    PubMed

    Franchini, Massimo; Lippi, Giuseppe; Veneri, Dino; Targher, Giovanni; Zaffanello, Marco; Guidi, Gian Cesare

    2008-01-01

    Inherited platelet disorders are a rare, but probably underdiagnosed, cause of symptomatic bleeding. They are characterized by abnormalities of platelet number (inherited thrombocytopenias), function (inherited disorders of platelet function) or both. This review briefly discusses the inherited platelet disorders with respect to molecular defects, diagnostic evaluation and treatment strategies.

  5. [Prophylactic platelet transfusions].

    PubMed

    Ilmakunnas, Minna; Remes, Kari; Hiippala, Seppo; Mäkisalo, Heikki; Åberg, Fredrik

    2016-01-01

    The consumption of platelet products in Finland is exceptionally high. For the most part, platelets are transfused pre-operatively to thrombocytopenic patients in order to prevent hemorrhage. Most of the minor procedures could, however, be conducted even if the patients'platelet levels would be lower than usual. In cardiac surgery, platelets are used because of the hemorrhagic diathesis associated with platelet inhibitors. Platelet inhibitors will, however, also bind to transfused platelets, whereby instead of prophylactic platelet transfusions it would be more sensible to leave the thorax open and not carry out ineffective platelet transfusions until the effect of the inhibitors has run out. We outline the prophylactic use of platelets based on recent international clinical practice guidelines. PMID:27400590

  6. Proplatelets and stress platelets.

    PubMed

    Tong, M; Seth, P; Penington, D G

    1987-02-01

    The process of platelet formation by the fragmentation of megakaryocyte pseudopodia, termed proplatelets, demonstrable in the marrow sinusoids is poorly understood. "Stress" platelets produced under conditions of stimulated platelet production differ from normal circulating platelets with respect to volume and a number of functional characteristics. To clarify the relationship of stress platelets to proplatelets, rats were injected with heterologous platelet antiserum. Nondiscoid platelet forms, some characteristically beaded in appearance, strongly resembling bone marrow proplatelets, can be recovered in the circulation of normal rats. During the early period of recovery from acute thrombocytopenia, there was a substantial increase in the proportion of these elongated platelets in the citrated platelet rich plasma. Exposure to EDTA rendered them spherical. Circulating proplatelets may contribute significantly to the prompt increase in platelet volume during recovery from acute thrombocytopenia at a time prior to significant increase in megakaryocyte size and ploidy. PMID:3801667

  7. Paying To Belong: When Does Rejection Trigger Ingratiation?

    PubMed Central

    Romero-Canyas, Rainer; Downey, Geraldine; Reddy, Kavita S.; Rodriguez, Sylvia; Cavanaugh, Timothy J.; Pelayo, Rosemary

    2010-01-01

    Societies and social scientists have long held the belief that exclusion induces ingratiation and conformity, an idea in contradiction with robust empirical evidence linking rejection with hostility and aggression. The classic literatures on ingratiation and conformity help resolve this contradiction by identifying circumstances under which rejection may trigger efforts to ingratiate. Jointly, findings from these literatures suggest that when people are given an opportunity to impress their rejecters, ingratiation is likely after rejection experiences that are harsh and that occur in important situations that threaten the individual’s self-definition. Four studies tested the hypothesis that people high in rejection sensitivity, and therefore dispositionally concerned about rejection, will utilize opportunities to ingratiate after harsh rejection in situations that are self-defining. In three studies of situations that are particularly self-defining for men, rejection predicted ingratiation among men (but not women) who were high in rejection sensitivity. In a fourth study, harsh rejection in a situation particularly self-defining for women predicted ingratiation among highly rejection-sensitive women (but not men). These findings help identify the specific circumstances under which people are willing to act in socially desirable ways toward those who have rejected them harshly. PMID:20649367

  8. Inhibition of platelet-activating factor- and zymosan-activated serum-induced chemotaxis of human neutrophils by nedocromil sodium, BN 52021 and sodium cromoglycate.

    PubMed Central

    Bruijnzeel, P. L.; Warringa, R. A.; Kok, P. T.

    1989-01-01

    1. Inflammatory cells such as eosinophils and neutrophils are thought to contribute actively to the pathogenesis of asthma since they infiltrate into the lung tissue. These cells are mobilized by lipid-like and protein-like chemotactic factors. As illustrative examples of both groups, platelet-activating-factor (Paf) and zymosan-activated-serum (ZAS) were used in this study. The inhibitory effects of nedocromil sodium, the Paf antagonist BN 52021 and sodium cromoglycate on Paf- and ZAS-induced neutrophil chemotaxis were evaluated. 2. All tested drugs inhibited Paf-induced neutrophil chemotaxis with approximately the same potency (IC50 approximately 1 nM). 3. Nedocromil sodium and sodium cromoglycate were equally potent in inhibiting ZAS-induced neutrophil chemotaxis (IC50 = 0.1-1 microM), whereas BN 52021 was considerably less potent (IC30 = 10 microM). 4. To find out whether the drugs tested could inhibit early events in cell activation, their capacity to inhibit Paf- and ZAS-induced cytosolic free Ca2+-mobilization was investigated. BN 52021, at a concentration of 100 microM, completely inhibited Paf-induced Ca2+-mobilization and inhibited ZAS-induced Ca2+-mobilization by about 50%. Nedocromil sodium and sodium cromoglycate were ineffective. PMID:2551444

  9. Platelets and infections - complex interactions with bacteria.

    PubMed

    Hamzeh-Cognasse, Hind; Damien, Pauline; Chabert, Adrien; Pozzetto, Bruno; Cognasse, Fabrice; Garraud, Olivier

    2015-01-01

    Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-like receptors but also integrins conventionally described in the hemostatic response, such as GPIIb-IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet-bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis, or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of neutrophil extracellular traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet-bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the aggregation response

  10. The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

    PubMed Central

    Siciliano, Camilla; Chimenti, Isotta; Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Peruzzi, Mariangela; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  11. The potential of GMP-compliant platelet lysate to induce a permissive state for cardiovascular transdifferentiation in human mediastinal adipose tissue-derived mesenchymal stem cells.

    PubMed

    Siciliano, Camilla; Chimenti, Isotta; Bordin, Antonella; Ponti, Donatella; Iudicone, Paola; Peruzzi, Mariangela; Rendina, Erino Angelo; Calogero, Antonella; Pierelli, Luca; Ibrahim, Mohsen; De Falco, Elena

    2015-01-01

    Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs. PMID:26495284

  12. Platelet function in the postprandial period

    PubMed Central

    2012-01-01

    Background Postprandial hyperlipidemia and hyperglycemia have been related to cardiovascular events. Among different underlying mechanisms platelet activation seems to be responsible too. No comparable data between various tests in normo- vs. hyperlipidemics before and at different time intervals are available after a fat meal. We aimed to compare 9 of them within the same patients at several time points in postprandial hyperlipidemia. Results For some tests baseline values between the groups were significantly different (TXB2, platelet sensitivity, sedimentation and WU-test). However, hyperlipidemia revealed a variable influence on the tests examined. Some of the available tests apparently sensitive to show platelet activation reflect the increase in triglycerides (TG), such as the sedimentation index. ADP-induced platelet aggregatory activity in count adjusted washed isolated platelet samples during postprandial hyperlipidemia indicates mildly enhanced platelet activity, but does not seem to induce significant changes in aggregation. In patients with severe hypertriglyceridemia (> 400 mg/dl fasting) changes in platelet function are more pronounced due to delayed decay and may last up to 16 hours paralleling TG reaching the prevalue. The overwhelming majority of platelet function tests do not significantly respond to postprandial hyperlipidemia. The correlation between the tests applied is poor. For standardization purpose, platelet aggregation tests, aimed to examine proaggregatory capacity in atherosclerosis, should only be performed at the same time of the day after a fasting period > 6 hours. The great variation in preanalytical work-up on comparison of various tests, large number of platelet tests available and their respective potential value are discussed. Conclusions At present, the suspicion that platelet function is significantly activated in the postprandial period cannot be supported by any of the tests used. The information provided is valuable to

  13. Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction.

    PubMed

    Bragado, M J; Gil, M C; Garcia-Marin, L J

    2011-12-01

    Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction. PMID:22023717

  14. "Science" Rejects Postmodernism.

    ERIC Educational Resources Information Center

    St. Pierre, Elizabeth Adams

    2002-01-01

    The National Research Council report, "Scientific Research in Education," claims to present an inclusive view of sciences in responding to federal attempts to legislate educational research. This article asserts that it narrowly defines science as positivism and methodology as quantitative, rejecting postmodernism and omitting other theories. Uses…

  15. Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

    NASA Technical Reports Server (NTRS)

    Reed, G. L.; Matsueda, G. R.; Haber, E.

    1992-01-01

    Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

  16. Interactions of human blood-platelets with vaccinia

    SciTech Connect

    Vernon, C.E.B.

    1989-01-01

    These investigations were conducted to determine whether vaccinia (strain WR) adsorbs to the human platelet and alters specific platelet activities, namely, the uptake of {sup 14}C-serotonin, the release of {sup 14}C-serotonin and also the release of {sup 14}C-serotonin stimulated by thrombin. Vaccinia did not alter the platelet uptake of {sup 14}C-serotonin. To determine if vaccinia induces a release of {sup 14}C-serotonin from platelets, vaccinia was added to washed or unwashed {sup 14}C-serotonin labeled platelets, and the release of {sup 14}C-Serotonin into the supernatant was measured. Less than 8% of the {sup 14}C-Serotonin was released. The action of vaccinia to alter the platelet release of {sup 14}C-serotonin induced by thrombin was monitored by measuring the radioactivity released from thrombin stimulated {sup 14}C-serotonin labeled platelets incubated with or without vaccinia. Vaccinia inhibited the thrombin induced release of {sup 14}C-serotonin from platelets at a virus to platelet ratios of 5 through 80 plaque forming units (p.f.u.)/platelet. The inhibition was dose dependent. The binding of virus to platelets was determined by a plaque assay of a washed mixture of vaccinia virus and platelets. After inoculation of mixture onto a monolayer of BSC40 cells at a virus to platelet ratio of 0.1 p.f.u./platelet, 50 cell-bound-virus per 130,000-150,000 platelets were enumerated. Vaccinia was observed to inhibit the thrombin induced clot formation of plasma by a thrombin clotting time test. Scanning electron micrographs of the clot formed in the presence of vaccinia revealed a close packed fibrous structure lacking the cross-linked mesh-like pattern seen in a normal clot. Transmission electron micrographs showed an increase in the length and a close packing of the fibrin threads.

  17. Anucleate platelets generate progeny

    PubMed Central

    Schwertz, Hansjörg; Köster, Sarah; Kahr, Walter H. A.; Michetti, Noemi; Kraemer, Bjoern F.; Weitz, David A.; Blaylock, Robert C.; Kraiss, Larry W.; Greinacher, Andreas; Zimmerman, Guy A.

    2010-01-01

    Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and α-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signal-dependent expression of surface P-selectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream. PMID:20086251

  18. Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells.

    PubMed

    Wagner, Brent; Gorin, Yves

    2014-01-01

    The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4. PMID:24197068

  19. Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells

    PubMed Central

    Gorin, Yves

    2013-01-01

    The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2′,7′-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4. PMID:24197068

  20. The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway.

    PubMed

    Yu, Chuanjin; Fan, Lili; Gao, Jinxin; Wang, Meng; Wu, Qiong; Tang, Jun; Li, Yaqian; Chen, Jie

    2015-01-01

    Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen. PMID:26273755

  1. Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart

    PubMed Central

    Ylä-Herttuala, Seppo; Betsholtz, Christer; Andrae, Johanna

    2016-01-01

    Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRβ) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRβ agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses. PMID:27513343

  2. The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway.

    PubMed

    Yu, Chuanjin; Fan, Lili; Gao, Jinxin; Wang, Meng; Wu, Qiong; Tang, Jun; Li, Yaqian; Chen, Jie

    2015-01-01

    Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

  3. Platelets as immune cells in infectious diseases.

    PubMed

    Speth, Cornelia; Löffler, Jürgen; Krappmann, Sven; Lass-Flörl, Cornelia; Rambach, Günter

    2013-11-01

    Platelets have been shown to cover a broad range of functions. Besides their role in hemostasis, they have immunological functions and thus participate in the interaction between pathogens and host defense. Platelets have a broad repertoire of receptor molecules that enable them to sense invading pathogens and infection-induced inflammation. Consequently, platelets exert antimicrobial effector mechanisms, but also initiate an intense crosstalk with other arms of the innate and adaptive immunity, including neutrophils, monocytes/macrophages, dendritic cells, B cells and T cells. There is a fragile balance between beneficial antimicrobial effects and detrimental reactions that contribute to the pathogenesis, and many pathogens have developed mechanisms to influence these two outcomes. This review aims to highlight aspects of the interaction strategies between platelets and pathogenic bacteria, viruses, fungi and parasites, in addition to the subsequent networking between platelets and other immune cells, and the relevance of these processes for the pathogenesis of infections.

  4. Role of anti-vimentin antibodies in allograft rejection.

    PubMed

    Rose, Marlene L

    2013-11-01

    Production of anti-vimentin antibodies (AVA) after solid organ transplantation are common. Although classically thought to be expressed mainly within the cytosol, recent evidence demonstrates that extracellular or cell surface expression of vimentin is not unusual. This review examines the evidence to assess whether AVA contribute to allograft pathology. Clinical studies suggest that AVA are associated with cardiac allograft vasculopathy in heart transplant recipients. Studies in non-human primates confirm that production of AVA after renal and heart transplantation are not inhibited by Cyclosporine. Experimental studies have demonstrated that mice pre-immunised with vimentin undergo accelerated acute rejection and vascular intimal occlusion of cardiac allografts. Adoptive transfer of hyperimmune sera containing AVA into B-cell-knock-out mice caused accelerated rejection of allografted hearts, this is clear evidence that antibodies to vimentin accelerate rejection. AVA act in concert with the alloimmune response and AVA do not damage syngeneic or native heart allografts. Confocal microscopy of allografted organs in vimentin immunised mice shows extensive expression of vimentin on endothelial cells, apoptotic leukocytes and platelet/leukocyte conjugates, co-localising with C4d. One explanation for the ability of AVA to accelerate rejection would be fixation of complement within the graft and subsequent pro-inflammatory effects; there may also be interactions with platelets within the vasculature.

  5. Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity

    PubMed Central

    Miao, Xinyan; Zhang, Wei; Huang, Zhangsen

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities. PMID:27612088

  6. Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity.

    PubMed

    Miao, Xinyan; Zhang, Wei; Huang, Zhangsen; Li, Nailin

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities. PMID:27612088

  7. Platelet generation in vivo and in vitro.

    PubMed

    Wang, Biao; Zheng, Jiansheng

    2016-01-01

    Platelet (PLT) transfusion, which is the primary cell therapy for thrombocytopenia, has been a source of concern in recent years due to its limitations of donor-dependent supply and soaring costs. In vitro platelet generation on an industrial scale is a possible solution requiring exploration. The technology of platelet generation ex vivo has been widely studied across the world, though the mechanisms of physiological thrombopoiesis and platelet biology function in vivo still remain elusive today. Various culture systems have been studied, most of which proved quite inefficient in generating functional platelets ex vivo, so there is still a long way to reach our ultimate goal of generating a fully functional platelet in vitro on an industrial scale. This review integrates the latest research into physiological platelet biogenesis and ex vivo-platelet/megakaryocyte (MK) generation protocols with a focus on the ability to generate PLT/MK in large quantities, summarizes current culture systems based on induced human pluripotent stem cells and adipose-derived stem cells, and discusses significant challenges that must be overcome for these approaches to be perfected. PMID:27390629

  8. Titanium surface hydrophilicity enhances platelet activation.

    PubMed

    Alfarsi, Mohammed A; Hamlet, Stephen M; Ivanovski, Saso

    2014-01-01

    Titanium implant surface modification is a key strategy used to enhance osseointegration. Platelets are the first cells that interact with the implant surface whereupon they release a wide array of proteins that influence the subsequent healing process. This study therefore investigated the effect of titanium surface modification on the attachment and activation of human platelets. The surface characteristics of three titanium surfaces: smooth (SMO), micro-rough (SLA) and hydrophilic micro-rough (SLActive) and the subsequent attachment and activation of platelets following exposure to these surfaces were determined. The SLActive surface showed the presence of significant nanoscale topographical features. While attached platelets appeared to be morphologically similar, significantly fewer platelets attached to the SLActive surface compared to both the SMO and SLA surfaces. The SLActive surface however induced the release of the higher levels of chemokines β-thromboglobulin and platelet factor 4 from platelets. This study shows that titanium surface topography and chemistry have a significant effect on platelet activation and chemokine release.

  9. Electrospun silk fibroin/poly (L-lactide-ε-caplacton) graft with platelet-rich growth factor for inducing smooth muscle cell growth and infiltration

    PubMed Central

    Yin, Anlin; Bowlin, Gary L.; Luo, Rifang; Zhang, Xingdong; Wang, Yunbing; Mo, Xiumei

    2016-01-01

    The construction of a smooth muscle layer for blood vessel through electrospinning method plays a key role in vascular tissue engineering. However, smooth muscle cells (SMCs) penetration into the electrospun graft to form a smooth muscle layer is limited due to the dense packing of fibers and lack of inducing factors. In this paper, silk fibroin/poly (L-lactide-ε-caplacton) (SF/PLLA-CL) vascular graft loaded with platelet-rich growth factor (PRGF) was fabricated by electrospinning. The in vitro results showed that SMCs cultured in the graft grew fast, and the incorporation of PRGF could induce deeper SMCs infiltrating compared to the SF/PLLA-CL graft alone. Mechanical properties measurement showed that PRGF-incorporated graft had proper tensile stress, suture retention strength, burst pressure and compliance which could match the demand of native blood vessel. The success in the fabrication of PRGF-incorporated SF/PLLA-CL graft to induce fast SMCs growth and their strong penetration into graft has important application for tissue-engineered blood vessels. PMID:27482466

  10. Electrospun silk fibroin/poly (L-lactide-ε-caplacton) graft with platelet-rich growth factor for inducing smooth muscle cell growth and infiltration.

    PubMed

    Yin, Anlin; Bowlin, Gary L; Luo, Rifang; Zhang, Xingdong; Wang, Yunbing; Mo, Xiumei

    2016-12-01

    The construction of a smooth muscle layer for blood vessel through electrospinning method plays a key role in vascular tissue engineering. However, smooth muscle cells (SMCs) penetration into the electrospun graft to form a smooth muscle layer is limited due to the dense packing of fibers and lack of inducing factors. In this paper, silk fibroin/poly (L-lactide-ε-caplacton) (SF/PLLA-CL) vascular graft loaded with platelet-rich growth factor (PRGF) was fabricated by electrospinning. The in vitro results showed that SMCs cultured in the graft grew fast, and the incorporation of PRGF could induce deeper SMCs infiltrating compared to the SF/PLLA-CL graft alone. Mechanical properties measurement showed that PRGF-incorporated graft had proper tensile stress, suture retention strength, burst pressure and compliance which could match the demand of native blood vessel. The success in the fabrication of PRGF-incorporated SF/PLLA-CL graft to induce fast SMCs growth and their strong penetration into graft has important application for tissue-engineered blood vessels. PMID:27482466

  11. Phloretin Inhibits Platelet-derived Growth Factor-BB-induced Rat Aortic Smooth Muscle Cell Proliferation, Migration, and Neointimal Formation After Carotid Injury.

    PubMed

    Wang, Dong; Wang, Qingjie; Yan, Gaoliang; Qiao, Yong; Tang, Chengchun

    2015-05-01

    Abnormal vascular smooth muscle cell proliferation and migration are key factors in many cardiovascular diseases. Here, we investigated the effects of phloretin on platelet-derived growth factor homodimer (PDGF-BB)-induced rat aortic smooth muscle cell (RASMC) proliferation, migration, and neointimal formation after carotid injury. Phloretin significantly inhibited the PDGF-BB-stimulated RASMC proliferation in a concentration-dependent manner (10-100 μM). Also, PDGF-BB-stimulated RASMC migration was inhibited by phloretin at 50 μM. Pretreating RASMC with phloretin dose-dependently inhibited PDGF-BB-induced Akt and p38 mitogen-activated protein kinases activation. Furthermore, phloretin increased p27 and decreased cyclin-dependent kinase 2, CDK4 expression, and p-Rb activation in PDGF-BB-stimulated RASMC in a concentration-dependent manner (10-50 μM). PDGF-BB-induced cell adhesion molecules and matrix metalloproteinase-9 expression were blocked by phloretin at 50 μM. Preincubation with phloretin dose-dependently reduced the intracellular reactive oxygen species production. In vivo study showed that phloretin (20 mg/kg) significantly reduced neointimal formation 14 days after carotid injury in rats. Thus, phloretin may have potential as a treatment against atherosclerosis and restenosis after vascular injury. PMID:25945863

  12. Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

    SciTech Connect

    Dalal, K.B.; Ebbe, S.; Mazoyer, E.; Carpenter, D.; Yee, T. )

    1990-02-01

    This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700-1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55 +/- 0.011 to 0.89 +/- 0.016 (P less than 0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of (14C)serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of (14C)serotonin in HC and normal platelets ranged from 78-94%. The percent of (14C)serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 microM acetylsalicylic acid into the aggregating medium suppressed the release of (14C)serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.

  13. Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma

    PubMed Central

    Liang, Pei; Cheng, Suk Hang; Cheng, Chi Keung; Lau, Kin Mang; Lin, Shek Ying; Chow, Eudora Y.D.; Chan, Natalie P.H.; Ip, Rosalina K.L.; Wong, Raymond S.M.; Ng, Margaret H. L.

    2013-01-01

    Platelet factor 4 (PF4) is an angiostatic chemokine that suppresses tumor growth and metastasis. We previously revealed frequent transcriptional silencing of PF4 in multiple myeloma, but the functional roles of this chemokine are still unknown. We studied the apoptotic effects of PF4 on myeloma cell lines and primary myeloma in vitro, and investigated the involved signaling pathway. The in vivo effects were also studied using a mouse model. PF4 not only suppressed myeloma-associated angiogenesis, but also inhibited growth and induced apoptosis in myeloma cells. We found that PF4 negatively regulated STAT3 and concordantly inhibited constitutive and interleukin-6-induced phosphorylation of STAT3, and down-regulated the expression of STAT3 target genes (Mcl-1, survivin and VEGF). Overexpression of constitutively activated STAT3 could rescue PF4-induced apoptotic effects. Furthermore, we found that PF4 induced the expression of SOCS3, a STAT3 inhibitor, and gene silencing of SOCS3 abolished its ability to inhibit STAT3 activation, suggesting a critical role of SOCS3 in PF4-induced STAT3 inhibition. Knockdown of LRP1, a putative PF4 receptor, could also abolish PF4-induced apoptosis and STAT3 inhibition. Finally, the tumor growth inhibitory effect of PF4 was confirmed by in vivo mouse models. Immunostaining of rabbit bone xenografts from PF4-treated mice showed induction of apoptosis of myeloma cells and inhibition of angiogenesis, which was associated with suppression of STAT3 activity. Together, our preclinical data indicate that PF4 may be a potential new targeting agent for the treatment of myeloma. PMID:22929979

  14. The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

    PubMed

    Lee, Hae-Ri; Jeon, Jun Ho; Park, Ok-Kyu; Chun, Jeong-Hoon; Park, Jungchan; Rhie, Gi-Eun

    2015-12-01

    The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively.

  15. Soothing the Sting of Rejection.

    ERIC Educational Resources Information Center

    Campbell, Joan Daniels

    1990-01-01

    Preventing rejection of a student by his/her peers and helping the child to cope with such rejection are ever-present challenges for teachers. Suggestions are given by teachers who have successfully dealt with students who were rejected by classmates. (IAH)

  16. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

    PubMed

    Modjeski, Kristina L; Ture, Sara K; Field, David J; Cameron, Scott J; Morrell, Craig N

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  17. Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation

    PubMed Central

    Modjeski, Kristina L.; Ture, Sara K.; Field, David J.; Cameron, Scott J.; Morrell, Craig N.

    2016-01-01

    Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium. PMID:27631377

  18. Monitoring Pharmacologically Induced Immunosuppression by Immune Repertoire Sequencing to Detect Acute Allograft Rejection in Heart Transplant Patients: A Proof-of-Concept Diagnostic Accuracy Study

    PubMed Central

    Valantine, Hannah A.; Penland, Lolita; Luikart, Helen; Strehl, Calvin; Cohen, Garrett; Khush, Kiran K.; Quake, Stephen R.

    2015-01-01

    Background It remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation. Methods and Findings In this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient’s net state of immunosuppression (correlation with tacrolimus level, r = −0.867, 95% CI −0.968 to −0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several

  19. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  20. Reduction of CTRP9, a novel anti-platelet adipokine, contributes to abnormal platelet activity in diabetic animals.

    PubMed

    Wang, Wenqing; Lau, Wayne Bond; Wang, Yajing; Ma, Xinliang; Li, Rong

    2016-01-11

    Platelet hyper-reactivity is a crucial cause of accelerated atherosclerosis increasing risk of thrombotic vascular events in diabetic patients. The mechanisms leading to abnormal platelet activity during diabetes are complex and not fully defined. The current study attempted to clarify the role of CTRP9, a novel adiponectin paralog, in enhanced platelet activity and determined whether CTRP9 may inhibit platelet activity. Adult male C57BL/6 J mice were randomized to receive high-fat diet (HFD) or normal diet (ND). 8 weeks after HFD, animals were sacrificed, and both plasma CTRP9 and platelet aggregation were determined. HFD-fed animals increased weight gain significantly, and became hyperglycemic and hyperinsulinemic 8 weeks post-HFD. Compared to ND animals, HFD animals exhibited significantly decreased plasma CTRP9 concentration and increased platelet response to ADP, evidenced by augmented aggregation amplitude, steeper aggregation slope, larger area under the curve, and shorter lag time (P < 0.01). A significant negative correlation between plasma CTRP9 concentration and platelet aggregation amplitude was observed. More importantly, in vitro pre-treatment with CTRP9 significantly inhibited ADP-stimulated platelet activation in platelet samples from both ND and HFD animals. Taken together, our results suggest reduced plasma CTRP9 concentration during diabetes plays a causative role in platelet hyper-activity, contributing to platelet-induced cardiovascular damage during this pathologic condition. Enhancing CTRP9 production and/or exogenous supplementation of CTRP9 may protect against diabetic cardiovascular injury via inhibition of abnormal platelet activity.

  1. Heat rejection system

    DOEpatents

    Smith, Gregory C.; Tokarz, Richard D.; Parry, Jr., Harvey L.; Braun, Daniel J.

    1980-01-01

    A cooling system for rejecting waste heat consists of a cooling tower incorporating a plurality of coolant tubes provided with cooling fins and each having a plurality of cooling channels therein, means for directing a heat exchange fluid from the power plant through less than the total number of cooling channels to cool the heat exchange fluid under normal ambient temperature conditions, means for directing water through the remaining cooling channels whenever the ambient temperature rises above the temperature at which dry cooling of the heat exchange fluid is sufficient and means for cooling the water.

  2. CLEC-2 expression is maintained on activated platelets and on platelet microparticles.

    PubMed

    Gitz, Eelo; Pollitt, Alice Y; Gitz-Francois, Jerney J; Alshehri, Osama; Mori, Jun; Montague, Samantha; Nash, Gerard B; Douglas, Michael R; Gardiner, Elizabeth E; Andrews, Robert K; Buckley, Christopher D; Harrison, Paul; Watson, Steve P

    2014-10-01

    The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

  3. Rhesus monkey platelets

    SciTech Connect

    Harbury, C.B.

    1986-03-01

    The purpose of this abstract is to describe the adenine nucleotide metabolism of Rhesus monkey platelets. Nucleotides are labelled with /sup 14/C-adenine and extracted with EDTA-ethanol (EE) and perchlorate (P). Total platelet ATP and ADP (TATP, TADP) is measured in the Holmsen Luciferase assay, and expressed in nanomoles/10/sup 8/ platelets. TR=TATP/TADP. Human platelets release 70% of their TADP, with a ratio of released ATP/ADP of 0.7. Rhesus platelets release 82% of their TADP, with a ratio of released ATP/ADP of 0.33. Thus, monkey platelets contain more ADP than human platelets. Thin layer chromatography of EE gives a metabolic ratio of 11 in human platelets and 10.5 in monkey platelets. Perchlorate extracts metabolic and actin bound ADP. The human and monkey platelets ratios were 5, indicating they contain the same proportion of actin. Thus, the extra ADP contained in monkey platelets is located in the secretory granules.

  4. Aspirin decreases platelet uptake on Dacron vascular grafts in baboons

    SciTech Connect

    Mackey, W.C.; Connolly, R.J.; Callow, A.D.; Keough, E.M.; Ramberg-Laskaris, K.; McCullough, J.L.; O'Donnell, T.F. Jr.; Melaragno, A.; Valeri, C.R.; Weiblen, B.

    1984-07-01

    The influence of a single dose of aspirin (5.4-7.4 mg/kg) on platelet uptake on 4-mm Dacron interposition grafts was studied in a baboon model using gamma camera scanning for 111-Indium labeled platelets. In vitro assessment of platelet function after aspirin administration revealed that in the baboon, as in the human, aspirin abolished arachidonic acid-induced platelet aggregation, prolonged the lag time between exposure to collagen and aggregation, and decreased plasma thromboxane B2 levels. Aspirin also prolonged the template bleeding time. Scans for 111-Indium labeled platelets revealed that pretreatment with a single dose of aspirin decreased platelet uptake on 4-mm Dacron carotid interposition grafts. This decrease in platelet uptake was associated with a significant improvement in 2-hour graft patency and with a trend toward improved 2-week patency.

  5. B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.

    PubMed Central

    Westermark, B; Siegbahn, A; Heldin, C H; Claesson-Welsh, L

    1990-01-01

    Porcine aorta endothelial cells are devoid of receptors for platelet-derived growth factor (PDGF). We have transfected such cells with cDNA for the PDGF B-type receptor, both the wild-type receptor and a mutant form of the receptor (K634A), in which the putative nucleotide-binding lysine of the protein-tyrosine domain has been changed to alanine. Immunoprecipitation studies of metabolically labeled cells showed that both types of receptors were synthesized and processed to the mature form of Mr 190,000. In cells expressing the wild-type receptor, PDGF-BB, the natural ligand for the B-type receptor, induced membrane ruffling and reorganization of actin. Such a response has previously been seen in cells expressing the natural PDGF B-type receptor in response to PDGF-BB. No such effect was induced in nontransfected cells or in cells expressing the K634A mutant receptor. PDGF was also shown to be chemotactic for cells expressing the wild-type receptor, whereas no chemotactic response was elicited in control cells or in cells expressing the K634A mutant receptor. Our study thus provides formal evidence that the PDGF B-type receptor mediates a motility response including actin reorganization and chemotaxis. Furthermore, the results establish a role for the receptor-associated protein-tyrosine kinase in the transduction of the chemotactic signal. Images PMID:2153283

  6. UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey

    2013-02-15

    Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.

  7. Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

    SciTech Connect

    Sotoca, Ana M.; Roelofs-Hendriks, Jose; Boeren, Sjef; Kraan, Peter M. van der; Vervoort, Jacques; Zoelen, Everardus J.J. van; Piek, Ester

    2013-10-15

    This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function. - Highlights: • PDGFs (C and D) significantly increased the number of multipotent undifferentiated hMSCs. • Enhanced proliferation did not impair the ability to undergo lineage-specific differentiation. • Proteomic analysis confirmed the overall signatures of the ‘intact’ cells.

  8. Protein Kinase A Regulates 3-Phosphatidylinositide Dynamics during Platelet-derived Growth Factor-induced Membrane Ruffling and Chemotaxis*S⃞

    PubMed Central

    Deming, Paula B.; Campbell, Shirley L.; Baldor, Linda C.; Howe, Alan K.

    2008-01-01

    Spatial regulation of the cAMP-dependent protein kinase (PKA) is required for chemotaxis in fibroblasts; however, the mechanism(s) by which PKA regulates the cell migration machinery remain largely unknown. Here we report that one function of PKA during platelet-derived growth factor (PDGF)-induced chemotaxis was to promote membrane ruffling by regulating phosphatidylinositol 3,4,5-trisphosphate (PIP3) dynamics. Inhibition of PKA activity dramatically altered membrane dynamics and attenuated formation of peripheral membrane ruffles in response to PDGF. PKA inhibition also significantly decreased the number and size of PIP3-rich membrane ruffles in response to uniform stimulation and to gradients of PDGF. This ruffling defect was quantified using a newly developed method, based on computer vision edge-detection algorithms. PKA inhibition caused a marked attenuation in the bulk accumulation of PIP3 following PDGF stimulation, without effects on PI3-kinase (PI3K) activity. The deficits in PIP3 dynamics correlated with a significant inhibition of growth factor-induced membrane recruitment of endogenous Akt and Rac activation in PKA-inhibited cells. Simultaneous inhibition of PKA and Rac had an additive inhibitory effect on growth factor-induced ruffling dynamics. Conversely, the expression of a constitutively active Rac allele was able to rescue the defect in membrane ruffling and restore the localization of a fluorescent PIP3 marker to membrane ruffles in PKA-inhibited cells, even in the absence of PI3K activity. These data demonstrate that, like Rac, PKA contributes to PIP3 and membrane dynamics independently of direct regulation of PI3K activity and suggest that modulation of PIP3/3-phosphatidylinositol (3-PI) lipids represents a major target for PKA in the regulation of PDGF-induced chemotactic events. PMID:18936099

  9. Platelet-derived growth factor and spatiotemporal cues induce development of vascularized bone tissue by adipose-derived stem cells.

    PubMed

    Hutton, Daphne L; Moore, Erika M; Gimble, Jeffrey M; Grayson, Warren L

    2013-09-01

    Vasculature is essential to the functional integration of a tissue-engineered bone graft to enable sufficient nutrient delivery and viability after implantation. Native bone and vasculature develop through intimately coupled, tightly regulated spatiotemporal cell-cell signaling. The complexity of these developmental processes has been a challenge for tissue engineers to recapitulate, resulting in poor codevelopment of both bone and vasculature within a unified graft. To address this, we cultured adipose-derived stromal/stem cells (ASCs), a clinically relevant, single cell source that has been previously investigated for its ability to give rise to vascularized bone grafts, and studied the effects of initial spatial organization of cells, the temporal addition of growth factors, and the presence of exogenous platelet-derived growth factor-BB (PDGF-BB) on the codevelopment of bone and vascular tissue structures. Human ASCs were aggregated into multicellular spheroids via the hanging drop method before encapsulation and subsequent outgrowth in fibrin gels. Cellular aggregation substantially increased vascular network density, interconnectivity, and pericyte coverage compared to monodispersed cultures. To form robust vessel networks, it was essential to culture ASCs in a purely vasculogenic medium for at least 8 days before the addition of osteogenic cues. Physiologically relevant concentrations of exogenous PDGF-BB (20 ng/mL) substantially enhanced both vascular network stability and osteogenic differentiation. Comparisons with the bone morphogenetic protein-2, another pro-osteogenic and proangiogenic growth factor, indicated that this potential to couple the formation of both lineages might be unique to PDGF-BB. Furthermore, the resulting tissue structure demonstrated the close association of mineral deposits with pre-existing vascular structures that have been described for developing tissues. This combination of a single cell source with a potent induction factor

  10. Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels in vivo and platelet aggregation in vitro

    PubMed Central

    Yuvaraju, Priya; Beegam, Sumaya; Ali, Badreldin H

    2015-01-01

    Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events in vivo and in vitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. In vitro, in whole blood samples collected from untreated mice (n = 3–5), betaine (0.01–1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen in vivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01–1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4–6). In conclusion, our data show that betaine protected against coagulation events in vivo and in vitro and decreased LPO in plasma. PMID:25662827

  11. Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels in vivo and platelet aggregation in vitro.

    PubMed

    Nemmar, Abderrahim; Yuvaraju, Priya; Beegam, Sumaya; Ali, Badreldin H

    2015-07-01

    Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events in vivo and in vitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. In vitro, in whole blood samples collected from untreated mice (n = 3-5), betaine (0.01-1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen in vivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01-1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4-6). In conclusion, our data show that betaine protected against coagulation events in vivo and in vitro and decreased LPO in plasma.

  12. Platelet antibody: review of detection methods

    SciTech Connect

    Schwartz, K.A.

    1988-10-01

    The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with /sup 125/I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated. 61 references.

  13. Nanoparticle biointerfacing by platelet membrane cloaking.

    PubMed

    Hu, Che-Ming J; Fang, Ronnie H; Wang, Kuei-Chun; Luk, Brian T; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H; Kroll, Ashley V; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina H; Zhu, Jie; Shi, William; Hofman, Florence M; Chen, Thomas C; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, Liangfang

    2015-10-01

    Development of functional nanoparticles can be encumbered by unanticipated material properties and biological events, which can affect nanoparticle effectiveness in complex, physiologically relevant systems. Despite the advances in bottom-up nanoengineering and surface chemistry, reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the preparation of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens associated with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an experimental rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, respectively, show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.

  14. [Mechanism of cooked blanched garlic leaves against platelet aggregation].

    PubMed

    Wang, Xin-Hua; Di, Yan-Hui

    2014-06-01

    This study was purposed to explore the mechanism of cooked blanched garlic leave juice against platelet aggregation. The juice of blanched garlic leaves was mixed with platelet rich plasma (PRP), the human platelet aggregation, the activation of human platelets induced by adenosine diphosphate (ADP) and collagen were observed; the expression levels of the activated platelets (Fib-R) and P-selectin (CD62P), and the amount of platelet fibrinogen binding were detected by flow cytometry; 10 rabbits were randomly divided into two groups, in addition to the normal diet, they were fed with physiologic saline and cooked blanched garlic leave juice respectively. After 1, 3, 5 , 8 weeks, the maximum ratio of rabbit platelet aggregation induced by ADP and collagen were observed . The results showed that the cooked blanched garlic leave juice could significantly inhibit human platelet aggregation induced by ADP and collagen (P < 0.05), the inhibitory ratio were 87.37% and 86.24% respectively; the juice could not inhibit activated platelets Fib-R and CD62P expression levels (P > 0.05), but was able to inhibit platelet fibrinogen binding capacity (P < 0.05); the rabbit platelet aggregation rate in the group given cooked blanched garlic leave juice was significantly lower than that in control group (P < 0.05). It is concluded that the cooked blanched garlic leave juice can inhibit platelet aggregation in vitro and in vivo, the inhibition of aggregation pathway mainly is blocking the combination of fibrinogen with Fib-R, which finally results in the inhibition of platelet aggregation. Therefore, regular consumption of cooked blanched garlic leaves may prevent cardiovascular thrombotic diseases. PMID:24989289

  15. [Mechanism of cooked blanched garlic leaves against platelet aggregation].

    PubMed

    Wang, Xin-Hua; Di, Yan-Hui

    2014-06-01

    This study was purposed to explore the mechanism of cooked blanched garlic leave juice against platelet aggregation. The juice of blanched garlic leaves was mixed with platelet rich plasma (PRP), the human platelet aggregation, the activation of human platelets induced by adenosine diphosphate (ADP) and collagen were observed; the expression levels of the activated platelets (Fib-R) and P-selectin (CD62P), and the amount of platelet fibrinogen binding were detected by flow cytometry; 10 rabbits were randomly divided into two groups, in addition to the normal diet, they were fed with physiologic saline and cooked blanched garlic leave juice respectively. After 1, 3, 5 , 8 weeks, the maximum ratio of rabbit platelet aggregation induced by ADP and collagen were observed . The results showed that the cooked blanched garlic leave juice could significantly inhibit human platelet aggregation induced by ADP and collagen (P < 0.05), the inhibitory ratio were 87.37% and 86.24% respectively; the juice could not inhibit activated platelets Fib-R and CD62P expression levels (P > 0.05), but was able to inhibit platelet fibrinogen binding capacity (P < 0.05); the rabbit platelet aggregation rate in the group given cooked blanched garlic leave juice was significantly lower than that in control group (P < 0.05). It is concluded that the cooked blanched garlic leave juice can inhibit platelet aggregation in vitro and in vivo, the inhibition of aggregation pathway mainly is blocking the combination of fibrinogen with Fib-R, which finally results in the inhibition of platelet aggregation. Therefore, regular consumption of cooked blanched garlic leaves may prevent cardiovascular thrombotic diseases.

  16. Escaping from Rejection

    PubMed Central

    Lynch, Raymond J.; Platt, Jeffrey L.

    2009-01-01

    Summary Those engaged in clinical transplantation and transplantation immunology have always taken as a central objective the elucidation of means to prevent graft rejection by the recipient immune system. Conceptually, such mechanisms stem from the concept of Paul Ehrlich that all organisms can selectively avoid autotoxicity; i.e. they exhibit horror autotoxicus. Some mechanisms of horror autotoxicus now understood. T lymphocytes and B lymphocytes recognize foreign antigens but not some auto-antigens. Clonal deletion generates lacunae in what is otherwise a virtually limitless potential to recognize antigens. We call this mechanism structural tolerance. Where imperfections in structural tolerance allow self-recognition, the full activation of lymphocytes and generation of effector activity depends on delivery of accessory signals generated by infection and/or injury. The absence of accessory signals prevents or even suppresses immunological responses. We call this dichotomy of responsiveness conditional tolerance. When, despite structural and conditional tolerance, effector activity perturbs autologous cells, metabolism changes in ways that protect against injury. We use the term accommodation to refer to this acquired protection against injury. Structural and conditional tolerance and accommodation overlap in such a way that potentially toxic products can be generated to control microorganisms and neutralize toxins without overly damaging adjacent cells. The central challenge in transplantation, then, should be the orchestration of structural and conditional tolerance and accommodation in such a way that toxic products can still be generated for defense while preserving graft function and survival. Since the earliest days of transplantation, immunobiologists have sought means by which to prevent recognition and rejection of foreign tissue. The goal of these strategies is the retention of recipient immune function while selectively avoiding graft injury. While

  17. Stimulus-response coupling in platelets

    SciTech Connect

    Huang, E.M.

    1986-01-01

    To understand the mechanism of stimulus-response coupling in platelets, the potentiating effect of succinate and lithium on platelet activation was examined. The action of succinate was immediate; preincubation with succinate did not lead to desensitization. Succinate was comparable to ADP in lowering cAMP levels previously elevated by PGl/sub 2/. Since inhibition of cAMP is not a prerequisite for platelet activation, the mechanism of potentiation of succinate remains undefined. Lithium has also been shown to inhibit adenylate cyclase in PGl/sub 2/-pretreated platelets. Lithium, however, can also inhibit inositol phosphate (InsP) phosphatase and lead to an accumulation of InsP. In human platelets, lithium also enhanced the thrombin-induced accumulation of (/sup 3/H)inositol-labelled inositol trisphosphate (InsP/sub 3/), and inositol bisphosphate (InsP/sub 2/). One hour after thrombin addition, all 3 inositol phosphates returned to near basal levels. In the presence of lithium, while labelled InsP/sub 2/ and InsP/sub 3/ returned to their respective basal levels, the InsP level remained elevated, consistent with the known inhibitory effect of lithium on InsP phosphatase. In thrombin-stimulated platelets prelabeled with (/sup 32/P)phosphate, lithium led to a decrease in labelled phosphatidylinositol 4-phosphate (PtdIns4P) as well as an enhanced production of labelled lysophosphatidylinositol, suggesting multiple effects of lithium on platelet phosphoinositide metabolism. These observed effects, however, occurred too slowly to be the mechanism by which lithium potentiated agonist-induced platelet activation. To study the agonist-receptor interaction, the effect of the specific, high affinity thrombin inhibitor, hirudin, on thrombin-induced accumulation of (/sup 3/H)inositol-labelled inositol phosphates was studied.

  18. Role of Siglec-7 in Apoptosis in Human Platelets

    PubMed Central

    Nguyen, Kim Anh; Hamzeh-Cognasse, Hind; Palle, Sabine; Anselme-Bertrand, Isabelle; Arthaud, Charles-Antoine; Chavarin, Patricia; Pozzetto, Bruno; Garraud, Olivier; Cognasse, Fabrice

    2014-01-01

    Background Platelets participate in tissue repair and innate immune responses. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are well-characterized I-type lectins, which control apoptosis. Methodology/Principal Findings We characterized the expression of Siglec-7 in human platelets isolated from healthy volunteers using flow cytometry and confocal microscopy. Siglec-7 is primarily expressed on α granular membranes and colocalized with CD62P. Siglec-7 expression was increased upon platelet activation and correlated closely with CD62P expression. Cross-linking Siglec-7 with its ligand, ganglioside, resulted in platelet apoptosis without any significant effects on activation, aggregation, cell morphology by electron microscopy analysis or secretion. We show that ganglioside triggered four key pathways leading to apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarization; (ii) elevated expression of pro-apoptotic Bax and Bak proteins with reduced expression of anti-apoptotic Bcl-2 protein; (iii) phosphatidylserine exposure and (iv), microparticle formation. Inhibition of NAPDH oxidase, PI3K, or PKC rescued platelets from apoptosis induced by Siglec-7 recruitment, suggesting that the platelet receptors P2Y1 and GPIIbIIIa are essential for ganglioside-induced platelet apoptosis. Conclusions/Significance The present work characterizes the role of Siglec-7 and platelet receptors in regulating apoptosis and death. Because some platelet pathology involves apoptosis (idiopathic thrombocytopenic purpura and possibly storage lesions), Siglec-7 might be a molecular target for therapeutic intervention/prevention. PMID:25230315

  19. Genetic engineering of platelets to neutralize circulating tumor cells.

    PubMed

    Li, Jiahe; Sharkey, Charles C; Wun, Brittany; Liesveld, Jane L; King, Michael R

    2016-04-28

    Mounting experimental evidence demonstrates that platelets support cancer metastasis. Within the circulatory system, platelets guard circulating tumor cells (CTCs) from immune elimination and promote their arrest at the endothelium, supporting CTC extravasation into secondary sites. Neutralization of CTCs in blood circulation can potentially attenuate metastases to distant organs. Therefore, extensive studies have explored the blockade of platelet-CTC interactions as an anti-metastatic strategy. Such an intervention approach, however, may cause bleeding disorders since the platelet-CTC interactions inherently rely on the blood coagulation cascade including platelet activation. On the other hand, platelets have been genetically engineered to correct inherited bleeding disorders in both animal models and human clinical trials. In this study, inspired by the physical association between platelets and CTCs, platelets were genetically modified to express surface-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine known to induce apoptosis specifically in tumor cells. The TRAIL-expressing platelets were demonstrated to kill cancer cells in vitro and significantly reduce metastases in a mouse model of prostate cancer metastasis. Our results suggest that using platelets to produce and deliver cancer-specific therapeutics can provide a Trojan-horse strategy of neutralizing CTCs to attenuate metastasis.

  20. Platelet receptor polymorphisms do not influence Staphylococcus aureus-platelet interactions or infective endocarditis.

    PubMed

    Daga, Shruti; Shepherd, James G; Callaghan, J Garreth S; Hung, Rachel K Y; Dawson, Dana K; Padfield, Gareth J; Hey, Shi Y; Cartwright, Robyn A; Newby, David E; Fitzgerald, J Ross

    2011-03-01

    Cardiac vegetations result from bacterium-platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen Pl(A1/A2) and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus-platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa Pl(A1/A1) genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa Pl(A1/A2) nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus-platelet interactions in vitro or the clinical course of infective endocarditis.

  1. Contrasting role of phospholipase C-{gamma}1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

    SciTech Connect

    Liao Hongjun; Santos, Josue de los; Carpenter, Graham . E-mail: graham.carpenter@vanderbilt.edu

    2006-04-01

    While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-{gamma}1 (PLC-{gamma}1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-{gamma}1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-{gamma}1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-{gamma}1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-{gamma}1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-{gamma}1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-{kappa}B.

  2. Pharmacological inhibition of eicosanoids and platelet-activating factor signaling impairs zymosan-induced release of IL-23 by dendritic cells.

    PubMed

    Rodríguez, Mario; Márquez, Saioa; Montero, Olimpio; Alonso, Sara; Frade, Javier García; Crespo, Mariano Sánchez; Fernández, Nieves

    2016-02-15

    The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid, and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter, thus mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans-activation and the cytokine signature induced by fungal patterns. PMID:26673542

  3. Pharmacological inhibition of eicosanoids and platelet-activating factor signaling impairs zymosan-induced release of IL-23 by dendritic cells.

    PubMed

    Rodríguez, Mario; Márquez, Saioa; Montero, Olimpio; Alonso, Sara; Frade, Javier García; Crespo, Mariano Sánchez; Fernández, Nieves

    2016-02-15

    The engagement of the receptors for fungal patterns induces the expression of cytokines, the release of arachidonic acid, and the production of PGE2 in human dendritic cells (DC), but few data are available about other lipid mediators that may modulate DC function. The combined antagonism of leukotriene (LT) B4, cysteinyl-LT, and platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) inhibited IL23A mRNA expression in response to the fungal surrogate zymosan and to a lower extent TNFA (tumor necrosis factor-α) and CSF2 (granulocyte macrophage colony-stimulating factor) mRNA. The combination of lipid mediators and the lipid extract of zymosan-conditioned medium increased the induction of IL23A by LPS (bacterial lipopolysaccharide), thus suggesting that unlike LPS, zymosan elicits the production of mediators at a concentration enough for optimal response. Zymosan induced the release of LTB4, LTE4, 12-hydroxyeicosatetraenoic acid (12-HETE), and PAF C16:0. DC showed a high expression and detectable Ser663 phosphorylation of 5-lipoxygenase in response to zymosan, and a high expression and activity of LPCAT1/2 (lysophosphatidylcholine acyltransferase 1 and 2), the enzymes that incorporate acetate from acetyl-CoA into choline-containing lysophospholipids to produce PAF. Pharmacological modulation of the arachidonic acid cascade and the PAF receptor inhibited the binding of P-71Thr-ATF2 (activating transcription factor 2) to the IL23A promoter, thus mirroring their effects on the expression of IL23A mRNA and IL-23 protein. These results indicate that LTB4, cysteinyl-LT, and PAF, acting through their cognate G protein-coupled receptors, contribute to the phosphorylation of ATF2 and play a central role in IL23A promoter trans-activation and the cytokine signature induced by fungal patterns.

  4. Agonist-independent desensitization and internalization of the human platelet-activating factor receptor by coumermycin-gyrase B-induced dimerization.

    PubMed

    Perron, Amelie; Chen, Zhang-Guo; Gingras, Denis; Dupre, Denis J; Stankova, Jana; Rola-Pleszczynski, Marek

    2003-07-25

    Platelet-activating factor (PAF) is a phospholipid with potent and diverse physiological actions, particularly as a mediator of inflammation. We have reported previously that mutant G protein-coupled receptors (GPCRs) affect the functional properties of coexpressed wild-type human PAF receptor (hPAFR) (Le Gouill, C., Parent, J. L., Caron, C. A., Gaudreau, R., Volkov, L., Rola-Pleszczynski, M., and Stankova, J. (1999) J. Biol. Chem. 274, 12548-12554). Increasing evidence suggests that dimerization of GPCRs may play an important role in the regulation of their biological activity. Additional data have also suggested that dimerization may be important in the subsequent internalization of the delta-opioid receptor. To investigate the specific role of dimerization in the internalization process of GPCRs, we generated a fusion protein of hPAFR and bacterial DNA gyrase B (GyrB), dimerized through the addition of coumermycin. We found that dimerization potentiates PAF-induced internalization of hPAFR-GyrB in Chinese hamster ovary cells stably expressing c-Myc-hPAFR-GyrB. Coumermycin-driven dimerization was also sufficient to induce an agonist-independent sequestration process in an arrestin- and clathrin-independent manner. Moreover, the protein kinase C inhibitors staurosporine and GF109203X blocked the coumermycin-induced desensitization of hPAFR-GyrB, suggesting the implication of protein kinase C in the molecular mechanism mediating the agonist-independent desensitization of the receptor. Taken together, these findings suggest a novel mechanism of GPCR desensitization and internalization triggered by dimerization. PMID:12756251

  5. What Is Vinculin Needed for in Platelets?

    PubMed Central

    Mitsios, John V.; Prévost, Nicolas; Kasirer-Friede, Ana; Gutierrez, Edgar; Groisman, Alex; Abrams, Charles S.; Wang, Yanfeng; Litvinov, Rustem I.; Zemljic-Harpf, Alice; Ross, Robert S.; Shattil, Sanford J.

    2010-01-01

    Summary Background Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective To determine whether vinculin is required for the functions of platelets and their major integrin, αIIbβ3. Methods The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on αIIbβ3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to αIIbβ3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, both under static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to wild-type littermates in response to carotid artery injury with FeCl3. Conclusion Despite promoting membrane cytoskeleton integrity when mechanical force is applied to αIIbβ3, vinculin is not required for the traditional functions of αIIbβ3 or the platelet actin cytoskeleton. PMID:20670372

  6. Platelets and primary haemostasis.

    PubMed

    Clemetson, Kenneth J

    2012-03-01

    Platelets have a critical role in haemostasis when vessel wall is injured. Platelet receptors are involved in sequence in this process by slowing platelets down via GPIb/von Willebrand factor to bring them into contact with exposed collagen, then activating them via GPVI to release granule contents and express integrins in a matrix protein binding state. More platelets are incorporated into the growing thrombus and a series of events are set off that finishes with the exposed subendothelium protected by a non-thrombogenic platelet surface and tissue repair underway and the blood flow through the vessel maintained. GPIb is also involved in thrombin activation and, together with GPVI, in the formation of COAT platelets. In thrombosis, pathological changes occur that may lead to life-threatening blockage of vessels. Prevention of thrombosis while maintaining haemostasis remains a major goal of medical research.

  7. Anger expression correlates with platelet aggregation.

    PubMed

    Wenneberg, S R; Schneider, R H; Walton, K G; MacLean, C R; Levitsky, D K; Mandarino, J V; Waziri, R; Wallace, R K

    1997-01-01

    Potential relationships between increased platelet aggregability and such psychological characteristics as hostility and anger were investigated as part of a larger intervention study investigating the potential efficacy of stress-reduction treatments. Participants performed 6-minute mental arithmetic tests under time pressure. Blood was sampled during the first minute of the task and whole blood platelet aggregation was measured in an aggregometer, using collagen and ADP. To assess anger and hostility, the authors used Spielberger's State-Trait Anger and Anger Expression scales together with the Cook-Medley Hostility Scale. The authors found positive correlations between collagen-induced platelet aggregation and outwardly expressed anger, as measured by the Anger Expression Scale. The findings suggested that modes of anger expression may be associated with increased platelet aggregation. If confirmed by future studies, this finding could provide a mechanism for the putative connection between anger/hostility and coronary heart disease.

  8. Radioisotope labeled platelets in medical diagnosis

    SciTech Connect

    Pope, C.F.; Sostman, H.D.

    1986-08-01

    The myriad of applications of indium-111 labeled platelets (/sup 111/In-P), both in biomedical research and clinical diagnostic imaging, in recent years is an index of the potential of this technology. Because many diseases involve the vascular system, a nontoxic platelet label suitable for imaging has immense potential for diagnosis. Presently confined to research centers, this technique is currently used in three main diagnostic situations: deep vein thrombosis, cardiac thrombi, and organ (renal) transplantation rejection. Future applications will proliferate when difficulties in achieving rapid labeling are overcome, and the period between study initiation and final diagnosis is diminished. This review emphasizes current clinical applications and the potential role of this technology in diagnostic imaging. 52 references.

  9. Platelet size in man.

    PubMed

    Paulus, J M

    1975-09-01

    The shape and parameters of platelet size distributions were studied in 50 normal persons and 97 patients in order to test the proposed thesis that platelet size heterogeneity results mainly from aging in the circulation. This thesis was contradicted (1) by size distributions of age-homogeneous, newly-born cell populations which were lognormal with increased (instead of decreased) dispersion of volumes and (2) by the macrothrombocytosis found in some populations with normal age distribution. For these reasons, thrombocytopoiesis appeared to play the major role in determining platelet size. A model was built in which the volume variation of platelet territories due to megakaryocyte growth and membrane demarcation at each step of maturation was a random proportion of the previous value of the volume. This model explains the lognormal shape of both newborn and circulating platelet size distributions. It also implies that (1) the mean and standard deviation of platelet logvolumes depend on the rates of volume change of the individual platelet territories (growth rate minus demarcation rate) as well as on megakaryocyte maturation time; (2) platelet hyperdestruction causes an increase in the mean and dispersion of the rates of territory volume change; (3) Mediterranean macrothrombocytosis and some hereditary macrothrombocytotic thrombocytopenias or dysthrombocytopoieses reflect a diminished rate of territory demarcation, and (4) platelet size heterogeneity is caused mainly by the variations in territory growth and demarcation and not by aging in the circulation.

  10. Fusaric acid, a mycotoxin, and its influence on blood coagulation and platelet function.

    PubMed

    Devaraja, Sannaningaiah; Girish, Kesturu S; Santhosh, Martin S; Hemshekhar, Mahadevappa; Nayaka, Siddaiah C; Kemparaju, Kempaiah

    2013-06-01

    The current study intended to explore the effect of fusaric acid on blood coagulation including plasma coagulation and platelet aggregation. Fusaric acid exhibited biphasic effects on citrated human plasma recalcification time. At concentrations below 50 ng, fusaric acid decreased the clotting time of plasma dose-dependently from 130 ± 3s control value to 32 ± 3s; however, above 50 ng, fusaric acid increased the clotting time from 32 ± 3s and reached a maximum of 152 s at 100 ng and remained unaltered thereafter for the increased dose of fusaric acid. Fusaric acid without damaging red blood cells and platelets, inhibited agonists such as collagen, ADP, thrombin, and epinephrine-induced aggregation of both platelet-rich plasma (PRP) and washed platelets preparations of human. Interestingly, fusaric acid showed biphasic effects only in thrombin-induced platelet aggregation of washed platelets, and at lower concentration (below 900 ng) it activated platelet aggregation; however, in increased concentration (above 900 ng) it inhibited the platelet aggregation of washed platelets. In addition, fusaric acid also inhibited the agonist ADP-induced platelet aggregation of washed platelet suspension but did not show biphasic effect. Further, fusaric acid did not induce the platelets to generate reactive oxygen species (ROS) that clearly suggests that the induction of platelet function could be the result of the fusaric acid-mediated receptor interaction but not through the morphological shape change.

  11. Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

    PubMed Central

    De Angelis, Valentina; Koekman, Arnold C.; Weeterings, Cees; Roest, Mark; de Groot, Philip G.; Herczenik, Eszter; Maas, Coen

    2014-01-01

    Background The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function. Objectives Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo. Methods and Results We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa. Conclusions Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function

  12. Platelets enhance neutrophil transendothelial migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflame...

  13. Effects of methaqualone on blood platelet function.

    PubMed

    Mills, D G

    1978-06-01

    To study the mechanism whereby toxic doses of methaqualone cause a bleeding tendency in humans, the effects of methaqualone, diphenhydramine, and the combination of methaqualone plus diphenhydramine on blood platelet function were investigated. Exposure of human platelets in platelet-rich plasma in vitro to final concentrations of methaqualone ranging from 1.1 to 4.5 X 10(-4)) M resulted in nearly complete inhibition of the secondary phase and significant inhibition of the primary phase of adenosine diphosphate (ADP)--induced aggregation. Both the slope and height of collagen-induced aggregation responses were reduced significantly in vitro by the drug. When methaqualone final concentrations of 1.1, 2.3, and 4.5 X 10(-4) M were studied in the presence of diphenhydramine (1.1, 2.3, and 4.5 X 10(-5) M, respectively), the degree of inhibition of ADP-induced aggregation was only slightly greater (not significant) than that observed with methaqualone. The platelets of rabbits injected intravenously with methaqualone, 10 mg/kg, demonstrated a significantly decreased ability to aggregate with ADP and collagen 30 and 60 min after administration of the drug. These results suggest that a drug-induced defect of blood platelet function may play a role in the bleeding associated with methaqualone toxicity.

  14. The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

    SciTech Connect

    Xu, Wei; Guo, Ting; Zhang, Yan; Jiang, Xiaohong; Zhang, Yongxian; Zen, Ke; Yu, Bo; Zhang, Chen-Yu

    2011-05-01

    Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration. Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.

  15. Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance.

    PubMed

    Jacob, Shancy Petsel; Lakshmikanth, Chikkamenahalli Lakshminarayana; Chaithra, Vyala Hanumanthareddy; Kumari, Titus Ruth Shantha; Chen, Chu-Huang; McIntyre, Thomas M; Marathe, Gopal Kedihitlu

    2016-01-01

    Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 μg/mouse) caused death within 15-20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention. PMID:27064683

  16. Lipopolysaccharide Cross-Tolerance Delays Platelet-Activating Factor-Induced Sudden Death in Swiss Albino Mice: Involvement of Cyclooxygenase in Cross-Tolerance

    PubMed Central

    Jacob, Shancy Petsel; Lakshmikanth, Chikkamenahalli Lakshminarayana; Chaithra, Vyala Hanumanthareddy; Kumari, Titus Ruth Shantha; Chen, Chu-Huang; McIntyre, Thomas M.; Marathe, Gopal Kedihitlu

    2016-01-01

    Lipopolysaccharide (LPS) signaling through Toll-like receptor-4 (TLR-4) has been implicated in the pathogenesis of many infectious diseases. Some believe that TLR-mediated pathogenicity is due, in part, to the lipid pro-inflammatory mediator platelet-activating factor (PAF), but this has been questioned. To test the direct contribution of PAF in endotoxemia in murine models, we injected PAF intraperitoneally into Swiss albino mice in the presence and absence of LPS. PAF alone (5 μg/mouse) caused death within 15–20 min, but this could be prevented by pretreating mice with PAF-receptor (PAF-R) antagonists or PAF-acetylhydrolase (PAF-AH). A low dose of LPS (5 mg/kg body wt) did not impair PAF-induced death, whereas higher doses (10 or 20 mg/kg body wt) delayed death, probably via LPS cross-tolerance. Cross-tolerance occurred only when PAF was injected simultaneously with LPS or within 30 min of LPS injection. Tolerance does not appear to be due to an abundant soluble mediator. Histologic examination of lungs and liver and measurement of circulating TNF-α and IL-10 levels suggested that the inflammatory response is not diminished during cross-tolerance. Interestingly, aspirin, a non-specific cyclooxygenase (COX) inhibitor, partially blocked PAF-induced sudden death, whereas NS-398, a specific COX-2 inhibitor, completely protected mice from the lethal effects of PAF. Both COX inhibitors (at 20 mg/kg body wt) independently amplified the cross-tolerance exerted by higher dose of LPS, suggesting that COX-derived eicosanoids may be involved in these events. Thus, PAF does not seem to have a protective role in endotoxemia, but its effects are delayed by LPS in a COX-sensitive way. These findings are likely to shed light on basic aspects of the endotoxin cross-tolerance occurring in many disease conditions and may offer new opportunities for clinical intervention. PMID:27064683

  17. Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

    PubMed Central

    Pescatore, Luciana A.; Bonatto, Diego; Forti, Fábio L.; Sadok, Amine; Kovacic, Hervé; Laurindo, Francisco R. M.

    2012-01-01

    Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration. PMID:22773830

  18. Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.

    PubMed

    Kim, Kyungho; Hahm, Eunsil; Li, Jing; Holbrook, Lisa-Marie; Sasikumar, Parvathy; Stanley, Ronald G; Ushio-Fukai, Masuko; Gibbins, Jonathan M; Cho, Jaehyung

    2013-08-01

    Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice. PMID:23788140

  19. Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T cells and down-regulates cardiac allograft rejection

    SciTech Connect

    Zheng, De-Hua; Dou, Li-Ping; Wei, Yu-Xiang; Du, Guo-Sheng; Zou, Yi-Ping; Song, Ji-Yong; Zhu, Zhi-Dong; Cai, Ming; Qian, Ye-Yong; Shi, Bing-Yi

    2010-05-14

    Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient's autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-{gamma} by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naive T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4{sup +}CD25{sup high}Foxp3{sup +} regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.

  20. Platelet protective efficacy of 3,4,5 trisubstituted isoxazole analogue by inhibiting ROS-mediated apoptosis and platelet aggregation.

    PubMed

    Jagadish, Swamy; Rajeev, Narasimhamurthy; NaveenKumar, Somanathapura K; Sharath Kumar, Kothanahally S; Paul, Manoj; Hegde, Mahesh; Basappa; Sadashiva, Marilinganadoddi P; Girish, Kesturu S; Rangappa, Kanchugarakoppal S

    2016-03-01

    Thrombocytopenia is a major hematological concern in oxidative stress-associated pathologies and chronic clinical disorders, where premature platelet destruction severely affects the normal functioning of thrombosis and hemostasis. In addition, frequent exposure of platelets to chemical entities and therapeutic drugs immensely contributes in the development of thrombocytopenia leading to huge platelet loss, which might be fatal sometimes. Till date, there are only few platelet protective molecules known to combat thrombocytopenia. Hence, small molecule therapeutics are extremely in need to relieve the burden on limited treatment strategies of thrombocytopenia. In this study, we have synthesized a series of novel 3,4,5 trisubstituted isoxazole derivatives, among which compound 4a [4-methoxy-N'-(5-methyl-3-phenylisoxazole-4-carbonyl) benzenesulfonohydrazide] was found to significantly ameliorate the oxidative stress-induced platelet apoptosis by restoring various apoptotic markers such as ROS content, cytosolic Ca(2+) levels, eIF2-α phosphorylation, mitochondrial membrane depolarization, cytochrome c release, caspase activation, PS externalization, and cytotoxicity markers. Additionally, compound 4a dose dependently inhibits collagen-induced platelet aggregation. Hence, compound 4a can be considered as a prospective molecule in the treatment regime of platelet activation and apoptosis and other clinical conditions of thrombocytopenia. Further studies might ensure the use of compound 4a as a supplementary therapeutic agent to treat, thrombosis and CVD-associated complications. Over all, the study reveals a platelet protective efficacy of novel isoxazole derivative 4a with a potential to combat oxidative stress-induced platelet apoptosis.

  1. Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

    PubMed

    Chu, L-X; Zhou, S-X; Yang, F; Qin, Y-Q; Liang, Z-S; Mo, C-G; Wang, X-D; Xie, J; He, L-P

    2016-01-01

    Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway. PMID:26950455

  2. Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries.

    PubMed

    Anselmo, Aaron C; Modery-Pawlowski, Christa Lynn; Menegatti, Stefano; Kumar, Sunny; Vogus, Douglas R; Tian, Lewis L; Chen, Ming; Squires, Todd M; Sen Gupta, Anirban; Mitragotri, Samir

    2014-11-25

    Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery. PMID:25318048

  3. The influence of bromelain on platelet count and platelet activity in vitro.

    PubMed

    Gläser, Doreen; Hilberg, Thomas

    2006-02-01

    Bromelain is a general name for a family of sulfhydryl-containing, proteolytic enzymes from the pineapple plant. The aim of the present study was to investigate the influence of bromelain on platelet count, platelet aggregation and platelet activity in vitro. Blood samples were taken from the antecubital vein of 10 healthy male non-smokers. Platelet count decreased after incubation with 2.5 and 5 mg bromelain/ml from 277 +/- 17 platelets/nl before to 256 +/- 21 and 247 +/- 19 platelets/nl after the treatment. The ADP and TRAP-6 induced platelet aggregation led to a significant decrease after the incubation with 2.5 mg (ADP: 48.6 +/- 25.7%; TRAP-6: 49.6 +/- 28.9%) or 5 mg (ADP: 5.0 +/- 4.6%; TRAP-6: 9.0 +/- 4.9%) bromelain/ml in comparison to control (ADP: 81.4 +/- 5.0%; TRAP-6: 77.4 +/- 10.4%). The percentage of unstimulated CD62P positive platelets which were investigated by flow cytometry was minimally higher after incubation with 5 mg bromelain/ml (0.57 +/- 0.48% PC) in comparison to control (0.22 +/- 0.11% PC), but after TRAP-6 stimulation the incubation with 5 mg bromelain/ml led to a remarkable decrease in comparison to the untreated control (50.4 +/- 20.2 to 0.9 +/- 0.8% PC). The changes of CD62P (TRAP-stimulated) and the results of platelet aggregation after incubation with bromelain in vitro may demonstrate the potential of bromelain as a substance for platelet inhibition. PMID:16308185

  4. Emotional responses to interpersonal rejection

    PubMed Central

    Leary, Mark R.

    2015-01-01

    A great deal of human emotion arises in response to real, anticipated, remembered, or imagined rejection by other people. Because acceptance by other people improved evolutionary fitness, human beings developed biopsychological mechanisms to apprise them of threats to acceptance and belonging, along with emotional systems to deal with threats to acceptance. This article examines seven emotions that often arise when people perceive that their relational value to other people is low or in potential jeopardy, including hurt feelings, jealousy, loneliness, shame, guilt, social anxiety, and embarrassment. Other emotions, such as sadness and anger, may occur during rejection episodes, but are reactions to features of the situation other than low relational value. The article discusses the evolutionary functions of rejection-related emotions, neuroscience evidence regarding the brain regions that mediate reactions to rejection, and behavioral research from social, developmental, and clinical psychology regarding psychological and behavioral concomitants of interpersonal rejection. PMID:26869844

  5. Emotional responses to interpersonal rejection.

    PubMed

    Leary, Mark R

    2015-12-01

    A great deal of human emotion arises in response to real, anticipated, remembered, or imagined rejection by other people. Because acceptance by other people improved evolutionary fitness, human beings developed biopsychological mechanisms to apprise them of threats to acceptance and belonging, along with emotional systems to deal with threats to acceptance. This article examines seven emotions that often arise when people perceive that their relational value to other people is low or in potential jeopardy, including hurt feelings, jealousy, loneliness, shame, guilt, social anxiety, and embarrassment. Other emotions, such as sadness and anger, may occur during rejection episodes, but are reactions to features of the situation other than low relational value. The article discusses the evolutionary functions of rejection-related emotions, neuroscience evidence regarding the brain regions that mediate reactions to rejection, and behavioral research from social, developmental, and clinical psychology regarding psychological and behavioral concomitants of interpersonal rejection.

  6. Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial.

    PubMed

    Spiel, Alexander O; Bartko, Johann; Schwameis, Michael; Firbas, Christa; Siller-Matula, Jolanta; Schuetz, Matthias; Weigl, Manuela; Jilma, Bernd

    2011-04-01

    Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events. PMID:21301783

  7. Nucleation and growth of platelets in hydrogen-ion-implanted silicon

    SciTech Connect

    Nastasi, Michael; Hoechbauer, Tobias; Lee, Jung-Kun; Misra, Amit; Hirth, John P.; Ridgway, Mark; Lafford, Tamzin

    2005-04-11

    H ion implantation into crystalline Si is known to result in the precipitation of planar defects in the form of platelets. Hydrogen-platelet formation is critical to the process that allows controlled cleavage of Si along the plane of the platelets and subsequent transfer and integration of thinly sliced Si with other substrates. Here we show that H-platelet formation is controlled by the depth of the radiation-induced damage and then develop a model that considers the influence of stress to correctly predict platelet orientation and the depth at which platelet nucleation density is a maximum.

  8. Interaction of the protein C activation peptide with platelets.

    PubMed

    Martínez-Cruz, Ruth; Canseco, María Del S Pina; Lopez-Martínez, Jael; Cruz, Pedro A Hernández; Pérez-Campos, Eduardo; Cruz, Margarito Martínez; Alva, Félix Córdoba; Majluf-Cruz, Abraham; Zenteno, Edgar; Ruiz-Argüelles, Alejandro

    2007-01-01

    The peptide NH(2)-DTEDQEDQVDPR-COOH is released during activation of protein C zymogen. We measured the effect of a synthetic peptide with an amino acid sequence similar to that of the natural peptide on platelets from healthy individuals using platelet aggregometry. We found that this synthetic peptide inhibits platelet aggregation induced by thrombin; furthermore, it diminishes mobilization of intraplatelet calcium. Molecular docking showed weak interaction between the synthetic peptide and thrombin. Our findings suggest that this synthetic peptide may interact with a receptor located on the platelet cell membrane.

  9. Amorphous silica nanoparticles aggregate human platelets: potential implications for vascular homeostasis

    PubMed Central

    Corbalan, J Jose; Medina, Carlos; Jacoby, Adam; Malinski, Tadeusz; Radomski, Marek W

    2012-01-01

    Background Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO−]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO−] balance and platelet aggregation. Methods Nanoparticle–platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO− released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy. Results Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO− leading to an unfavorably low [NO]/[ONOO−] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size. Conclusions The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO−] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets. PMID:22334785

  10. Platelet Function Tests

    MedlinePlus

    ... of the clotting process in the body ( in vivo ). A person with normal platelet function test results may still experience excessive bleeding or inappropriate clotting during and after a surgery. Most samples for platelet function testing are only stable for a very short period ...

  11. Gasotransmitters and platelets.

    PubMed

    Truss, Nicola J; Warner, Timothy D

    2011-11-01

    Platelets are essential to prevent blood loss and promote wound healing. Their activation comprises of several complex steps which are regulated by a range of mediators. Over the last few decades there has been intense interest in a group of gaseous mediators known as gasotransmitters; currently comprising nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H(2)S). Here we consider the action of gasotransmitters on platelet activity. NO is a well established platelet inhibitor which mediates its effects predominantly through activation of soluble guanylyl cyclase leading to a decrease in intraplatelet calcium. More recently CO has been identified as a gasotransmitter with inhibitory actions on platelets; CO acts through the same mechanism as NO but is less potent. The in vivo and platelet functions of the most recently identified gasotransmitter, H(2)S, are still the subject of investigations, but they appear generally inhibitory. Whilst there is evidence for the individual action of these mediators, it is also likely that combinations of these mediators are more relevant regulators of platelets. Furthermore, current evidence suggests that these mediators in combination alter the production of each other, and so modify the circulating levels of gasotransmitters. The use of gasotransmitters as therapeutic agents is also being explored for a range of indications. In conclusion, the importance of NO in the regulation of vascular tone and platelet activity has long been understood. Other gasotransmitters are now establishing themselves as mediators of vascular tone, and recent evidence suggests that these other gasotransmitters may also modulate platelet function.

  12. Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations.

    PubMed

    Augustine, Tanya N; van der Spuy, Wendy J; Kaberry, Lindsay L; Shayi, Millicent

    2016-06-01

    Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool. PMID:27329313

  13. The platelet serotonin-release assay.

    PubMed

    Warkentin, Theodore E; Arnold, Donald M; Nazi, Ishac; Kelton, John G

    2015-06-01

    Few laboratory tests are as clinically useful as The platelet serotonin-release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin-induced thrombocytopenia (HIT), a life- and limb-threatening prothrombotic disorder caused by anti-platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin-release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ∼4% of test sera) and potential for false-positive reactions. As only a subset of anti-PF4/heparin antibodies detectable by enzyme-immunoassay (EIA) are additionally platelet-activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false-positive SRA (since a negative EIA in a patient with a "positive" SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin-"independent" platelet activation is a marker of unusually severe HIT, including delayed-onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis. PMID:25775976

  14. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  15. Treatment of osteochondral injuries with platelet gel

    PubMed Central

    Danieli, Marcus Vinicius; da Rosa Pereira, Hamilton; de Sá Carneiro, Carlos Augusto; Felisbino, Sérgio Luiz; Deffune, Elenice

    2014-01-01

    OBJECTIVES: Treatments for injured articular cartilage have not advanced to the point that efficient regeneration is possible. However, there has been an increase in the use of platelet-rich plasma for the treatment of several orthopedic disorders, including chondral injuries. Our hypothesis is that the treatment of chondral injuries with platelet gel results in higher-quality repair tissue after 180 days compared with chondral injuries not treated with gel. METHODS: A controlled experimental laboratory study was performed on 30 male rabbits to evaluate osteochondral injury repair after treatment with or without platelet gel. Osteochondral injuries were surgically induced in both knees of each rabbit at the medial femoral condyle. The left knee injury was filled with the platelet gel, and the right knee was not treated. Microscopic analysis of both knee samples was performed after 180 days using a histological grading scale. RESULTS: The only histological evaluation criterion that was not significantly different between treatments was metachromasia. The group that was treated with platelet gel exhibited superior results in all other criteria (cell morphology, surface regularity, chondral thickness and repair tissue integration) and in the total score. CONCLUSION: The repair tissue was histologically superior after 180 days in the study group treated with platelet gel compared with the group of untreated injuries. PMID:25518022

  16. Effect of BN 52021, a specific antagonist of platelet activating factor (PAF-acether), on calcium movements and phosphatidic acid production induced by PAF-acether in human platelets

    SciTech Connect

    Simon, M.F.; Chap, H.; Braquet, P.; Douste-Blazy, L.

    1987-02-15

    /sup 32/P-labelled human platelets loaded with quin 2 and pretreated with aspirin were stimulated with 1-100 nM platelet activating factor (PAF-acether or 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in a medium containing the ADP-scavenging system creatine phosphate/creatine phosphokinase. Under these conditions, PAF-acether evoked a characteristic fluorescence change allowing to quantify elevations in cytoplasmic free Ca/sup 2 +/ from internal stores (Ca/sup 2 +/ mobilization) or from external medium (Ca/sup 2 +/ influx), as well as an increased production of phosphatidic acid, reflecting phospholipase C activation. These effects, which can be attributed to PAF-acether only and not to released products such as ADP or thromboxane A2, were strongly inhibited in a dose-dependent manner by BN 52021, a specific antagonist of PAF-acether isolated from Ginkgo biloba. As the drug remained inactive against the same effects elicited by thrombin, it is concluded that BN 52021 does not interfere directly with the mechanism of transmembrane signalling involving inositol-phospholipids or (and) some putative receptor-operated channels, but rather acts on the binding of PAF-acether to its presumed membrane receptor.

  17. Dengue virus binding and replication by platelets.

    PubMed

    Simon, Ayo Y; Sutherland, Michael R; Pryzdial, Edward L G

    2015-07-16

    Dengue virus (DENV) infection causes ∼200 million cases of severe flulike illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome in ∼500,000. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. As a probable initiating event, direct purified DENV-platelet binding was followed in the current study by quantitative reverse transcription-polymerase chain reaction and confirmed antigenically. Approximately 800 viruses specifically bound per platelet at 37°C. Fewer sites were observed at 25°C, the blood bank storage temperature (∼350 sites), or 4°C, known to attenuate virus cell entry (∼200 sites). Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and heparan sulfate proteoglycan were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Interestingly, at 37°C and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days. Further time course experiments demonstrated production of viral NS1 protein, which is known to be highly antigenic in patient serum. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. This was shown using a transcription inhibitor and confirmed by freeze-denatured platelets being incapable of replicating the DENV genome. For the first time, these data demonstrate that platelets directly bind DENV saturably and produce infectious virus. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism. These results furthermore draw attention to the possibility that platelets may produce permissive RNA viruses in addition to DENV.

  18. Effects of hesperetin on platelet-derived growth factor-BB-induced pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wei, Li; Deng, Wei; Cheng, Zhihong; Guo, Haipeng; Wang, Shihong; Zhang, Xiao; He, Yiyu; Tang, Qizhu

    2016-01-01

    Hesperetin is a natural flavonoid, which has been reported to exert various biological activities and positive health effects on mammalian cells. The present study aimed to investigate the effects of hesperetin on the proliferation of primary cultured rat pulmonary artery smooth muscle cells (PASMCs), and to elucidate the possible underlying molecular mechanisms. The results of the present study indicated that hesperetin was able to inhibit the proliferation and DNA synthesis of platelet‑derived growth factor‑BB (PDGF‑BB)‑induced PASMCs in a dose‑ and time‑dependent manner, without exerting cell cytotoxicity. In addition, hesperetin blocked the progression of the cell cycle from G0/G1 to S phase, which was correlated with the decreased mRNA expression levels of cyclin D1, cyclin E, cyclin‑dependent kinase (CDK)2 and CDK4, and the increased mRNA expression levels of p27. Furthermore, the anti‑proliferative effects of hesperetin were associated with suppression of the AKT/glycogen synthase kinase (GSK)3β and p38 signaling pathway, but were not associated with the extracellular signal‑regulated kinases 1/2 and c‑Jun N‑terminal kinases signaling pathways. These results suggested that hesperetin may inhibit PDGFa‑BB‑induced PASMC proliferation via the AKT/GSK3β signaling pathway, and that it may possess therapeutic potential for the treatment of pulmonary vascular remodeling diseases.

  19. Private Information and Insurance Rejections

    PubMed Central

    Hendren, Nathaniel

    2013-01-01

    Across a wide set of non-group insurance markets, applicants are rejected based on observable, often high-risk, characteristics. This paper argues that private information, held by the potential applicant pool, explains rejections. I formulate this argument by developing and testing a model in which agents may have private information about their risk. I first derive a new no-trade result that theoretically explains how private information could cause rejections. I then develop a new empirical methodology to test whether this no-trade condition can explain rejections. The methodology uses subjective probability elicitations as noisy measures of agents beliefs. I apply this approach to three non-group markets: long-term care, disability, and life insurance. Consistent with the predictions of the theory, in all three settings I find significant amounts of private information held by those who would be rejected; I find generally more private information for those who would be rejected relative to those who can purchase insurance; and I show it is enough private information to explain a complete absence of trade for those who would be rejected. The results suggest private information prevents the existence of large segments of these three major insurance markets. PMID:24187381

  20. Private Information and Insurance Rejections.

    PubMed

    Hendren, Nathaniel

    2013-09-01

    Across a wide set of non-group insurance markets, applicants are rejected based on observable, often high-risk, characteristics. This paper argues that private information, held by the potential applicant pool, explains rejections. I formulate this argument by developing and testing a model in which agents may have private information about their risk. I first derive a new no-trade result that theoretically explains how private information could cause rejections. I then develop a new empirical methodology to test whether this no-trade condition can explain rejections. The methodology uses subjective probability elicitations as noisy measures of agents beliefs. I apply this approach to three non-group markets: long-term care, disability, and life insurance. Consistent with the predictions of the theory, in all three settings I find significant amounts of private information held by those who would be rejected; I find generally more private information for those who would be rejected relative to those who can purchase insurance; and I show it is enough private information to explain a complete absence of trade for those who would be rejected. The results suggest private information prevents the existence of large segments of these three major insurance markets.

  1. Platelet morphologic changes and fibrinogen receptor localization. Initial responses in ADP-activated human platelets.

    PubMed

    Hensler, M E; Frojmovic, M; Taylor, R G; Hantgan, R R; Lewis, J C

    1992-09-01

    Platelet exposure to agonists results in rapid morphologic changes paralleled by fibrinogen binding and platelet aggregation. The current study used standardized stereology in conjunction with immunogold electron microscopy to correlate the initial morphologic changes with fibrinogen receptor localization on the surfaces of ADP-activated human platelets. A 45% increase in platelet circumference was observed after 3 seconds of activation (P = 0.001). Virtually all of this increase was due to a 13-fold increase in projection membrane, and the projections observed by stereo microscopy at this time were mostly blunt. Both blunt and long projections also accounted for the increase in platelet-platelet contacts at 10 seconds of activation. Immunogold electron microscopy using the monoclonal antibodies P2 and AP-2 against the fibrinogen receptor, glycoprotein IIb/IIIa (GP IIb/IIIa), showed relatively equivalent immunogold densities on projections compared with cell body during 30 seconds of activation. The activation-dependent anti-GP IIb/IIIa monoclonal antibody, 7E3, showed an immunogold density 37% greater on projections compared with cell body (P = 0.0001). Colocalization studies using 7E3 with a polyclonal antifibrinogen antibody showed bound fibrinogen in close proximity to the GP IIb/IIIa localized by 7E3 on projections. These studies support an important role for platelet projections during the earliest stages of fibrinogen binding and ADP-induced aggregation.

  2. Topical Application of a Platelet Activating Factor Receptor Agonist Suppresses Phorbol Ester-Induced Acute and Chronic Inflammation and Has Cancer Chemopreventive Activity in Mouse Skin

    PubMed Central

    Ocana, Jesus A.; DaSilva-Arnold, Sonia C.; Bradish, Joshua R.; Richey, Justin D.; Warren, Simon J.; Rashid, Badri; Travers, Jeffrey B.; Konger, Raymond L.

    2014-01-01

    Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development. PMID:25375862

  3. MicroRNA-145 regulates platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration by targeting CD40

    PubMed Central

    Li, Yumei; Huang, Jiangnan; Jiang, Zhiyuan; Zhong, Yuanli; Xia, Mingjie; Wang, Hui; Jiao, Yang

    2016-01-01

    The objective of this study is to investigate the expression of microRNA (miR)-145 in human aortic vascular smooth muscle cells (VSMCs) and the effect of miR-145 in the biological behavior and expression of CD40 in VSMCs. Cells were treated with either miR-145 or miR-145 inhibitor. Cell proliferation was analyzed by a colony formation assay and a methyl thiazolyl tetrazolium assay. Cell migration and invasion were assessed using a transwell assay, an invasion assay, and a wound healing assay. A luciferase reporter assay was used to detect the interaction between miR-145 and CD40. Expression of α-SMA, calponin, osteopontin (OPN), epiregulin, activator protein-1 (AP-1) and CD40 was measured using real-time RT-PCR for mRNA levels and Western blotting for protein levels. Overexpression of miR-145 significantly inhibited VSMC proliferation, invasion and migration. Furthermore, OPN, epiregulin, AP-1 and CD40 expression at the mRNA and protein levels was down-regulated by overexpression of miR-145. However, α-SMA and calponin expression at the mRNA and protein levels was up-regulated by overexpression of miR-145. In addition, the luciferase reporter assay showed that CD40 may be a direct target gene of miR-145 in VSMC initiation and development. Moreover, these data demonstrate that the up-regulation of CD40 is critical for miR-145-mediated inhibitory effects on platelet-derived growth factor-induced cell proliferation and migration in human VSMCs. In summary, CD40, a direct target of miR-145, reverses the inhibitory effects of miR-145. These results suggest that the specific modulation of miR-145 in human VSMCs may be an attractive approach for the treatment of proliferative vascular diseases. PMID:27186305

  4. Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

    SciTech Connect

    Sanchez, Elda E.; Galan, Jacob A.; Russell, William K.; Soto, Julio G.; Russell, David H.; Perez, John C. . E-mail: kfjcp00@tamuk.edu

    2006-04-01

    Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC{sub 5}s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.

  5. Selective inhibition of the platelet phosphoinositide 3-kinase p110beta as promising new strategy for platelet protection during extracorporeal circulation.

    PubMed

    Straub, Andreas; Wendel, Hans Peter; Dietz, Klaus; Schiebold, Daniela; Peter, Karlheinz; Schoenwaelder, Simone M; Ziemer, Gerhard

    2008-03-01

    Extracorporeal circulation (ECC) is used in cardiac surgery for cardiopulmonary bypass as well as in ventricular assist devices and for extracorporeal membrane oxygenation. Blood contact with the artificial surface and shear stress of ECC activates platelets and leukocytes resulting in a coagulopathy and proinflammatory events. Blockers of the platelet glycoprotein (GP) IIb/IIIa (CD41/CD61) can protect platelet function during ECC, a phenomenon called "platelet anaesthesia", but may be involved in post-ECC bleeding. We hypothesized that the new selective phosphoinositide 3-kinase p110beta inhibitor TGX-221 that inhibits shear-induced platelet activation without prolonging the bleeding time in vivo may also protect platelet function during ECC. Heparinized blood of healthy volunteers (n = 6) was treated in vitro with either the GP IIb/IIIa blocker tirofiban, TGX-221 or as control and circulated in an ECC model. Before and after 30 minutes circulation CD41 expression on the ECC-tubing as measure for platelet-ECC binding and generation of the platelet activation marker beta-thromboglobulin were determined using ELISA. Platelet aggregation and platelet-granulocyte binding were analysed in flow cytometry. After log-transforming the data statistical evaluation was performed using multifactor ANOVA in combination with Tukey's HSD test (global alpha = 5%). Tirofiban and TGX-221 inhibited platelet-ECC interaction, platelet aggregation and platelet-granulocyte binding. Tirofiban also inhibited ECC-induced beta-thromboglobulin release. The observed inhibition of platelet-ECC interaction and platelet activation by tirofiban contributes to explain the mechanism of "platelet anaesthesia". TGX-221 represents a promising alternative to GP IIb/IIIa blockade and should be further investigated for use during ECC in vivo.

  6. Multimodal physiological sensor for motion artefact rejection.

    PubMed

    Goverdovsky, Valentin; Looney, David; Kidmose, Preben; Mandic, Danilo P

    2014-01-01

    This work introduces a novel physiological sensor, which combines electrical and mechanical modalities in a co-located arrangement, to reject motion-induced artefacts. The mechanically sensitive element consists of an electret condenser microphone containing a light diaphragm, allowing it to detect local mechanical displacements and disregard large-scale whole body movements. The electrically sensitive element comprises a highly flexible membrane, conductive on one side and insulating on the other. It covers the sound hole of the microphone, thereby forming an isolated pocket of air between the membrane and the diaphragm. The co-located arrangement of the modalities allows the microphone to sense mechanical disturbances directly through the electrode, thus providing an accurate proxy to artefacts caused by relative motion between the skin and the electrode. This proxy is used to reject such artefacts in the electrical physiological signals, enabling enhanced recording quality in wearable health applications.

  7. Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

    PubMed Central

    Liu, L X; Weller, P F

    1992-01-01

    The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids. Images PMID:1313445

  8. Do Scientists Really Reject God?

    ERIC Educational Resources Information Center

    Scott, Eugenie C.

    1998-01-01

    Suggests that the title of the recent Larson and Witham article in the journal Nature, "Leading Scientists Still Reject God", is premature and without reliable data upon which to base it. (Author/CCM)

  9. Inhibition of PAR-4 and P2Y12 receptor-mediated platelet activation produces distinct hepatic pathologies in experimental xenobiotic-induced cholestatic liver disease.

    PubMed

    Joshi, Nikita; Kopec, Anna K; Ray, Jessica L; Luyendyk, James P

    2016-07-15

    Emerging evidence supports a protective effect of platelets in experimental cholestatic liver injury and cholangiofibrosis. Coagulation-mediated platelet activation has been shown to inhibit experimental chronic cholestatic liver necrosis and biliary fibrosis. This occurs through thrombin-mediated activation of protease activated receptor-4 (PAR-4) in mice. However, it is not known whether other pathways of platelet activation, such as adenosine diphosphate (ADP)-mediated receptor P2Y12 activation is also protective. We tested the hypothesis that inhibition of P2Y12-mediated platelet activation exacerbates hepatic injury and cholangiofibrosis, and examined the impact of P2Y12 inhibition in both the presence and absence of PAR-4. Treatment of wild-type mice with the P2Y12 receptor antagonist clopidogrel increased biliary hyperplasia and cholangiofibrosis in wild-type mice exposed to the xenobiotic alpha-naphthylisothiocyanate (ANIT) for 4 weeks compared to vehicle-treated mice exposed to ANIT. Interestingly, this effect of clopidogrel occurred without a corresponding increase in hepatocellular necrosis. Whereas biliary hyperplasia and cholangiofibrosis were increased in PAR-4(-/-) mice, clopidogrel treatment failed to further increase these pathologies in PAR-4(-/-) mice. The results indicate that inhibition of receptor P2Y12-mediated platelet activation exacerbates bile duct fibrosis in ANIT-exposed mice, independent of hepatocellular necrosis. Moreover, the lack of an added effect of clopidogrel administration on the exaggerated pathology in ANIT-exposed PAR-4(-/-) mice reinforces the prevailing importance of coagulation-mediated platelet activation in limiting this unique liver pathology. PMID:27475285

  10. ENDOTHELIAL CELLS IN ALLOGRAFT REJECTION

    PubMed Central

    Al-Lamki, Rafia S.; Bradley, John R.; Pober, Jordan S.

    2008-01-01

    In organ transplantation, blood borne cells and macromolecules (e.g. antibodies) of the host immune system are brought into direct contact with the endothelial cell (EC) lining of graft vessels. In this location, graft ECs play several roles in allograft rejection, including the initiation of rejection responses by presentation of alloantigen to circulating T cells; the development of inflammation and thrombosis; and as targets of injury and agents of repair. PMID:19034000

  11. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways

    SciTech Connect

    Zhang, Feng; Ni, Chunyan; Kong, Desong; Zhang, Xiaoping; Zhu, Xiaojing; Chen, Li; Lu, Yin; Zheng, Shizhong

    2012-11-15

    Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H{sub 2}O{sub 2}), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H{sub 2}O{sub 2} at 5 μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H{sub 2}O{sub 2}-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H{sub 2}O{sub 2} stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H{sub 2}O{sub 2}-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis. Highlights: ► Ligustrazine inhibits oxidative stress-induced HSC activation.

  12. Onion (Allium cepa L.) peel extract has anti-platelet effects in rat platelets.

    PubMed

    Ro, Ju-Ye; Ryu, Jin-Hyeob; Park, Hwa-Jin; Cho, Hyun-Jeong

    2015-01-01

    The effects of onion peel extract (OPE) in collagen (5 μg/mL)-stimulated washed rat platelet aggregation were investigated. OPE inhibited platelet aggregation via inhibition of aggregation-inducing molecules, intracellular Ca(2+) and thromboxane A2 (TXA2) by blocking cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS) activities in a dose-dependent manner. In addition, OPE elevated the formation of cyclic adenosine monophosphate (cAMP), aggregation-inhibiting molecule, but not cyclic guanosine monophosphate (cGMP). High performance liquid chromatography (HPLC) analysis of OPE revealed that OPE contains quercetin, one of the major flavonoids, which has anti-platelet effect. In conclusion, we suggest that OPE is an effective inhibitor of collagen-stimulated platelet aggregation in vitro. Therefore, it can be a promising and safe strategy for anti-cardiovascular diseases. PMID:25628983

  13. Membrane rejection of nitrogen compounds

    NASA Technical Reports Server (NTRS)

    Lee, S.; Lueptow, R. M.

    2001-01-01

    Rejection characteristics of nitrogen compounds were examined for reverse osmosis, nanofiltration, and low-pressure reverse osmosis membranes. The rejection of nitrogen compounds is explained by integrating experimental results with calculations using the extended Nernst-Planck model coupled with a steric hindrance model. The molecular weight and chemical structure of nitrogen compounds appear to be less important in determining rejection than electrostatic properties. The rejection is greatest when the Donnan potential exceeds 0.05 V or when the ratio of the solute radius to the pore radius is greater than 0.8. The transport of solute in the pore is dominated by diffusion, although convective transport is significant for organic nitrogen compounds. Electromigration contributes negligibly to the overall solute transport in the membrane. Urea, a small organic compound, has lower rejection than ionic compounds such as ammonium, nitrate, and nitrite, indicating the critical role of electrostatic interaction in rejection. This suggests that better treatment efficiency for organic nitrogen compounds can be obtained after ammonification of urea.

  14. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race.

    PubMed

    Edelstein, Leonard C; Simon, Lukas M; Lindsay, Cory R; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E; Chen, Edward S; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A; Bray, Paul F

    2014-11-27

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.

  15. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race

    PubMed Central

    Edelstein, Leonard C.; Simon, Lukas M.; Lindsay, Cory R.; Kong, Xianguo; Teruel-Montoya, Raúl; Tourdot, Benjamin E.; Chen, Edward S.; Ma, Lin; Coughlin, Shaun; Nieman, Marvin; Holinstat, Michael; Shaw, Chad A.

    2014-01-01

    Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. PMID:25293779

  16. Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets.

    PubMed

    Etulain, Julia; Negrotto, Soledad; Carestia, Agostina; Pozner, Roberto Gabriel; Romaniuk, María Albertina; D'Atri, Lina Paola; Klement, Giannoula Lakka; Schattner, Mirta

    2012-01-01

    Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

  17. Platelet-delivered therapeutics.

    PubMed

    Lyde, R; Sabatino, D; Sullivan, S K; Poncz, M

    2015-06-01

    We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

  18. Platelet associated antibodies

    MedlinePlus

    ... of the following: For unknown reasons (idiopathic thrombocytopenic purpura, or ITP ) Side effect of certain drugs such ... 2012:chap 134. Read More Antibody Idiopathic thrombocytopenic purpura (ITP) Platelet count Serum globulin electrophoresis Thrombocytopenia Update ...

  19. Nanotechnology: Platelet mimicry

    NASA Astrophysics Data System (ADS)

    Farokhzad, Omid C.

    2015-10-01

    Cloaking drug-loaded nanoparticles with platelet membranes enhances the drugs' abilities to target desired cells and tissues. This technology might improve treatments for cardiovascular and infectious diseases. See Letter p.118

  20. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

    PubMed Central

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M.

    2010-01-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets. PMID:19965619

  1. p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo.

    PubMed

    Canault, Matthias; Duerschmied, Daniel; Brill, Alexander; Stefanini, Lucia; Schatzberg, Daphne; Cifuni, Stephen M; Bergmeier, Wolfgang; Wagner, Denisa D

    2010-03-01

    Platelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-alpha and GPV. We recently demonstrated that tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37 degrees C or 22 degrees C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

  2. Spontaneous restoration of transplantation tolerance after acute rejection.

    PubMed

    Miller, Michelle L; Daniels, Melvin D; Wang, Tongmin; Chen, Jianjun; Young, James; Xu, Jing; Wang, Ying; Yin, Dengping; Vu, Vinh; Husain, Aliya N; Alegre, Maria-Luisa; Chong, Anita S

    2015-01-01

    Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection.

  3. UV-induced graft copolymerization of monoacrylate-poly(ethylene glycol) onto poly(3-hydroxyoctanoate) to reduce protein adsorption and platelet adhesion.

    PubMed

    Kim, Hyung Woo; Chung, Chung Wook; Rhee, Young Ha

    2005-03-01

    Homogeneous solutions of poly(3-hydroxyoctanoate) (PHO) and the monoacrylate-poly(ethylene glycol) (PEGMA) monomer in chloroform were irradiated with UV light to obtain PEGMA-grafted PHO (PEGMA-g-PHO) copolymers. Variables affecting the degree of grafting (DG), such as the time of UV irradiation and the concentrations of the PEGMA monomer and initiator, were investigated. The PEGMA-g-PHO copolymers were characterized by measuring the water contact angle, molecular weight, thermal transition temperatures and mechanical properties, as well as by nuclear magnetic resonance spectroscopy. The results from all of these measurements indicate that PEGMA groups were present on the PHO polymer. The protein adsorption and platelet adhesion on the PEGMA-g-PHO surfaces were examined using poly(L-lactide) (PLLA) surfaces as the control. The proteins and platelets had a significantly lower tendency to adhere to the PEGMA-g-PHO copolymers than to PLLA. The graft copolymer with a high DG of PEGMA was very effective in reducing the protein adsorption and platelet adhesion and did not activate the platelets. The results obtained in this study suggest that PEGMA-g-PHO copolymers have the potential to be used as blood-contacting devices in a broad range of biomedical applications. PMID:15769515

  4. Autophagy in allografts rejection: A new direction?

    PubMed

    Sun, Hukui; Cheng, Dayan; Ma, Yuanyuan; Wang, Huaiquan; Liang, Ting; Hou, Guihua

    2016-03-18

    Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection is still a major risk for graft survival. Modulating the dosage of immunosuppressive drugs is not a good choice for all patients, new rejection mechanisms discovery are crucial to limit the inflammatory process and preserve the function of the transplant. Autophagy, a fundamental cellular process, can be detected in all subsets of lymphocytes and freshly isolated naive T lymphocytes. It is required for the homeostasis and function of T lymphocytes, which lead to cell survival or cell death depending on the context. T cell receptor (TCR) stimulation and costimulator signals induce strong autophagy, and autophagy deficient T cells leads to rampant apoptosis upon TCR stimulation. Autophagy has been proved to be activated during ischemia-reperfusion (I/R) injury and associated with grafts dysfunction. Furthermore, Autophagy has also emerged as a key mechanism in orchestrating innate and adaptive immune response to self-antigens, which relates with negative selection and Foxp3(+) Treg induction. Although, the role of autophagy in allograft rejection is unknown, current data suggest that autophagy indeed sweeps across both in the graft organs and recipients lymphocytes after transplantation. This review presents the rationale for the hypothesis that targeting the autophagy pathway could be beneficial in promoting graft survival after transplantation. PMID:26876576

  5. Autophagy in allografts rejection: A new direction?

    PubMed

    Sun, Hukui; Cheng, Dayan; Ma, Yuanyuan; Wang, Huaiquan; Liang, Ting; Hou, Guihua

    2016-03-18

    Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection is still a major risk for graft survival. Modulating the dosage of immunosuppressive drugs is not a good choice for all patients, new rejection mechanisms discovery are crucial to limit the inflammatory process and preserve the function of the transplant. Autophagy, a fundamental cellular process, can be detected in all subsets of lymphocytes and freshly isolated naive T lymphocytes. It is required for the homeostasis and function of T lymphocytes, which lead to cell survival or cell death depending on the context. T cell receptor (TCR) stimulation and costimulator signals induce strong autophagy, and autophagy deficient T cells leads to rampant apoptosis upon TCR stimulation. Autophagy has been proved to be activated during ischemia-reperfusion (I/R) injury and associated with grafts dysfunction. Furthermore, Autophagy has also emerged as a key mechanism in orchestrating innate and adaptive immune response to self-antigens, which relates with negative selection and Foxp3(+) Treg induction. Although, the role of autophagy in allograft rejection is unknown, current data suggest that autophagy indeed sweeps across both in the graft organs and recipients lymphocytes after transplantation. This review presents the rationale for the hypothesis that targeting the autophagy pathway could be beneficial in promoting graft survival after transplantation.

  6. HMGB1 binds to activated platelets via the receptor for advanced glycation end products and is present in platelet rich human coronary artery thrombi.

    PubMed

    Ahrens, Ingo; Chen, Yung-Chih; Topcic, Danijal; Bode, Michael; Haenel, David; Hagemeyer, Christoph E; Seeba, Hannah; Duerschmied, Daniel; Bassler, Nicole; Jandeleit-Dahm, Karin A; Sweet, Matthew J; Agrotis, Alex; Bobik, Alex; Peter, Karlheinz

    2015-11-01

    High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.

  7. Equid Herpesvirus Type 1 Activates Platelets

    PubMed Central

    Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

    2015-01-01

    Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in

  8. Equid herpesvirus type 1 activates platelets.

    PubMed

    Stokol, Tracy; Yeo, Wee Ming; Burnett, Deborah; DeAngelis, Nicole; Huang, Teng; Osterrieder, Nikolaus; Catalfamo, James

    2015-01-01

    Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in

  9. Effect of SJAMP on ATP release of platelet.

    PubMed

    Guo, T; Shen, D; Song, S; Wei, W

    1999-01-01

    The aggregation and ATP release of placelet of normal subjects were measured by platelet lumi-aggregometer. It was found that the aggregation curve induced by SJAMP at the concentration of 100 mg/L was a typical second phase aggregation. There existed a certain lag between platelet aggregation and secretion. The secretion actually began slightly after the second phase of aggregation, suggesting that the second phase aggregation induced by SJAMP is not dependent upon the release of contents of dense granule alone. If platelets were incubated with cyclo-oxygenase inhibitor, the second phase aggregation was inhibited and no ATP was released. The results indicated that the aggregation and release reaction induced by SJAMP were dependent upon the generation of prostaglandin endoperoxides and TXA2 in normal subjects. The amount of ATP release was 0.69 +/- 0.22 nmol/10(8) platelets as stimulated with SJAMP (100 mg/L). But the amount of ATP release were 1.60 +/- 0.25 and 1.37 +/- 0.15 nmol/10(8) platelets when platelets were stimulated with ADP (5 mumol/L) and collagen (5 mg/L). The amount of ATP release induced by SJAMP was significantly lower than that of ADP and collagen. These findings indicated that SJAMP was a weaker agonist than ADP in terms of platelets release reaction.

  10. Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation.

    PubMed

    Kuriyama, Shuhko; Kashiwagi, Hitoshi; Yuhki, Koh-ichi; Kojima, Fumiaki; Yamada, Takehiro; Fujino, Takayuki; Hara, Akiyoshi; Takayama, Koji; Maruyama, Takayuki; Yoshida, Akitoshi; Narumiya, Shuh; Ushikubi, Fumitaka

    2010-10-01

    The effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2-/- platelets) and EP4 (EP4-/- platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1-259 and AE1-329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2-/- and EP4-/- platelets. In human platelets, AE1-259 and AE1-329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1-329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1-259. AE1-329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3-208, an EP4 antagonist. In addition, AE1-329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

  11. Ajoene inhibition of platelet aggregation: possible mediation by a hemoprotein.

    PubMed

    Jamaluddin, M P; Krishnan, L K; Thomas, A

    1988-05-31

    Ajoene, an organosulfur compound derived from garlic, was found by spectral measurements, to interact, cooperatively, with a purified hemoprotein implicated, previously, in platelet activation. It modified the binding interactions of the protein with ligands, deemed to be physiologically relevant as effectors. The characteristics of the modifications were found to parallel those of ajoene induced modifications of agonist-induced aggregation kinetics of gel-filtered calf platelets.

  12. Acidic-store depletion is required for human platelet aggregation.

    PubMed

    Amor, Nidhal Ben; Zbidi, Hanene; Bouaziz, Aicha; Jardin, Isaac; Isaac, Jardin; Hernández-Cruz, Juan M; Salido, Ginés M; Rosado, Juan A; Bartegi, Aghleb

    2009-10-01

    Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets. PMID:19587585

  13. Influence of biofouling on pharmaceuticals rejection in NF membrane filtration.

    PubMed

    Botton, Sabrina; Verliefde, Arne R D; Quach, Nhut T; Cornelissen, Emile R

    2012-11-15

    The effects of biomass attachment and growth on the surface characteristics and organic micropollutants rejection performance of nanofiltration membranes were investigated in a pilot installation. Biomass growth was induced by dosing of a readily biodegradable carbon source resulting in the formation of a biofouling in the investigated membrane elements. Surface properties and rejection behaviour of a biofouled and virgin membrane were investigated and compared in terms of surface charge, surface energy and hydrophobicity. The last two were accomplished by performing contact angle measurements on fully hydrated membrane surfaces, in order to mimic the operating conditions of a membrane in contact with water. Compared to a virgin membrane, deposition and growth of biofilm did slightly alter the surface charge, which became more negative, and resulted in a higher hydrophilicity of the membrane surface. In addition, the presence of the negatively charged biofilm induced accumulation of positively charged pharmaceuticals within the biomass layer, which probably also hindered back diffusion. This caused a reduction in rejection efficiency of positively charged solutes but did not alter rejection of neutral and negatively charged pharmaceuticals. Pharmaceuticals rejection was found to positively correlate with the specific free energy of interaction between virgin or biofouled membranes and pharmaceuticals dissolved in the water phase. The rejection values obtained with both virgin and biofouled membranes were compared and found in good agreement with the predictions calculated with a solute transport model earlier developed for high pressure filtration processes. PMID:22960036

  14. Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible.

    PubMed

    Rochat, Sophie; Alberio, Lorenzo

    2015-01-01

    Strong platelet activation results in a redistribution of negatively charged phospholipids from the cytosolic to the outer leaflet of the cellular membrane. Annexin V has a high affinity to negatively charged phospholipids and can be used to identify procoagulant platelets. Formaldehyde fixation can cause factitious Annexin V binding. Our aim was to evaluate a method for fixing platelets avoiding additional Annexin V binding. We induced expression of negatively charged phospholipids on the surface of a fraction of platelets by combined activation with convulxin and thrombin in the presence of Annexin V-fluorescein isothiocyanate and calcium. Aliquots of resting and activated platelets were fixed with a low concentration, calcium-free formaldehyde solution. Both native platelets and fixed platelets were analyzed by flow cytometry immediately and after a 24-h storage at 4°C. We observed that the percentage of Annexin V positive resting platelets ranged from 1.5 to 9.3% for the native samples and from 0.4 to 12.8% for the fixed samples (P=0.706, paired t-test). The amount of Annexin V positive convulxin/thrombin activated platelets varied from 12.9 to 35.4% without fixation and from 15.3 to 36.3% after formalin fixation (P=0.450). After a 24-h storage at 4°C, Annexin V positive platelets significantly increased both in the resting and in the convulxin/thrombin activated samples of native platelets (both P<0.001), while results for formalin fixed platelets did not differ from baseline values (P=0.318 for resting fixed platelets; P=0.673 for activated fixed platelets). We conclude that platelet fixation with a low concentration, calcium-free formaldehyde solution does not alter the proportion of Annexin V positive platelets. This method can be used to investigate properties of procoagulant platelets by multicolor flow-cytometric analysis requiring fixation steps.

  15. Clinical uses of radiolabeled platelets

    SciTech Connect

    Datz, F.L.; Christian, P.E.; Baker, W.J.

    1985-12-01

    Platelets were first successfully radiolabeled in 1953. At that time, investigators were primarily interested in developing a technique to accurately measure platelet life span in both normal and thrombocytopenic patients. Studies using platelets labeled with /sup 51/Cr have shown shortened platelet survival times in a number of diseases including idiopathic thrombocytopenic purpura, coronary artery disease, and diabetes mellitus. More recently, labels such as /sup 111/In have been developed that allow in vivo imaging of platelets. Indium-111 platelets are being used to better understand the pathophysiology of atherosclerosis, thrombophlebitis, pulmonary embolism and clotting disorders, and to improve the clinical diagnosis of these diseases.

  16. Platelet transfusions in platelet consumptive disorders are associated with arterial thrombosis and in-hospital mortality.

    PubMed

    Goel, Ruchika; Ness, Paul M; Takemoto, Clifford M; Krishnamurti, Lakshmanan; King, Karen E; Tobian, Aaron A R

    2015-02-26

    While platelets are primary mediators of hemostasis, there is emerging evidence to show that they may also mediate pathologic thrombogenesis. Little data are available on risks and benefits associated with platelet transfusions in thrombotic thrombocytopenic purpura (TTP), heparin-induced thrombocytopenia (HIT) and immune thrombocytopenic purpura (ITP). This study utilized the Nationwide Inpatient Sample to evaluate the current in-hospital platelet transfusion practices and their association with arterial/venous thrombosis, acute myocardial infarction (AMI), stroke, and in-hospital mortality over 5 years (2007-2011). Age and gender-adjusted odds ratios (adjOR) associated with platelet transfusions were calculated. There were 10 624 hospitalizations with TTP; 6332 with HIT and 79 980 with ITP. Platelet transfusions were reported in 10.1% TTP, 7.1% HIT, and 25.8% ITP admissions. Platelet transfusions in TTP were associated with higher odds of arterial thrombosis (adjOR = 5.8, 95%CI = 1.3-26.6), AMI (adjOR = 2.0, 95%CI = 1.2-3.3) and mortality (adjOR = 2.0,95%CI = 1.3-3.0), but not venous thrombosis. Platelet transfusions in HIT were associated with higher odds of arterial thrombosis (adjOR = 3.4, 95%CI = 1.2-9.5) and mortality (adjOR = 5.2, 95%CI = 2.6-10.5) but not venous thrombosis. Except for AMI, all relationships remained significant after adjusting for clinical severity and acuity. No associations were significant for ITP. Platelet transfusions are associated with higher odds of arterial thrombosis and mortality among TTP and HIT patients.

  17. Platelet thrombopathy in asthmatic patients with elevated immunoglobulin e.

    PubMed

    Maccia, C A; Gallagher, J S; Ataman, G; Glueck, H I; Brooks, S M; Bernstein, I L

    1977-02-01

    Abnormalities of second-wave platelet aggregation were demonstrated in 17 of 33 asthmatic patients in whom drug and diet intake were controlled in the hospital. Mean abnormal responses were significantly greater after epinephrine- (p less than 0.001), adenosine diphosphate-(less than 0.001), collagen- (p = 0.01), and thrombin- (p less than 0.001) induced platelet aggregation in patients with immunologically mediated asthma and serum IgE levels greater than 250 U/ml as compared to patients without immunologic factors and/or normal controls. Mean pollen-specific radioallergosorbent (RAST) binding was also significantly higher in patients with abnormal aggregation as compared to normal platelet responders (p = 0.02). Release of serotonin generally reflected abnormal aggregation patterns in asthmatic patients. Platelet factor 4 release was significantly decreased in the same groups of patients. These results suggest that the allergic state may affect platelet membrane responsiveness to multiple aggregating agents.

  18. Modulating platelet reactivity through control of RGS18 availability.

    PubMed

    Ma, Peisong; Ou, Kristy; Sinnamon, Andrew J; Jiang, Hong; Siderovski, David P; Brass, Lawrence F

    2015-12-10

    Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

  19. Platelets and Infections – Complex Interactions with Bacteria

    PubMed Central

    Hamzeh-Cognasse, Hind; Damien, Pauline; Chabert, Adrien; Pozzetto, Bruno; Cognasse, Fabrice; Garraud, Olivier

    2015-01-01

    Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-like receptors but also integrins conventionally described in the hemostatic response, such as GPIIb–IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet-bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis, or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of neutrophil extracellular traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet–bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the aggregation response

  20. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

    SciTech Connect

    Derian, C.K.; Friedman, P.A.

    1988-04-01

    The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

  1. Investigation of platelet function and platelet disorders using flow cytometry.

    PubMed

    Rubak, Peter; Nissen, Peter H; Kristensen, Steen D; Hvas, Anne-Mette

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  2. Platelets and platelet-like particles mediate intercellular RNA transfer

    PubMed Central

    Risitano, Antonina; Beaulieu, Lea M.; Vitseva, Olga

    2012-01-01

    The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis. PMID:22596260

  3. Platelet size does not correlate with platelet age

    SciTech Connect

    Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

    1983-08-01

    The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with /sup 75/Se-methionine, (2) in vitro labeling with /sup 51/Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of /sup 75/Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using /sup 51/Cr and /sup 14/C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

  4. H-induced platelet and crack formation in hydrogenated epitaxial Si/Si{sub 0.98}B{sub 0.02}/Si structures

    SciTech Connect

    Shao Lin; Lin Yuan; Swadener, J.G.; Lee, J.K.; Jia, Q.X.; Wang, Y.Q.; Nastasi, M.; Thompson, Phillip E.; Theodore, N. David; Alford, T.L.; Mayer, J.W.; Chen Peng; Lau, S.S.

    2006-01-09

    An approach to transfer a high-quality Si layer for the fabrication of silicon-on-insulator wafers has been proposed based on the investigation of platelet and crack formation in hydrogenated epitaxial Si/Si{sub 0.98}B{sub 0.02}/Si structures grown by molecular-beam epitaxy. H-related defect formation during hydrogenation was found to be very sensitive to the thickness of the buried Si{sub 0.98}B{sub 0.02} layer. For hydrogenated Si containing a 130 nm thick Si{sub 0.98}B{sub 0.02} layer, no platelets or cracking were observed in the B-doped region. Upon reducing the thickness of the buried Si{sub 0.98}B{sub 0.02} layer to 3 nm, localized continuous cracking was observed along the interface between the Si and the B-doped layers. In the latter case, the strains at the interface are believed to facilitate the (100)-oriented platelet formation and (100)-oriented crack propagation.

  5. A Zero-Reject System.

    ERIC Educational Resources Information Center

    Rowley, Jo Anne

    The handbook describes the Project FIND Zero-Reject Model for identifying and serving handicapped children in Texas's Gregory-Portland Independent School District. A Flow Chart of the system is provided, and the following components are discussed (sample subtopics in parentheses): needs assessment, staffing patterns (responsibilities of directors,…

  6. Augmented orbiter heat rejection study

    NASA Technical Reports Server (NTRS)

    Hixon, C. W.

    1981-01-01

    Spacecraft radiator concepts are presented that relieve attitude restrictions required by the shuttle orbiter space radiator for baseline and extended capability STS missions. Cost effective heat rejection kits are considered which add additional capability in the form of attached spacelab radiators or a deployable radiator module.

  7. Evidence that plasma fibrinogen and platelet membrane GPIIb-IIIa are involved in the adhesion of platelets to an artificial surface exposed to plasma.

    PubMed

    Nagai, H; Handa, M; Kawai, Y; Watanabe, K; Ikeda, Y

    1993-09-15

    We investigated the molecular mechanism(s) by which platelets adhere to an artificial surface exposed to plasma, using polystyrene microtiter plates pretreated with plasma. Washed platelets labelled with 51Cr were incubated with the plates under static conditions. Prostaglandin E1(PGE1) was added to the platelets to prevent platelet-platelet interactions. Adhesion required the presence of a divalent cation such as Mg++ or Ca++. Polyclonal anti-fibrinogen antibody inhibited adhesion by 70%. Polyclonal antibodies against fibronectin, vitronectin, von Willebrand's Factor, and the Fc portion of human IgG, had no effect on adhesion. Platelets adhered normally to a surface pretreated with plasma from a patient with severe von Willebrand's disease. No platelet adhesion occurred when the surface was pretreated with an afibrinogenemic plasma. Monoclonal antibodies against platelet membrane GPIIb-IIIa, potent inhibitors of ADP-induced fibrinogen binding to platelets, completely inhibited adhesion. Monoclonal antibodies against the GPIb alpha subunit and GPIc(VLA alpha 5) showed no inhibitory effects on adhesion. Platelets from a patient with Glanzmann's thrombasthenia (type I) did not adhere to the surface pretreated with normal plasma. These results suggest that plasma fibrinogen adsorbed onto the surface and that platelet membrane glycoprotein(GP)IIb-IIIa were responsible for adhesion in an activation-independent manner.

  8. Platelets and the immune continuum.

    PubMed

    Semple, John W; Italiano, Joseph E; Freedman, John

    2011-04-01

    Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunologically savvy than we once thought.

  9. The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

    PubMed Central

    Baker, Sandra M.; Loren, Cassandra P.; Haley, Kristina M.; Itakura, Asako; Pang, Jiaqing; Greenberg, Daniel L.; David, Larry L.; Manser, Ed; Chernoff, Jonathan; McCarty, Owen J. T.

    2013-01-01

    Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets. PMID:23784547

  10. Individual dosing of ASA prophylaxis by controlling platelet aggregation.

    PubMed

    Syrbe, G; Redlich, H; Weidlich, B; Ludwig, J; Kopitzsch, S; Göckefitz, A; Herzog, T

    2001-07-01

    Acetylsalicylic acid is widely used in the primary and secondary prevention of cardiovascular diseases. In the current study, we used platelet aggregation ex vivo in platelet-rich plasma induced with arachidonic acid as a routine method for the determination of the individual dose of acetylsalicylic acid necessary to inhibit platelet aggregation in 108 patients with cardiovascular diseases. In 40% of all patients studied, a dose of 30 mg/day was sufficient to block the arachidonic acid-induced platelet aggregation nearly completely. In 50% of all patients, a dose of 100 mg/day was necessary. In 10% of all patients, the dose had to be further increased to 300 mg/day or even to 500 mg/day to inhibit platelet aggregation nearly completely. These results demonstrate that platelet aggregation can be used as a simple routine laboratory method to control acetylsalicylic acid treatment in patients with cardiovascular diseases and to determine individual doses of acetylsalicylic acid for a nearly complete inhibition of platelet aggregation. With a standard dose of 100 mg/day, 10% of the patients were nonresponders. PMID:11441981

  11. In vitro effects of ethanol on the pathways of platelet aggregation

    SciTech Connect

    Rand, M.L.; Kinlough-Rathbone, R.L.; Packham, M.A.; Mustard, J.F.

    1986-03-01

    Ethanol is reported to inhibit platelet aggregation in vivo and in vitro, but the mechanisms of its action on stimulus-response coupling in platelets is unknown. Platelet aggregation to thrombin occurs through at least three pathways: released ADP; thromboxane A/sub 2/ (TXA/sub 2/); and a third pathway(s). Aggregation of rabbit platelets in citrated platelet-rich plasma (PRP) or washed suspensions to ADP (0.5-10 ..mu..M) was not affected by ethanol, at concentrations up to 5 mg/ml (lethal). Primary ADP-induced (5 ..mu..M) aggregation of human platelets in PRP was also unaffected by ethanol, but secondary aggregation and release of /sup 14/C-serotonin, due to TXA/sub 2/ formation, was inhibited by ethanol (2 and 4 mg/ml). Since arachidonate (AA)-induced (25-250 ..mu..M) aggregation and release by washed rabbit platelets was unaltered by ethanol, it may inhibit mobilization of AA from platelet membrane phospholipids. Ethanol (2-4 mg/ml) inhibited rabbit platelet aggregation and release to low concentrations of thrombin (< 10 mU/ml) or collagen, and also inhibited aggregation and release of aspirin-treated (500 ..mu.. M) rabbit platelets (that cannot form TXA/sub 2/) to low concentrations of thrombin (< 10 mU/ml). Thus, ethanol does not inhibit the mobilization of AA, and partially inhibits the third pathway(s) of platelet aggregation.

  12. Platelets and coagulation in infection

    PubMed Central

    Davis, Rachelle P; Miller-Dorey, Sarah; Jenne, Craig N

    2016-01-01

    Disseminated intravascular coagulation (DIC) is a frequent complication in sepsis that is associated with worse outcomes and higher mortality in patients. In addition to the uncontrolled generation of thrombi throughout the patient's vasculature, DIC often consumes large quantities of clotting factors leaving the patient susceptible to hemorrhaging. Owing to these complications, patients often receive anticoagulants to treat the uncontrolled clotting, often with mixed outcomes. This lack of success with the current ar