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Sample records for activated partial thrombin

  1. Relationship between short activated partial thromboplastin times, thrombin generation, procoagulant factors and procoagulant phospholipid activity.

    PubMed

    Mina, Ashraf; Favaloro, Emmanuel J; Koutts, Jerry

    2012-04-01

    Short activated partial thromboplastin times (APTTs) are associated with thrombosis. However, what short APTTs actually represent in terms of possible mechanistic pathways is not well characterized. We have assessed thrombin generation as compared with levels of procoagulant factor (fibrinogen, V, VIII, IX, XI and XII) activities, von Willebrand factor level and activity using collagen binding, as well as procoagulant phospholipid activity, in 113 consecutive samples exhibiting a short APTT compared with an equal number of age-matched and sex-matched samples yielding a normal APTT. We found a significant difference in peak thrombin generation, velocity index and area under the curve between the two groups, and that thrombin generation markers correlated with the APTT, procoagulant phospholipid activity and several procoagulant clotting factors. We conclude that short APTTs represent a procoagulant milieu, as represented by heightened thrombin generation and several other heightened procoagulant activities, which may help explain the association with thrombosis.

  2. The effect of dabigatran on the activated partial thromboplastin time and thrombin time as determined by the Hemoclot thrombin inhibitor assay in patient plasma samples.

    PubMed

    Hapgood, Greg; Butler, Jenny; Malan, Erica; Chunilal, Sanjeev; Tran, Huyen

    2013-08-01

    Dabigatran is an oral direct thrombin inhibitor that does not require routine laboratory monitoring. However, an assessment of its anticoagulant effect in certain clinical settings is desirable. We examined the relationship between dabigatran levels, as determined by the Hemoclot thrombin inhibitor assay (HTI), the thrombin time (TT) and the activated partial thromboplastin time (aPTT) using different reagents. We describe these parameters with the clinical outcomes of patients receiving dabigatran. Seventy-five plasma samples from 47 patients were analysed. The HTI assay was established to measure dabigatran level. aPTTs were performed using TriniCLOT aPTT S reagent (TC) and three additional aPTT reagents. From linear regression lines, we established the aPTT ranges corresponding to the therapeutic drug levels for dabigatran (90-180 ng/ml). The aPTT demonstrated a modest correlation with the dabigatran level (r= 0.80) but the correlation became less reliable at higher dabigatran levels. The therapeutic aPTT ranges for reagents were clinically and statistically different compared with our reference reagent (46-54 s (TC) vs 51-60 s (SP), 54-64 s (SS) and 61-71 s (Actin FS) (p<0.05)). The TT was sensitive to the presence of dabigatran with a level of 60 ng/ml resulting in a TT > 300 s. In conclusion, the aPTT demonstrated a modest correlation with the dabigatran level and was less responsive with supra-therapeutic levels. aPTT reagents differed in their responsiveness, suggesting individual laboratories must determine their own therapeutic range for their aPTT reagent. The TT is too sensitive to quantify dabigatran levels, but a normal TT suggests minimal or no plasma dabigatran.

  3. [Increased activated partial thrombin time: analysis of 250 cases discovered at laboratory].

    PubMed

    Freyburger, G; Janvier, G; De Cacqueray, C; Dief, S; Negrery, H; Drouillard, B

    1991-01-01

    This study evaluates and discusses the potential utility (clinical value) of complementary coagulation tests performed in cases with a prolonged aPTT of no obvious etiology from a total of 85,500 routine coagulation tests carried out in our general hospital. aPTT was measured using Actin F.S.L. (Dade, plant-derived and rabbit phospholipids complex with ellagic acid as activator) and Diagen (Biotrol, rabbit phospholipids with kaolin). Tests for acquired anticoagulants and endogenous pathway factors (XII, XI, IX, VIII) were assayed if the aPTT was prolonged by 7 sec or more. 250 abnormal aPTT of previously unknown etiology were found over a 14 months period. 46% of them were without any obvious cause, and were considered "spontaneous" increases: 2/3 of these spontaneous increases were 7-9 (group A), and 1/3 were 10-19 (group B). The diagnoses found in group A were mostly deficits in the contact system, while group B contained mostly cases with acquired anticoagulants and deficits in the contact system. In group C (increases over 20"), an etiology could be established in all cases, with a predominance of acquired anticoagulants and some deficits in factors VIII and XII.

  4. Relation between dabigatran concentration, as assessed using the direct thrombin inhibitor assay, and activated clotting time/activated partial thromboplastin time in patients with atrial fibrillation.

    PubMed

    Okubo, Kenji; Kuwahara, Taishi; Takagi, Katsumasa; Takigawa, Masateru; Nakajima, Jun; Watari, Yuji; Nakashima, Emiko; Yamao, Kazuya; Fujino, Tadashi; Tsutsui, Hiroyuki; Takahashi, Atsushi

    2015-06-15

    Dabigatran is a direct thrombin inhibitor that has been approved for preventing stroke in patients with atrial fibrillation. In this study, we aimed to assess the associations between the dabigatran concentration (calculated through plasma-diluted thrombin time, as assessed using the Hemoclot assay) and the activated partial thromboplastin time (aPTT) and activated clotting time (ACT). We recruited 137 patients with atrial fibrillation who were receiving a normal dose of dabigatran (300 mg/d) or a reduced dose of dabigatran (220 mg/d, usually administered to patients who were elderly, had moderate renal dysfunction, or who were also receiving verapamil). We then assessed the aPTT, ACT, and Hemoclot results of the patients and calculated the plasma dabigatran concentration. The mean plasma concentration of dabigatran was 127 ± 88 ng/ml, although no significant differences in dabigatran concentration, ACT, or aPTT were observed when we compared the 2 doses of dabigatran (300 or 220 mg/d). The dabigatran concentration was within the therapeutic levels in most patients, although a high value (>300 ng/ml) was observed in several patients, which indicated a high risk of bleeding. The dabigatran concentration was strongly and positively correlated with ACT and aPTT (r = 0.87, p <0.001; and r = 0.76, p <0.001; respectively). Multivariate analysis revealed that verapamil use was independently associated with elevated dabigatran concentrations (p <0.001). Therefore, ACT and aPTT may be useful for bedside assessment of the anticoagulant activity of dabigatran, and verapamil use may be a risk factor for elevated dabigatran concentrations.

  5. A method to measure thrombin activity in a mixture of fibrinogen and thrombin powders

    PubMed Central

    DeAnglis, Ashley P.; Nur, Israel; Gorman, Anne J.; Meidler, Roberto

    2017-01-01

    Thrombin and fibrinogen powders are the active components of advanced surgical hemostasis products including the EVARREST Fibrin Sealant Patch. Measuring the enzymatic activity of thrombin in the presence of fibrinogen is challenging, as hydration of the powders in a neutral aqueous environment will cause the enzyme to rapidly react with the fibrinogen to form a fibrin clot, which in turn binds and entraps the enzyme thus preventing subsequent measurement of thrombin activity. A novel approach has been developed to overcome this challenge. After isolation of the mixture of powders, an alkaline carbonate solution is used to solubilize the proteins, while reversibly inhibiting the activity of thrombin and preventing clot formation. Once the powders have been fully solubilized, thrombin activity can be restored by neutralization in a buffered fibrinogen solution resulting in fibrin clot formulation. The rate of clot formation can be quantified in a coagulometer to determine the thrombin activity of the original powder. Samples coated with powders containing fibrinogen and varying amounts of thrombin were tested using the method described herein. The results demonstrated that the method could consistently measure the activity of (alpha) thrombin in the presence of fibrinogen over a broad range of thrombin activity levels. The test was successfully validated according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines and thus is suitable for use as part of a commercial manufacturing process. A method has been developed that enables thrombin activity to be measured in a mixture of fibrinogen and thrombin powders. PMID:26991860

  6. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer.

    PubMed

    Kemkes-Matthes, Bettina; Fischer, Ronald; Peetz, Dirk

    2011-04-01

    This study evaluates the effect of whole blood storage on common coagulation parameters in order to confirm or revise acceptable storage limits as defined by current guidelines and diverse study reports. Aliquots were taken from the citrated whole blood of inpatients and outpatients (n = 147) within 4 h after blood withdrawal and after extended storage of whole blood for 8 and 24 h at ambient temperature. Aliquots were centrifuged and analyzed for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fbg), antithrombin (AT), thrombin time (TT) and D-dimer. For each parameter, samples from 33-56 patients were investigated covering a wide range of normal and pathological values. Samples from patients receiving heparin were excluded from analyses of APTT and TT. All assays were performed using reagents and an analyzer from Siemens Healthcare Diagnostics Products GmbH. The mean percentage change after 8 and 24-h storage was below 10% for all parameters. Considering the changes in individual samples, all parameters can be reliably tested after 8-h storage, since less than 15% of the samples demonstrated individual changes of above 10%. The acceptable storage time can be extended to 24 h for PT, TT and D-dimer. Clinically relevant changes were detected after 24-h storage for APTT: 41% of the investigated samples demonstrated changes of above 10%. After 24-h storage, changes for Fbg and AT values were more than 15% in five out of 49 and in three out of 45 samples, respectively. This sporadic increase of values is clinically acceptable except for borderline samples.

  7. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

    PubMed Central

    Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

  8. Early detection of thrombin activity in neuroinflammatory disease

    PubMed Central

    Davalos, Dimitrios; Baeten, Kim M; Whitney, Michael A; Mullins, Eric S; Friedman, Beth; Olson, Emilia S; Ryu, Jae Kyu; Smirnoff, Dimitri S; Petersen, Mark A; Bedard, Catherine; Degen, Jay L; Tsien, Roger Y; Akassoglou, Katerina

    2014-01-01

    Although multiple sclerosis (MS) has been associated with the coagulation system, the temporal and spatial regulation of coagulation activity in neuroinflammatory lesions is unknown. Using a novel molecular probe, we characterized the activity pattern of thrombin, the central protease of the coagulation cascade, in experimental autoimmune encephalomyelitis. Thrombin activity preceded onset of neurological signs, increased at disease peak, and correlated with fibrin deposition, microglial activation, demyelination, axonal damage, and clinical severity. Mice with a genetic deficit in prothrombin confirmed the specificity of the thrombin probe. Thrombin activity might be exploited for developing sensitive probes for preclinical detection and monitoring of neuroinflammation and MS progression. PMID:24740641

  9. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro.

    PubMed

    Golderman, Valery; Shavit-Stein, Efrat; Tamarin, Ilia; Rosman, Yossi; Shrot, Shai; Rosenberg, Nurit; Maggio, Nicola; Chapman, Joab; Eisenkraft, Arik

    Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain.

  10. The Organophosphate Paraoxon and Its Antidote Obidoxime Inhibit Thrombin Activity and Affect Coagulation In Vitro

    PubMed Central

    Golderman, Valery; Shavit-Stein, Efrat; Tamarin, Ilia; Rosman, Yossi; Shrot, Shai; Rosenberg, Nurit

    2016-01-01

    Organophosphates (OPs) are potentially able to affect serine proteases by reacting with their active site. The potential effects of OPs on coagulation factors such as thrombin and on coagulation tests have been only partially characterized and potential interactions with OPs antidotes such as oximes and muscarinic blockers have not been addressed. In the current study, we investigated the in vitro interactions between coagulation, thrombin, the OP paraoxon, and its antidotes obidoxime and atropine. The effects of these substances on thrombin activity were measured in a fluorescent substrate and on coagulation by standard tests. Both paraoxon and obidoxime but not atropine significantly inhibited thrombin activity, and prolonged prothrombin time, thrombin time, and partial thromboplastin time. When paraoxon and obidoxime were combined, a significant synergistic effect was found on both thrombin activity and coagulation tests. In conclusion, paraoxon and obidoxime affect thrombin activity and consequently alter the function of the coagulation system. Similar interactions may be clinically relevant for coagulation pathways in the blood and possibly in the brain. PMID:27689805

  11. Toward understanding allosteric activation of thrombin: a conjecture for important roles of unbound Na(+) molecules around thrombin.

    PubMed

    Kurisaki, Ikuo; Takayanagi, Masayoshi; Nagaoka, Masataka

    2015-03-05

    We shed light on important roles of unbound Na(+) molecules in enzymatic activation of thrombin. Molecular mechanism of Na(+)-activation of thrombin has been discussed in the context of allostery. However, the recent challenge to redesign K(+)-activated thrombin revealed that the allosteric interaction is insufficient to explain the mechanism. Under these circumstances, we have examined the roles of unbound Na(+) molecule in maximization of thrombin-substrate association reaction rate. We performed all-atomic molecular dynamics (MD) simulations of thrombin in the presence of three different cations; Li(+), Na(+), and Cs(+). Although these cations are commonly observed in the vicinity of the S1-pocket of thrombin, smaller cations are distributed more densely and extensively than larger ones. This suggests the two observation rules: (i) thrombin surrounded by Na(+) is at an advantage in the initial step of association reaction, namely, the formation of an encounter complex ensemble, and (ii) the presence of Na(+) molecules does not necessarily have an advantage in the final step of association reaction, namely, the formation of the stereospecific complex. In conclusion, we propose a conjecture that unbound Na(+) molecules also affect the maximization of rate constant of thrombin-substrate association reaction through optimally forming an encounter complex ensemble.

  12. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin

  13. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  14. The platelet strip. II. Pharmacomechanical coupling in thrombin-activated human platelets.

    PubMed

    Salganicoff, L; Sevy, R W

    1985-09-01

    A model of contracted, irreversibly aggregated thrombin-activated human platelets relaxes when treated with ethyleneglycol-bis(beta-aminoethylether-N,N'-tetraacetic acid (EGTA) in the presence of Mg2+. Inhibition of the cyclooxygenase or blockade of the thromboxane A2 receptor decreases the tension partially, but EGTA treatment is needed for full relaxation. After a stable relaxation has been achieved (3-4 h), Ca2+ addition in a cumulative manner does not reinduce contraction. Whether in the absence or presence of external Ca2+, the relaxed preparation contracts when stimulated with ADP, epinephrine, thromboxane A2 or its analogues, or thrombin. At supramaximal doses, each of the agonists activates only a partial amount of the total tension capable of being generated. Addition of an agonist of a different class to the partially contracted preparation further increases its force. The contractile responses are reversible on washout, with kinetics dependent on the class of agonist and time of contact with the preparation. The contraction induced by the prolonged simultaneous stimulation with ADP, arachidonate, and thrombin reverts very slowly on washout of the agonists and for all practical purposes reproduces the initial state of irreversible platelet contraction.

  15. Contact system activation and high thrombin generation in hyperthyroidism.

    PubMed

    Kim, Namhee; Gu, Ja-Yoon; Yoo, Hyun Ju; Han, Se Eun; Kim, Young Il; Nam-Goong, Il Seong; Kim, Eun Sook; Kim, Hyun Kyung

    2017-01-30

    Background Hyperthyroidism is associated with increased thrombotic risk. Since contact system activation through formation of neutrophil extracellular traps (NET) has emerged as an important trigger of thrombosis, we hypothesized that the contact system is activated along with active NET formation in hyperthyroidism and that their markers correlate with disease severity. Materials and Methods In 61 patients with hyperthyroidism and 40 normal controls, the levels of coagulation factors (fibrinogen, and factor VII, VIII, IX, XI, and XII), D-dimer, thrombin generation assay (TGA) markers, NET formation markers (histone-DNA complex, double-stranded DNA, neutrophil elastase), and contact system markers (activated factor XII [XIIa], high-molecular-weight kininogen [HMWK], prekallikrein, and bradykinin) were measured. Results Patients with hyperthyroidism showed higher levels of fibrinogen [median (interquartile range), 315 (280-344) versus 262 (223-300), P=0.001], D-dimer [103.8 (64.8-151.5) versus 50.7 (37.4-76.0), P<0.001], peak thrombin [131.9 (102.2-159.4) versus 31.6 (14.8-83.7), P<0.001] and endogenous thrombin potential [649 (538-736) versus 367 (197-1147), P=0.021] in TGA with 1 pM tissue factor, neutrophil elastase [1.10 (0.39-2.18) versus 0.23 (0.20-0.35), P<0.001], factor XIIa [66.9 (52.8-87.0) versus 73.0 (57.1-86.6), P<0.001], HMWK [6.11 (4.95-7.98) versus 3.83 (2.60-5.68), P<0.001], prekallikrein [2.15 (1.00-6.36) versus 1.41 (0.63-2.22), P=0.026], and bradykinin [152.4 (137.6-180.4) versus 118.3 (97.1-137.9), P<0.001] than did normal controls. In age- and sex-adjusted logistic regression analysis, fibrinogen, factor VIII, IX, and XIIa, D-dimer, peak thrombin, neutrophil elastase, HMWK, and bradykinin showed significant odds ratios representing hyperthyroidism's contribution to coagulation and contact system activation. Free T4 was significantly correlated with factors VIII and IX, D-dimer, double-stranded DNA, and bradykinin. Conclusion This study

  16. The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.

    PubMed Central

    Vijayalakshmi, J.; Padmanabhan, K. P.; Mann, K. G.; Tulinsky, A.

    1994-01-01

    The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with

  17. A novel biologic activity of thrombin: stimulation of monocyte chemotactic protein production

    PubMed Central

    1994-01-01

    Thrombin is a serine protease that is released at sites of vascular injury and exerts a variety of biologic effects on different cell types. Thrombin is postulated to play a role in the pathogenesis of a number of diseases including atherosclerosis, since it activates vascular smooth muscle and endothelial cells. Thrombin mediates these effects through a specific receptor that is upregulated in vascular cells in atherosclerosis. Atherosclerosis and glomerulosclerosis are characterized by the presence of monocyte-macrophages in the lesions. Monocyte chemotactic protein (MCP-1) is believed to be an important mediator of monocyte recruitment to the tissue and can be induced in a broad variety of cells including mesangial cells. We studied the effect of thrombin on MCP-1 production and gene expression in well- characterized human mesangial cells, vascular pericytes that play a central role in fibrosis of the glomerular microvascular bed. alpha thrombin stimulates MCP-1 production and gene expression in mesangial cells in a dose- and time-dependent manner. Experiments with diisopropylfluorophosphate thrombin and gamma thrombin demonstrate that this thrombin effect requires both receptor binding as well as catalytic activity, features consistent with the known properties of the recently characterized and cloned thrombin receptor. Moreover, a human thrombin receptor activating peptide (TRAP1-7) also stimulates MCP-1 production. Northern blot analysis demonstrated that mesangial cells express an mRNA transcript that hybridizes with labeled human thrombin receptor cDNA. These data describe a novel biologic activity of thrombin and suggest an additional mechanism by which this coagulation factor may participate in the progression of glomerulosclerosis, and by analogy, atherosclerosis. PMID:8163952

  18. Atherosclerosis proceeds independently of thrombin-induced platelet activation in ApoE-/- mice

    PubMed Central

    Hamilton, J.R.; Cornelissen, I.; Mountford, J.K.; Coughlin, S.R.

    2009-01-01

    Platelet activation has long been postulated to contribute to the development of atherosclerotic plaques, although the mechanism by which this might occur remains unknown. Thrombin is a potent platelet activator and transfusion of thrombin-activated platelets into mice increases plaque formation, suggesting that thrombin-induced platelet activation might contribute to platelet-dependent atherosclerosis. Platelets from protease-activated receptor 4-deficient (Par4-/-) mice fail to respond to thrombin. To determine whether thrombin-activated platelets play a necessary role in a model of atherogenesis, we compared plaque formation and progression in Par4+/+ and Par4-/- mice in the atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) background. Littermate Par4+/+ and Par4-/- mice, all ApoE-/-, were placed on a Western diet (21% fat, 0.15% cholesterol) for 5 or 10 weeks. The percent of aortic lumenal surface covered by plaques in Par4+/+ and Par4-/- mice was not different at either time point (2.2 ± 0.3% vs. 2.5 ± 0.2% and 5.1 ± 0.4% vs. 5.6 ± 0.4% after 5 and 10 weeks, respectively). Further, no differences were detected in the cross-sectional area of plaques measured at the aortic root (1.53 ± 0.17 vs. 1.66 ± 0.16 × 105 μm2 and 12.56 ± 1.23 vs. 13.03 ± 0.55 × 105 μm2 after 5 and10 weeks, respectively). These findings indicate that thrombin-mediated platelet activation is not required for the early development of atherosclerotic plaques in the ApoE-/- mouse model and suggest that, if platelet activation is required for plaque formation under these experimental conditions, platelet activators other than thrombin suffice. PMID:19217621

  19. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

    PubMed

    Citron, Bruce A; Ameenuddin, Syed; Uchida, K; Suo, William Z; SantaCruz, Karen; Festoff, Barry W

    2016-07-15

    Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies.

  20. Interaction of hirudin with thrombin: Identification of a minimal binding domain of hirudin that inhibits clotting activity

    SciTech Connect

    Mao, S.J.T.; Yates, M.T.; Owen, T.J.; Krstenansky, J.L. )

    1988-10-18

    Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, the authors have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace {sup 125}I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. They show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, they found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.

  1. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    PubMed

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

  2. Systems Biology of Coagulation Initiation: Kinetics of Thrombin Generation in Resting and Activated Human Blood

    PubMed Central

    Chatterjee, Manash S.; Denney, William S.; Jing, Huiyan; Diamond, Scott L.

    2010-01-01

    Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (Ti) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces Ti to ∼20 minutes. Since current kinetic models fail to generate thrombin in the absence of added TF, we implemented a Platelet-Plasma ODE model accounting for: the Hockin-Mann protease reaction network, thrombin-dependent display of platelet phosphatidylserine, VIIa function on activated platelets, XIIa and XIa generation and function, competitive thrombin substrates (fluorogenic detector and fibrinogen), and thrombin consumption during fibrin polymerization. The kinetic model consisting of 76 ordinary differential equations (76 species, 57 reactions, 105 kinetic parameters) predicted the clotting of resting and convulxin-activated human blood as well as predicted Ti of human blood under 50 different initial conditions that titrated increasing levels of TF, Xa, Va, XIa, IXa, and VIIa. Experiments with combined anti-XI and anti-XII antibodies prevented thrombin production, demonstrating that a leak of XIIa past saturating amounts of CTI (and not “blood-borne TF” alone) was responsible for in vitro initiation without added TF. Clotting was not blocked by antibodies used individually against TF, VII/VIIa, P-selectin, GPIb, protein disulfide isomerase, cathepsin G, nor blocked by the ribosome inhibitor puromycin, the Clk1 kinase inhibitor Tg003, or inhibited VIIa (VIIai). This is the first model to predict the observed behavior of CTI-treated human blood, either resting or stimulated with platelet activators. CTI-treated human blood will clot in vitro due to the combined activity of XIIa and XIa, a process enhanced by platelet activators and which proceeds in the

  3. Platelet high-density lipoprotein activates transferrin-derived phagocytosis activators, MAPPs, following thrombin digestion.

    PubMed

    Sakamoto, Haruhiko; Wu, Bin; Nagai, Yumiko; Tanaka, Sumiko; Onodera, Masayuki; Ogawa, Takafumi; Ueno, Masaki

    2011-01-01

    Macromolecular activators of phagocytosis from platelets (MAPPs), transferrin-derived phagocytosis activators released from platelets, activate leukocytic phagocytosis via Fcγ receptors. It has been found that MAPPs can be prepared using stored platelets or their lysate. Using this artificial MAPP production system, it has been found that they can be produced from precursors (tetrameric and dimeric transferrins) following reaction with a low-molecular-weight (LMW) activator of MAPPs, which is liberated from a high-molecular-weight activator of MAPP (HMW activator) by reaction with thrombin. In this study, the HMW activator in platelet lysate was characterized by assaying phagocytosis of washed neutrophils. In an ultracentrifugation study of the platelet lysate, HMW activator activity was observed in the fraction corresponding to the density of high-density lipoprotein (HDL). The activity was observed in the apolipoproteins obtained from the HDL fraction. Among the apolipoproteins tested only apolipoprotein CIII showed the activity to produce MAPP in vitro. Affinity chromatography of the apolipoproteins from the HDL fraction of the platelet lysate using an anti-apolipoprotein CIII column revealed that the substance that binds with the antibody showed MAPP-forming activity. In a gel filtration study of thrombin-treated apolipoprotein CIII, a peak of LMW activator activity was observed for fractions with a molecular size smaller than that of apolipoprotein CIII. Finally, MAPP-forming activity of HDL obtained from the plasma was examined. MAPP was formed only when delipidized HDL was used. In conclusion, it is suggested that platelet HDL is the HMW activator and that this activation is achieved via apolipoprotein CIII after thrombin reaction in platelets.

  4. A Cannabinoid Receptor 2 Agonist Prevents Thrombin-Induced Blood-Brain Barrier Damage via the Inhibition of Microglial Activation and Matrix Metalloproteinase Expression in Rats.

    PubMed

    Li, Lin; Tao, Yihao; Tang, Jun; Chen, Qianwei; Yang, Yang; Feng, Zhou; Chen, Yujie; Yang, Liming; Yang, Yunfeng; Zhu, Gang; Feng, Hua; Chen, Zhi

    2015-12-01

    Thrombin mediates the life-threatening cerebral edema and blood-brain barrier (BBB) damage that occurs after intracerebral hemorrhage (ICH). We previously found that the selective cannabinoid receptor 2 (CB2R) agonist JWH-133 reduced brain edema and neurological deficits following germinal matrix hemorrhage (GMH). We explored whether CB2R stimulation ameliorated thrombin-induced brain edema and BBB permeability as well as the possible molecular mechanism involved. A total of 144 Sprague-Dawley (S-D) rats received a thrombin (20 U) injection in the right basal ganglia. JWH-133 (1.5 mg/kg) or SR-144528 (3.0 mg/kg) and vehicle were intraperitoneally (i.p.) injected 1 h after surgery. Brain water content measurement, Evans blue (EB) extravasation, Western blot, and immunofluorescence were used to study the effects of a CB2R agonist 24 h after surgery. The results demonstrated that JWH-133 administration significantly decreased thrombin-induced brain edema and reduced the number of Iba-1-positive microglia. JWH-133 also decreased the number of P44/P42(+)/Iba-1(+) microglia, lowered Evans blue extravasation, and inhibited the elevated matrix metallopeptidase (MMP)-9 and matrix metallopeptidase (MMP)-12 activities. However, a selective CB2R antagonist (SR-144528) reversed these effects. We demonstrated that CB2R stimulation reduced thrombin-induced brain edema and alleviated BBB damage. We also found that matrix metalloproteinase suppression may be partially involved in these processes.

  5. Cleavage at Arg-1689 influences heavy chain cleavages during thrombin-catalyzed activation of factor VIII.

    PubMed

    Newell, Jennifer L; Fay, Philip J

    2009-04-24

    The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

  6. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    PubMed

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  7. Evaluation of Antithrombotic Activity of Thrombin DNA Aptamers by a Murine Thrombosis Model

    PubMed Central

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4–7.1 µmol/kg (14–70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors. PMID:25192011

  8. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation

    PubMed Central

    Stopa, Jack D.; Neuberg, Donna; Puligandla, Maneka; Furie, Bruce; Zwicker, Jeffrey I.

    2017-01-01

    BACKGROUND: Protein disulfide isomerase (PDI) is required for thrombus formation. We previously demonstrated that glycosylated quercetin flavonoids such as isoquercetin inhibit PDI activity and thrombus formation in animal models, but whether extracellular PDI represents a viable anticoagulant target in humans and how its inhibition affects blood coagulation remain unknown. METHODS: We evaluated effects of oral administration of isoquercetin on platelet-dependent thrombin generation in healthy subjects and patients with persistently elevated anti-phospholipid antibodies. RESULTS: Following oral administration of 1,000 mg isoquercetin to healthy adults, the measured peak plasma quercetin concentration (9.2 μM) exceeded its IC50 for inhibition of PDI by isoquercetin in vitro (2.5 ± 0.4 μM). Platelet-dependent thrombin generation decreased by 51% in the healthy volunteers compared with baseline (P = 0.0004) and by 64% in the anti-phospholipid antibody cohort (P = 0.015) following isoquercetin ingestion. To understand how PDI affects thrombin generation, we evaluated substrates of PDI identified using an unbiased mechanistic-based substrate trapping approach. These studies identified platelet factor V as a PDI substrate. Isoquercetin blocked both platelet factor Va and thrombin generation with an IC50 of ~5 μM. Inhibition of PDI by isoquercetin ingestion resulted in a 53% decrease in the generation of platelet factor Va (P = 0.001). Isoquercetin-mediated inhibition was reversed with addition of exogenous factor Va. CONCLUSION: These studies show that oral administration of isoquercetin inhibits PDI activity in plasma and diminishes platelet-dependent thrombin generation predominantly by blocking the generation of platelet factor Va. These pharmacodynamic and mechanistic observations represent an important step in the development of a novel class of antithrombotic agents targeting PDI. TRIAL REGISTRATION: Clinicaltrials.gov (NCT01722669) FUNDING: National Heart

  9. Thrombin activation and liver inflammation in advanced hepatitis C virus infection

    PubMed Central

    González-Reimers, Emilio; Quintero-Platt, Geraldine; Martín-González, Candelaria; Pérez-Hernández, Onán; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-01-01

    Hepatitis C virus (HCV) infection is associated with increased thrombotic risk. Several mechanisms are involved including direct endothelial damage by the HCV virus, with activation of tissue factor, altered fibrinolysis and increased platelet aggregation and activation. In advanced stages, chronic HCV infection may evolve to liver cirrhosis, a condition in which alterations in the portal microcirculation may also ultimately lead to thrombin activation, platelet aggregation, and clot formation. Therefore in advanced HCV liver disease there is an increased prevalence of thrombotic phenomena in portal vein radicles. Increased thrombin formation may activate hepatic stellate cells and promote liver fibrosis. In addition, ischemic changes derived from vascular occlusion by microthrombi favor the so called parenchymal extinction, a process that promotes collapse of hepatocytes and the formation of gross fibrous tracts. These reasons may explain why advanced HCV infection may evolve more rapidly to end-stage liver disease than other forms of cirrhosis. PMID:27182154

  10. Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

    PubMed

    Södergren, Anna L; Svensson Holm, Ann-Charlotte B; Ramström, Sofia; Lindström, Eva G; Grenegård, Magnus; Öllinger, Karin

    2016-01-01

    Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

  11. Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

    PubMed

    Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

    1990-04-01

    The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

  12. Urokinase has direct catalytic activity against fibrinogen and renders it less clottable by thrombin.

    PubMed Central

    Weitz, J I; Leslie, B

    1990-01-01

    Recently, we demonstrated that tissue plasminogen activator directly releases fibrinopeptides A and B (FPA and FPB) from fibrinogen. The purpose of this study was to determine whether urokinase has similar activity. Incubation of urokinase with fibrinogen or heparinized plasma results in concentration-dependent FPB release unaccompanied by FPA cleavage. For equivalent amidolytic activity, high molecular weight urokinase releases twofold more FPB than the low molecular weight species. In contrast, prourokinase does not release FPB until activated to urokinase. Contaminating thrombin or plasma is not responsible for urokinase-mediated FPB release because this activity is unaccompanied by FPA or B beta 1-42 cleavage, and is unaffected by heparin, hirudin, a monospecific antibody against thrombin, aprotinin, or alpha 2-antiplasmin. FPB release reflects a direct action of urokinase on fibrinogen because release is completely inhibited by a monospecific antibody against the enzyme. Further, urokinase releases FPB from the FPB-containing substrate B beta 1-42, thus confirming its specificity for the B beta 14 (Arg)-B beta 15 (Gly) bond. In addition to FPB release, SDS-PAGE analysis of the time course of urokinase-mediated fibrinogenolysis indicates progressive proteolysis of both the A alpha- and B beta-chains of fibrinogen that occurs after FPB release is completed. As a consequence of urokinase-mediated fibrinogenolysis, there is progressive prolongation of the thrombin clotting time. These studies indicate that urokinase has direct catalytic activity against fibrinogen. By releasing FPB, a potent chemoattractant, and by rendering fibrinogen less clottable by thrombin, urokinase may participate in processes extending beyond fibrinolysis. Images PMID:2365816

  13. Effect of resveratrol, a natural polyphenolic compound, on platelet activation induced by endotoxin or thrombin.

    PubMed

    Olas, Beata; Wachowicz, Barbara; Saluk-Juszczak, Joanna; Zieliński, Tomasz

    2002-08-15

    Resveratrol (3, 4', 5-trihydroxystilbene), a natural polyphenol, is found in some plants that are used in human nutrition. Grapes are a major source for resveratrol, and a significant amount can also be found in red wine. Several experimental studies have demonstrated biological properties of resveratrol, especially its anti-inflammatory, antioxidant, anti-platelet and antitumor effects. In the present study, we investigated the first step of platelet activation-platelet adhesion stimulated by lipopolysaccharide (LPS) from Proteus mirabilis (weak stimulator) and thrombin (strong activator) in the presence of resveratrol. Our studies show that endotoxin (0.3 microg/10(8) platelets), like thrombin (0.2 U/10(8) platelets), induced the adhesion of platelets (expressed as absorbance of cell attached proteins) to collagen and fibrinogen. Preincubation of washed platelets with resveratrol at physiological plasma concentrations (25-100 microg/ml, 30 min, 37 degrees C) had an inhibitory effect on adhesion of platelets to collagen after activation by LPS alone or LPS with thrombin. The strongest effect on this process was caused by resveratrol at the concentration of 100 microg/ml. Pretreatment of platelets with resveratrol (25-100 microg/ml, 30 min, 37 degrees C) had also inhibitory effects on adhesion of platelets to fibrinogen after stimulation of these cells by LPS alone or by LPS with thrombin at the same concentration. In conclusion, we suggest that resveratrol present in human diet may be an important compound responsible for the reduction of platelet adhesion and changed reactivity of blood platelets in inflammatory process.

  14. Activated partial thromboplastin time.

    PubMed

    Ignjatovic, Vera

    2013-01-01

    Activated partial thromboplastin time (APTT) is a commonly used coagulation assay that is easy to perform, is affordable, and is therefore performed in most coagulation laboratories, both clinical and research, worldwide. The APTT is based on the principle that in citrated plasma, the addition of a platelet substitute, factor XII activator, and CaCl2 allows for formation of a stable clot. The time required for the formation of a stable clot is recorded in seconds and represents the actual APTT result.

  15. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    SciTech Connect

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P. )

    1989-09-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO{sub 4}/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated {sup 125}I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

  16. Synthesis, characterization and in vitro activity of thrombin-binding DNA aptamers with triazole internucleotide linkages.

    PubMed

    Varizhuk, Anna M; Tsvetkov, Vladimir B; Tatarinova, Olga N; Kaluzhny, Dmitry N; Florentiev, Vladimir L; Timofeev, Edward N; Shchyolkina, Anna K; Borisova, Olga F; Smirnov, Igor P; Grokhovsky, Sergei L; Aseychev, Anton V; Pozmogova, Galina E

    2013-09-01

    A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.

  17. Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.

    PubMed

    Nielsen, Vance G; Boyer, Leslie V; Redford, Daniel T; Ford, Paul

    2016-06-16

    Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

  18. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

    NASA Astrophysics Data System (ADS)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  19. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  20. Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential.

    PubMed

    Wirsching, Frank; Luge, Cornelia; Schwienhorst, Andreas

    2002-03-01

    In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis.

  1. Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.

    PubMed

    Morgan, Lloyd T; Thomas, Christopher P; Kühn, Hartmut; O'Donnell, Valerie B

    2010-10-01

    Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets.

  2. Rational Design of Potent, Small, Synthetic Allosteric Inhibitors of Thrombin

    PubMed Central

    Sidhu, Preetpal Singh; Liang, Aiye; Mehta, Akul Y.; Abdel Aziz, May H.; Zhou, Qibing; Desai, Umesh R.

    2011-01-01

    Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of potent, small allosteric inhibitors of thrombin. A sulfated benzofuran library of 15 sulfated monomers and 13 sulfated dimers with different charged, polar and hydrophobic substituents was studied in this work. Synthesis of the sulfated benzofurans was achieved through a multiple step, highly branched strategy, which culminated with microwave-assisted chemical sulfation. Of the 28 potential inhibitors, eleven exhibited reasonable inhibition of human α-thrombin at pH 7.4. Structure activity relationship analysis indicated that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. A t-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC50 of 7.3 μM under physiologically relevant conditions. Michaelis-Menten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of allosteric inhibitors of thrombin. PMID:21714536

  3. Thrombin generation testing for monitoring hemophilia treatment: a clinical perspective.

    PubMed

    Salvagno, Gian Luca; Berntorp, Erik

    2010-10-01

    Thrombin generation is a key process that determines the extent of a hemostatic plug or a thrombotic process. The ensuing thrombin burst is crucial for the formation of a stable fibrin clot. During its active life, thrombin exerts a multitude of highly regulated actions on the blood and the vessel wall, among which is the clotting of fibrinogen. The inappropriate generation of thrombin may lead to pathological processes, foremost of which are hemorrhagic or thrombotic diseases. The coagulation system is usually investigated by means of two in vitro classical clotting tests, the activated partial thromboplastin time and prothrombin time. These assays assess only the time taken to form a clot and do not entirely reflect global hemostatic balance. They permit identification of connectivity between the component activities identified as required for plasma coagulation and define the concept of intrinsic and extrinsic coagulation pathways, which converge at the step of formation of the prothrombinase complex. However, the mechanisms established by in vitro tests are not always mirrored in the human pathologies associated with bleeding or thrombosis. The recent development of newer tests based on the continuous registration of thrombin generation (TG) under in vitro conditions that mimic more closely what occurs in vivo prompted us to reinvestigate the balance between procoagulants and anticoagulants in patients. Thrombin generation assays (TGA) not only provide an overall assessment of hemostasis, but they also target potential extrahemostatic effects of the generated thrombin, a potent agonist of a multitude of cellular activation pathways. Moreover, estimation of an individual's thrombin generation potential may correlate more closely with a hyper- or hypocoagulable phenotype, compared with traditional coagulation tests. In this review, we discuss to what extent TG can be expected to reflect the clotting function of blood, the development and use of different TGA

  4. Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

    PubMed Central

    Switzer, Mary Ellen; McKee, Patrick A.

    1977-01-01

    When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl2, the protein and vWF activity appear in the void volume, but most of the FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl2. To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl2 on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl2. When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl2, the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl2, the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl2. When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIII/vWF protein was filtered in 0.25 M CaCl2, the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h. The procoagulant peak isolated by filtering nonthrombin-activated FVIII/vWF protein on agarose in 0.25 M CaCl2 was compared to that isolated from thrombin-activated FVIII/vWF protein. Both procoagulant activity peak proteins had about the same specific vWF activity as the corresponding void volume protein. Before reduction, the sodium dodecyl sulfate gel patterns for the two procoagulant activity peak proteins were the same. After reduction, the gel pattern for the nonthrombin-activated procoagulant

  5. Anticoagulant Activity of a Unique Sulfated Pyranosic (1→3)-β-l-Arabinan through Direct Interaction with Thrombin*

    PubMed Central

    Fernández, Paula V.; Quintana, Irene; Cerezo, Alberto S.; Caramelo, Julio J.; Pol-Fachin, Laercio; Verli, Hugo; Estevez, José M.; Ciancia, Marina

    2013-01-01

    A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans. PMID:23161548

  6. Anticoagulant activity of a unique sulfated pyranosic (1->3)-β-L-arabinan through direct interaction with thrombin.

    PubMed

    Fernández, Paula V; Quintana, Irene; Cerezo, Alberto S; Caramelo, Julio J; Pol-Fachin, Laercio; Verli, Hugo; Estevez, José M; Ciancia, Marina

    2013-01-04

    A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans.

  7. Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance.

    PubMed

    Crowley, Maeve P; Kevane, Barry; O'Shea, Susan I; Quinn, Shane; Egan, Karl; Gilligan, Oonagh M; Ní Áinle, Fionnuala

    2016-09-01

    The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P < .05). In the presence of APC, endogenous thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies.

  8. Proteomic analysis of the porcine platelet proteome and alterations induced by thrombin activation.

    PubMed

    Esteso, Gloria; Mora, María Isabel; Garrido, Juan José; Corrales, Fernando; Moreno, Angela

    2008-12-02

    Platelets are enucleated cells derived from bone marrow megakaryocytes and defects in platelet functions could be involved in many cardiovascular diseases. Proteomics can be used to provide a new insight in the study of these platelet functions and can help to identify the biochemical events underlying platelet activation. In this study, we have obtained a reference 2-DE map of porcine platelet proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Other proteins implicated in the cell signalling process, transport or apoptosis were also identified. Moreover, we have analysed, by 2D-DIGE methodology, quantitative modifications of platelet proteins following their activation by thrombin. 26 spots exhibited statistically significant differences, and a total of 16 spots corresponding to 13 different proteins were successfully identified. Using Ingenuity Pathway Analysis, the association of the deregulated proteins with canonical pathways highlighted two major pathways; coagulation system and integrin signalling. These results confirm that this proteomic approach (based on 2D-DIGE, mass spectrometry and bioinformatic and pathway databases) has proved to be a powerful tool when applied to studying signalling pathways that could play a relevant role in the activation of platelets.

  9. Imaging the elastic modulus of human platelets during thrombin-induced activation using scanning ion conductance microscopy.

    PubMed

    Rheinlaender, Johannes; Vogel, Sebastian; Seifert, Jan; Schächtele, Marc; Borst, Oliver; Lang, Florian; Gawaz, Meinrad; Schäffer, Tilman E

    2015-02-01

    Platelet activation plays a critical role in haemostasis and thrombosis. It is well-known that platelets generate contractile forces during activation. However, their mechanical material properties have rarely been investigated. Here, we use scanning ion conductance microscopy (SICM) to visualise morphological and mechanical properties of live human platelets at high spatial resolution. We found that their mean elastic modulus decreases during thrombin-induced activation by about a factor of two. We observed a similar softening of platelets during cytochalasin D-induced cytoskeleton depolymerisation. However, thrombin-induced temporal and spatial modulations of the elastic modulus were substantially different from cytochalasin D-mediated changes. We thereby provide new insights into the mechanics of haemostasis and establish SICM as a novel imaging platform for the ex vivo investigation of the mechanical properties of live platelets.

  10. Role of thrombin-PAR1-PKCθ/δ axis in brain pericytes in thrombin-induced MMP-9 production and blood-brain barrier dysfunction in vitro.

    PubMed

    Machida, Takashi; Dohgu, Shinya; Takata, Fuyuko; Matsumoto, Junichi; Kimura, Ikuya; Koga, Mariko; Nakamoto, Keiko; Yamauchi, Atsushi; Kataoka, Yasufumi

    2017-03-24

    Thrombin, an essential component in the coagulation cascade, participates in the pathogenesis of brain diseases, such as ischemic stroke, intracerebral hemorrhage, Alzheimer's disease and Parkinson's disease through blood-brain barrier (BBB) dysfunction. It is thought that the thrombin-matrix metalloproteinase (MMP)-9 axis is an important process in the pathogenesis of neurovascular disease, such as BBB dysfunction. We recently reported that brain pericytes are the most MMP-9-releasing cells in response to thrombin stimulation among the BBB-constituting cells. This thrombin-induced MMP-9 release is partially due to protease-activated receptor (PAR1), one of the specific thrombin receptors. Then, we evaluated the intracellular signaling pathways involved in MMP-9 release and the contribution of thrombin-reactive brain pericytes to BBB dysfunction. PKC activator evoked MMP-9 release from brain pericytes. The thrombin-induced MMP-9 release was inhibited by U0126, LY294002, Go6976, and Go6983. However, Go6976 decreased phosphorylation levels of PKCθ and Akt, and Go6983 decreased phosphorylation levels of PKCδ and extracellular signal-regulated kinase (ERK). Additionally, treatment of pericytes with thrombin or PAR1-activating peptide stimulated PKCδ/θ signaling. These substances impaired brain endothelial barrier function in the presence of brain pericytes. Brain pericytes function through two independent downstream signaling pathways via PAR1 activation to release MMP-9 in response to thrombin - the PKCθ-Akt pathway and the PKCδ-ERK1/2 pathway. These pathways participate in PAR1-mediated MMP-9 release from pericytes, which leads to BBB dysfunction. Brain pericytes and their specific signaling pathways could provide novel therapeutic targets for thrombin-induced neurovascular diseases.

  11. Thrombin enhances NGF-mediated neurite extension via increased and sustained activation of p44/42 MAPK and p38 MAPK.

    PubMed

    Mufti, Rania E; Sarker, Krishna; Jin, Yan; Fu, Songbin; Rosales, Jesusa L; Lee, Ki-Young

    2014-01-01

    Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.

  12. Thrombin generation by activated factor VII on platelet activated by different agonists. Extending the cell-based model of hemostasis

    PubMed Central

    Altman, Raul; Scazziota, Alejandra Silvia; Herrera, Maria de Lourdes; Gonzalez, Claudio

    2006-01-01

    Background Platelet activation is crucial in normal hemostasis. Using a clotting system free of external tissue factor, we investigated whether activated Factor VII in combination with platelet agonists increased thrombin generation (TG) in vitro. Methods and results TG was quantified by time parameters: lag time (LT) and time to peak (TTP), and by amount of TG: peak of TG (PTG) and area under thrombin formation curve after 35 minutes (AUC→35min) in plasma from 29 healthy volunteers using the calibrated automated thrombography (CAT) technique. TG parameters were measured at basal conditions and after platelet stimulation by sodium arachidonate (AA), ADP, and collagen (Col). In addition, the effects of recombinant activated FVII (rFVIIa) alone or combined with the other platelet agonists on TG parameters were investigated. We found that LT and TTP were significantly decreased (p < 0.05) and PTG and AUC→35min were significantly increased (p < 0.05) in platelet rich plasma activated with AA, ADP, Col, and rFVIIa compared to non-activated platelet rich plasma from normal subjects (p = 0.01). Furthermore platelet rich plasma activated by the combined effects of rFVIIa plus AA, ADP or Col had significantly reduced LT and TTP and increased AUC→35min (but not PTG) when compared to platelet rich plasma activated with agonists in the absence of rFVIIa. Conclusion Platelets activated by AA, ADP, Col or rFVIIa triggered TG. This effect was increased by combining rFVIIa with other agonists. Our intrinsic coagulation system produced a burst in TG independent of external tissue factor activity an apparent hemostatic effect with little thrombotic capacity. Thus we suggest a modification in the cell-based model of hemostasis. PMID:16630353

  13. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA.

    PubMed

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure-activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes.

  14. Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA

    PubMed Central

    Mena-Ulecia, Karel; Tiznado, William; Caballero, Julio

    2015-01-01

    Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyanofluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure–activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations. We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. PMID:26599107

  15. Label-free fluorescent detection of thrombin activity based on a recombinant enhanced green fluorescence protein and nickel ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles.

    PubMed

    Wang, Ming; Lei, Chunyang; Nie, Zhou; Guo, Manli; Huang, Yan; Yao, Shouzhuo

    2013-11-15

    Herein, a novel label-free fluorescent assay has been developed to detect the activity of thrombin and its inhibitor, based on a recombinant enhanced green fluorescence protein (EGFP) and Ni(2+) ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni(2+)-NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni(2+)-NTA MNPs through Ni(2+)-hexahistidine interaction, and dragged out of the solution by magnetic separation. Thrombin can selectively digest EGFP accompanied by His-tag peptide sequence leaving, and the resulting EGFP cannot be captured by Ni(2+)-NTA MNPs and kept in supernatant. Hence the fluorescence change of supernatant can clearly represent the activity of thrombin. Under optimized conditions, such assay showed a relatively low detection limit (3.0×10(-4) U mL(-1)), and was also used to detect the thrombin inhibitor, Hirudin, and further applied to detect thrombin activity in serum. Combined with the satisfactory reusability of Ni(2+)-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.

  16. Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge

    PubMed Central

    Mahmud, Shawn A.; Lambert, Michele P.; Poncz, Mortimer; Slungaard, Arne

    2007-01-01

    Pharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4−/−) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C–deficient (PC+/−) mice. Transgenic mice overexpressing human PF4 (hPF4+) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC+/− mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4+ and PC+/−/hPF4+ mice had improved survival. Further, infusion of hPF4+ platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting. PMID:17540840

  17. Endogenous platelet factor 4 stimulates activated protein C generation in vivo and improves survival after thrombin or lipopolysaccharide challenge.

    PubMed

    Kowalska, M Anna; Mahmud, Shawn A; Lambert, Michele P; Poncz, Mortimer; Slungaard, Arne

    2007-09-15

    Pharmacologic infusion of activated protein C (APC) improves survival in severe sepsis, and platelet factor 4 (PF4) accelerates APC generation in a primate thrombin-infusion model. We now tested whether endogenous platelet PF4 content affects APC generation. Mice completely deficient in PF4 (mPF4(-/-)) had impaired APC generation and survival after thrombin infusion, similar to the impairment seen in heterozygote protein C-deficient (PC(+/-)) mice. Transgenic mice overexpressing human PF4 (hPF4(+)) had increased plasma APC generation. Overexpression of platelet PF4 compensated for the defect seen in PC(+/-) mice. In both a thrombin and a lipopolysaccharide (LPS) survival model, hPF4(+) and PC(+/-)/hPF4(+) mice had improved survival. Further, infusion of hPF4(+) platelets improved survival of wild-type mice after an LPS challenge. These studies suggest that endogenous PF4 release may have biologic consequences for APC generation and survival in clinical sepsis. Infusions of PF4-rich platelets may be an effective strategy to improve outcome in this setting.

  18. Influenza virus H1N1 activates platelets through FcγRIIA signaling and thrombin generation.

    PubMed

    Boilard, Eric; Paré, Guillaume; Rousseau, Matthieu; Cloutier, Nathalie; Dubuc, Isabelle; Lévesque, Tania; Borgeat, Pierre; Flamand, Louis

    2014-05-01

    Platelets play crucial functions in hemostasis and the prevention of bleeding. During H1N1 influenza A virus infection, platelets display activation markers. The platelet activation triggers during H1N1 infection remain elusive. We observed that H1N1 induces surface receptor activation, lipid mediator synthesis, and release of microparticles from platelets. These activation processes require the presence of serum/plasma, pointing to the contribution of soluble factor(s). Considering that immune complexes in the H1N1 pandemic were reported to play a pathogenic role, we assessed their contribution in H1N1-induced platelet activation. In influenza-immunized subjects, we observed that the virus scaffolds with immunoglobulin G (IgG) to form immune complexes that promote platelet activation. Mechanistically, this activation occurs through stimulation of low-affinity type 2 receptor for Fc portion of IgG (FcγRIIA), a receptor for immune complexes, independently of thrombin. Using a combination of in vitro and in vivo approaches, we found that the antibodies from H3N2-immunized mice activate transgenic mouse platelets that express FcγRIIA when put in the presence of H1N1, suggesting that cross-reacting influenza antibodies suffice. Alternatively, H1N1 can activate platelets via thrombin formation, independently of complement and FcγRIIA. These observations identify both the adaptive immune response and the innate response against pathogens as 2 intertwined processes that activate platelets during influenza infections.

  19. Ionophore A23187 induces a refractory state in thrombin-activated release of inositol phosphates.

    PubMed Central

    Moscat, G; Moreno, F; Iglesias, S; Garcia-Barreno, P; Municio, A M

    1986-01-01

    The phosphatidylinositol cycle has been proposed to be involved in the regulation of platelet functionality through the control of cytoplasmic Ca2+ levels. However, the requirements of phospholipase C for Ca2+ has not yet been elucidated in intact platelets. The primary purpose of the present study was to investigate the Ca2+ requirements of this enzyme in platelets from miniature swine by taking advantage of the permeabilizing properties of the ionophore A23187. Our results strongly suggest that the treatment of platelets with A23187 induces a refractory state in thrombin-stimulated release of inositol phosphates while 5-hydroxytryptamine (serotonin)-secretory capacity in response to thrombin remained constant. This refractory state seems to be dependent on some cytochalasin-inhibitable cytoskeletal phenomena. PMID:3099773

  20. 'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex.

    PubMed

    Shivaprasad, Holenarasipura V; Rajaiah, Rajesh; Frey, Brigitte M; Frey, Felix J; Vishwanath, Bannikuppe S

    2010-03-01

    Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

  1. Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation.

    PubMed

    Thomas, Christopher P; Morgan, Lloyd T; Maskrey, Benjamin H; Murphy, Robert C; Kühn, Hartmut; Hazen, Stanley L; Goodall, Alison H; Hamali, Hassan A; Collins, Peter W; O'Donnell, Valerie B

    2010-03-05

    Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

  2. Facile fabrication of an aptasensor for thrombin based on graphitic carbon nitride/TiO2 with high visible-light photoelectrochemical activity.

    PubMed

    Fan, Dawei; Guo, Cuijuan; Ma, Hongmin; Zhao, Di; Li, Yina; Wu, Dan; Wei, Qin

    2016-01-15

    A novel aptasensor for thrombin with high visible-light activity was facilely fabricated based on graphitic carbon nitride/TiO2 (g-C3N4/TiO2) photoelectrochemical (PEC) composite. Crystallization of TiO2 nanoparticles (NPs) and their strong interaction with g-C3N4 sheet were confirmed by high-resolution transmission electron microscope (HR-TEM), both of which contributed to the high photocurrent intensity under visible-light irradiation. Carboxyl functionalized thrombin aptamers were first successfully bound to the g-C3N4/TiO2 modified electrode as proven by photoelectrochemical test and electrochemical impedance spectroscopy (EIS) analysis. Ascorbic acid was utilized as the electron donor for scavenging photo-generated holes and inhibiting light driven electron-hole pair recombination. The specific recognition between thrombin aptamer and thrombin led to the linear decrease of photocurrent with the increase of logarithm of thrombin concentration in the range of 5.0×10(-13)molL(-1) to 5.0×10(-9)molL(-1) with a detection limit of 1.2×10(-13)molL(-1). This proposed low-cost, convenient and sensitive aptasensor showed promising applications in biosensor and photoelectrochemical analysis.

  3. Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo

    SciTech Connect

    Nieman, M T; Burke, F; Warnock, M; Zhou, Y; Sweigart, J; Chen, A; Ricketts, D; Lucchesi, B R; Chen, Z; Cera, E Di; Hilfinger, J; Kim, J S; Mosberg, H I; Schmaier, A H

    2008-04-29

    Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D-isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin - RPPGF. These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α-thrombin-induced cleavage of a chromogenic substrate at 4.4--8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa-TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p-Me)], blocks α-thrombin-induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 μm. FM19 achieved 100% inhibition of threshold α- or γ-thrombin-induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N-terminal D-Arg retrobinds into the S1 pocket, its second residue Oic interacts with His-57, Tyr-60a and Trp-60d, and its C-terminal p-methyl Phe engages thrombin's aryl binding site composed of Ile-174, Trp-215, and Leu-99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add-on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

  4. Zinc modulates thrombin adsorption to fibrin

    SciTech Connect

    Hopmeier, P.; Halbmayer, M.; Fischer, M.; Marx, G. )

    1990-05-01

    Human thrombin with high affinity to Sepharose insolubilized fibrin monomers (high-affinity thrombin) was used to investigate the effect of Zn(II) on the thrombin adsorption to fibrin. Results showed that at Zn(II) concentrations exceeding 100 mumols/l, thrombin binding to fibrin was decreased concomitant with the Zn(II) concentration and time; at lower Zn(II) concentrations, thrombin adsorption was enhanced. Experimental results were identical by using 125I-labelled high-affinity alpha-thrombin or by measuring the thrombin activity either by chromogenic substrate or by a clotting time method. In contrast, Ca(II) alone (final conc. 3 mmol/l) or in combination with Zn(II) was not effective. However, at higher Ca(II) concentrations (7.5-15 mmol/l), thrombin adsorption was apparently decreased. Control experiments revealed that Zn(II) had no impact on the clottability of fibrinogen, and that the results of the experiments with Ca(II) were not altered by possible cross-linking of fibrin. We conclude that unlike Ca(II), Zn(II) is highly effective in modulating thrombin adsorption to fibrin.

  5. Two sites on P-selectin (the lectin and epidermal growth factor-like domains) are involved in the adhesion of monocytes to thrombin-activated endothelial cells.

    PubMed Central

    Murphy, J F; McGregor, J L

    1994-01-01

    P-selectin, also known as GMP-140, PADGEM or CD62, is expressed on the surface of thrombin-activated platelets and endothelial cells (EC). It is a member of the selectin family of adhesion molecules that regulate leucocyte interactions with the blood vessel wall. In this study we have found that peptides derived from both the lectin (residues 19-34 and 51-61) and epidermal growth factor (EGF)-like (residues 127-139) domains inhibit the adhesion of peripheral blood mononuclear cells (PBMC), elutriated monocytes and a monocytic cell line (U937) to thrombin-activated EC. This inhibition occurred in a concentration-dependent manner and the peptide most active at the lowest concentrations was the one derived from the EGF-like motif (127-139). The scrambled forms of these peptides, identical in amino acid composition to the authentic peptides but with altered sequences, were not inhibitory. Thrombin-activated platelets supported adhesion of U937 cells and this adhesion was dramatically inhibited by the two peptides derived from the lectin-like domain (residues 19-34 and 51-61). All three peptides, when conjugated to BSA and coated on plastic plates, mediated U937 cell adhesion. This study shows, for the first time, that two sites on P-selectin, the lectin and EGF-like domains, are involved in the adhesion of monocytes to thrombin-activated EC. PMID:7526845

  6. Label-free monitoring of DNA polymerase activity based on a thrombin-binding aptamer G-quadruplex.

    PubMed

    Wang, Jing; Liu, Haisheng; Ma, Changbei; Wang, Jun; Zhong, Linxiu; Wu, Kefeng

    2017-04-01

    We have developed a label-free assay for the detection of DNA polymerase activity based on a thrombin-binding aptamer (TBA) G-quadruplex. In the presence of DNA polymerase, the 3'-OH termini of the hairpin substrate are immediately elongated to replace the TBA, which can be recognized quickly by the ThT dye and results in an increase of fluorescence. This method is highly sensitive with a detection limit of 0.1 U/mL. It is simple and cost-effective without any requirement of labeling with a fluorophore-quencher pair. Furthermore, the proposed method can also be applied to analyze the inhibition of DNA polymerase, which clearly indicates that the proposed method can be applied for screening of potential DNA polymerase inhibitors.

  7. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

    PubMed Central

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H. J.; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  8. Differential effect of the inhibition of Grb2-SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement.

    PubMed Central

    Saci, Abdelhafid; Liu, Wang-Qing; Vidal, Michel; Garbay, Christiane; Rendu, Francine; Bachelot-Loza, Christilla

    2002-01-01

    The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2-Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2-SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2-SH2 domain formed an association with the beta3 subunit of the alphaIIb-beta3 integrin (GPIIb-IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcgammaRIIa) or in resting platelets. Grb2-SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2-SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced 'inside-out' signalling. Platelet aggregation induced by direct activation via alphaIIb-beta3 ('outside-in' signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2-SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and alphaIIb-beta3-mediated platelet activation. PMID:11964172

  9. Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.

    PubMed

    Shirakawa, Hisashi; Sakimoto, Shinya; Nakao, Kenji; Sugishita, Aiko; Konno, Masakazu; Iida, Shota; Kusano, Ayaka; Hashimoto, Emina; Nakagawa, Takayuki; Kaneko, Shuji

    2010-09-29

    Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own

  10. Thrombin inhibitor design.

    PubMed

    Sanderson, P E; Naylor-Olsen, A M

    1998-08-01

    Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

  11. Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

    SciTech Connect

    Maraganore, J.M.; Bourdon, P.; Jablonski, J.; Ramachandran, K.L. ); Fenton, J.W. II )

    1990-07-31

    A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.

  12. Structure and behavior of human α-thrombin upon ligand recognition: thermodynamic and molecular dynamics studies.

    PubMed

    Silva, Vivian de Almeira; Cargnelutti, Maria Thereza; Giesel, Guilherme M; Palmieri, Leonardo C; Monteiro, Robson Q; Verli, Hugo; Lima, Luis Mauricio T R

    2011-01-01

    Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors.

  13. Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways.

    PubMed Central

    Thomas, C P; Dunn, M J; Mattera, R

    1995-01-01

    The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway. Images Figure 5 Figure 6 Figure 7 Figure 9 PMID:7492305

  14. 49 CFR 234.106 - Partial activation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Partial activation. 234.106 Section 234.106..., DEPARTMENT OF TRANSPORTATION GRADE CROSSING SIGNAL SYSTEM SAFETY AND STATE ACTION PLANS Response to Reports of Warning System Malfunction § 234.106 Partial activation. Upon receipt of a credible report of...

  15. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

    PubMed Central

    Shimada, K; Ozawa, T

    1985-01-01

    It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin

  16. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  17. An evaluation of platelet-rich plasma without thrombin activation with or without anorganic bone mineral in the treatment of human periodontal intrabony defects.

    PubMed

    Rodrigues, Silvia V; Acharya, Anirudh B; Thakur, Srinath L

    2011-01-01

    The efficacy of platelet-rich plasma (PRP) in periodontal regeneration is not well understood and the definite clinical viability of blood derived platelets lacks clarity. Also, the use of thrombin for platelet activation is disputed. Hence, the purpose of this study was to evaluate the efficacy of blood derived platelets without thrombin activation, alone or in combination with bovine anorganic bone mineral (ABM), in the treatment of human periodontal intrabony defects. PRP was prepared using a simple tabletop centrifuge and activated using calcium chloride without the addition of thrombin. This PRP was used alone (in Group A) and in combination with bovine ABM (in Group B) in the treatment of human periodontal angular defects. Both the control and the test groups showed definite improvement in clinical parameters. On comparison, however, there was a statistically significant improvement in the probing pocket depths and relative attachment level in Group B over Group A at 3 and 6 months intervals, whereas at the end of 9 months this difference was not statistically significant. There was no statistically significant difference between the groups with respect to the relative defect depth. Within the limitations of this study and the type of PRP used, i.e. without thrombin mediated activation, it can be concluded that both PRP and PRP combined with bovine ABM results in significant clinical improvement. Albeit statistically insignificant, there is a preponderance of better clinical results with the addition of ABM to PRP. Further studies need to be carried out on a larger sample size to confirm the results of the present study.

  18. Prostaglandin E1-induced deconsolidation of thrombin-activated platelet aggregates I: ultrastructure-computer image analysis.

    PubMed

    Salganicoff, L; Russo, M A; Sevy, R W

    1999-06-15

    We have compared, at an ultrastructural-computer image morphometric level, the relaxation induced by Mg-ethylene-bis-oxyethylenenitrilo-tetracetic acid and prostaglandin E1 on a model of a thrombin-activated platelet aggregate. Mg-ethylene-bisoxyethylenenitrilo-tetracetic acid produced a small increase of 5.0% of the intercellular space over the control levels, and a decrease of 10.0+/-1.3% of the cross-sectional area of the platelets, with no apparent cytoskeletal alterations. In contrast, the prostaglandin El-treated preparation shows a 360% increase in the intercellular space and a decrease of the average platelet cross-sectional area of 30.0+/-2.0% with marked cytoskeletal alterations. We use the term "deconsolidation" to describe this effect. The enlargement of the intercellular space allows the observation of two types of contacts: (1) a type S (segmental) complex, of approximately 200-nm length that maintains a narrow interplatelet gap of 20-30 nm, filled with a dense intercellular material, and (2) a type R (reticular) complex, formed by scant focal regions of the plasma membrane from opposing platelets that are connected through a mesh of fibrillar or granular material contained within a variable-size space. We hypothesize that deconsolidation is caused by fluid loss from the platelets into the intercellular space. As a result, platelet volume decreases and intercellular space increases.

  19. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

    PubMed

    Lin, Jibin; He, Shaolin; Sun, Xinghui; Franck, Gregory; Deng, Yihuan; Yang, Dafeng; Haemmig, Stefan; Wara, A K M; Icli, Basak; Li, Dazhu; Feinberg, Mark W

    2016-09-01

    Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and

  20. Evaluation of the bone healing process utilizing platelet-rich plasma activated by thrombin and calcium chloride: a histologic study in rabbit calvaria.

    PubMed

    Betoni-Junior, Walter; Dechichi, Paula; Esteves, Jônatas Caldeira; Zanetta-Barbosa, Darceny; Magalhães, Aparecido Eurípedes Onório

    2013-02-01

    To evaluate the bone healing of defects filled with particulate bone graft in combination with platelet-rich plasma (PRP), added with a mixture of calcium chloride and thrombin or just calcium chloride. Two 5-mm bone defects were created in the calvaria of 24 rabbits. Each defect was filled with particulate bone graft and PRP. In one defect the PRP was activated by a mixture of calcium chloride and thrombin; in the other, PRP was activated by calcium chloride only. The animals were euthanized 1, 2, 4, and 8 weeks after the surgeries, and the calvaria was submitted to histologic processing for histomorphometric analysis. The qualitative analysis has shown that both defects presented the same histologic characteristics so that a better organized, more mature, and well-vascularized bone tissue was noticed in the eighth week. A good bone repair was achieved using either the mixture of calcium chloride and thrombin or the calcium chloride alone as a restarting agent of the coagulation process.

  1. Thrombin-induced CCAAT/enhancer-binding protein β activation and IL-8/CXCL8 expression via MEKK1, ERK, and p90 ribosomal S6 kinase 1 in lung epithelial cells.

    PubMed

    Lin, Chien-Huang; Nai, Po-Ling; Bien, Mauo-Ying; Yu, Chung-Chi; Chen, Bing-Chang

    2014-01-01

    Thrombin, a serine protease, is a well-known coagulation factor generated during vascular injury and plays an important role in lung inflammation. We previously showed that the c-Src- and Rac/PI3K/Akt-dependent NF-κB pathways are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells (A549). In this study, we investigated the role of the MEK kinase (MEKK)1/ERK/p90 ribosomal S6 kinase (RSK)1-dependent C/EBPβ signaling pathway in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by small interfering RNA (siRNA) of C/EBPβ and by cells transfected with the C/EBPβ site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced κB-luciferase activity was also inhibited by C/EBPβ siRNA. The thrombin-induced increases in IL-8/CXCL8 release and IL-8/CXCL8-luciferase were also inhibited by MEKK1 siRNA, PD98059 (an MEK inhibitor), U0126 (an ERK inhibitor), and RSK1 siRNA. Treatment of cells with thrombin caused an increase in C/EBPβ phosphorylation at Thr(235), C/EBPβ-luciferase activity, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation. Furthermore, thrombin-mediated C/EBPβ phosphorylation and C/EBPβ-luciferase activity were inhibited by MEKK1 siRNA, PD98059, and RSK1 siRNA. Stimulation of cells with thrombin resulted in an increase in RSK1 phosphorylation at Thr(359)/Ser(363), and this effect was inhibited by MEKK1 siRNA and PD98059. The thrombin-induced increase in ERK activation was inhibited by MEKK1 siRNA. These results imply that thrombin activates the MEKK1/ERK/RSK1 signaling pathway, which in turn initiates C/EBPβ activation, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.

  2. Is the activated partial thromboplastin time suitable to screen for von Willebrand factor deficiencies?

    PubMed

    Lippi, Giuseppe; Franchini, Massimo; Poli, Giovanni; Salvagno, Gian Luca; Montagnana, Martina; Guidi, Gian Cesare

    2007-06-01

    The diagnostic approach to von Willebrand factor deficiencies is challenging and requires discretionary use of laboratory resources. Although extensive preoperative testing is not recommended, the activated partial thromboplastin time may be useful, especially in selected categories of patients. To establish the diagnostic sensitivity of this test to identify isolate von Willebrand factor deficiencies, 204 consecutive patients underwent a routine preoperative screening consisting of activated partial thromboplastin time, von Willebrand factor antigen, intrinsic pathway clotting factors activity, lupus anticoagulants and thrombin time. Thirty-seven patients were diagnosed with haemostasis disturbances other than von Willebrand factor deficiencies and were excluded from the evaluation. Isolated von Willebrand factor deficiency was diagnosed in 11 of the remaining 167 patients. A significant correlation was observed between von Willebrand factor antigen and activated partial thromboplastin time. Receiver operating characteristic curve analysis showed an area under the curve of 0.982 (95% confidence interval: 0.972-0.992; P < 0.001). At the 1.17 upper limit of the activated partial thromboplastin time, sensitivity and specificity were 100 and 85%, respectively, with negative and positive predictive values of 100 and 31%, respectively. These results demonstrate that activated partial thromboplastin time has an excellent diagnostic sensitivity and a satisfactory specificity for identifying isolated von Willebrand factor deficiencies.

  3. Thrombin regulates the function of human blood dendritic cells

    SciTech Connect

    Yanagita, Manabu; Kobayashi, Ryohei; Kashiwagi, Yoichiro; Shimabukuro, Yoshio; Murakami, Shinya E-mail: ipshinya@dent.osaka-u.ac.jp

    2007-12-14

    Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.

  4. Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

    PubMed Central

    Weinstein, M J; Chute, L E

    1984-01-01

    We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity. Images PMID:6421875

  5. Activation of tumour cell ECM degradation by thrombin-activated platelet membranes: potentially a P-selectin and GPIIb/IIIa-dependent process.

    PubMed

    Pang, J H; Coupland, L A; Freeman, C; Chong, B H; Parish, Christopher R

    2015-06-01

    The promotion of tumour metastasis by platelets may occur through several mechanisms including the induction of a more metastatic phenotype in tumour cells and assisted extravasation of circulating tumour cells. Whilst the mechanisms underlying platelet-assisted extravasation have been extensively studied, much less attention has been paid to the mechanisms underlying platelet promotion of an aggressive phenotype within a tumour cell population. Herein, we demonstrate in vitro that MDA-MB-231 breast carcinoma cells incubated with washed thrombin-activated platelet membranes adopt a Matrigel-degrading phenotype in a dose- and contact time-dependent manner. The same phenotypic change was observed with three other human tumour cell lines of diverse anatomical origin. Moreover, tumour cell lines that had been cultured with washed thrombin-activated platelet membranes had a greater metastatic capacity when injected into mice. This in vivo effect was reliant upon a co-incubation period of >2 h implying a mechanism involving more than platelet membrane binding that occurred within 5 min. Upon further investigation it was found that simultaneous blocking of the platelet-membrane proteins P-selectin and GPIIb/IIIa prevented interactions between platelet membranes and MDA-MB-231 cells but also significantly reduced the ability of tumour cells to degrade Matrigel. These results confirm that platelets induce a more aggressive phenotype in tumour cells but also identify the platelet proteins involved in this effect. P-selectin and GPIIb/IIIa also play a role in assisting tumour cell extravasation and, thus, are ideal targets for the therapeutic intervention of both stages of platelet-assisted metastasis.

  6. Thrombin-induced increase in albumin permeability across the endothelium

    SciTech Connect

    Garcia, J.G.; Siflinger-Birnboim, A.; Bizios, R.; Del Vecchio, P.J.; Fenton, J.W. 2d.; Malik, A.B.

    1986-07-01

    We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium.

  7. Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin.

    PubMed Central

    Festoff, B. W.; D'Andrea, M. R.; Citron, B. A.; Salcedo, R. M.; Smirnova, I. V.; Andrade-Gordon, P.

    2000-01-01

    BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar

  8. Combination of thrombin-antithrombin complex, plasminogen activator inhibitor-1, and protein C activity for early identification of severe coagulopathy in initial phase of sepsis: a prospective observational study

    PubMed Central

    2014-01-01

    Introduction Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis. Methods A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α2-plasmin inhibitor (PI), plasmin-α2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days. Results A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three

  9. Abnormal activated partial thromboplastin time and malignancy.

    PubMed

    Delicata, M; Hambley, H

    2011-08-01

    Malignancy often results in clotting abnormalities. The aetiology of haemostasis problems in cancer is complex, and is still not completely understood. We describe a case of a patient with malignant mesothelioma, who was found to have elevated activated partial thromboplastin time, due to lupus anticoagulant. We suggest that patients with malignancy should have their coagulation checked prior to any invasive procedures.

  10. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships.

    PubMed Central

    Bode, W.; Turk, D.; Karshikov, A.

    1992-01-01

    Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70

  11. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

    PubMed Central

    Boggio, Elena; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α4β1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases. PMID:27478856

  12. Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo.

    PubMed

    Boggio, Elena; Dianzani, Chiara; Gigliotti, Casimiro Luca; Soluri, Maria Felicia; Clemente, Nausicaa; Cappellano, Giuseppe; Toth, Erika; Raineri, Davide; Ferrara, Benedetta; Comi, Cristoforo; Dianzani, Umberto; Chiocchetti, Annalisa

    2016-01-01

    Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α 4 β 1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases.

  13. Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

    PubMed

    Halldórsson, Haraldur; Thors, Brynhildur; Thorgeirsson, Gudmundur

    2015-01-01

    To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD.

  14. Activated partial thromboplastin time and minor coagulopathies.

    PubMed

    Hathaway, W E; Assmus, S L; Montgomery, R R; Dubansky, A S

    1979-01-01

    Five commercially available activated partial thromboplastin time (APTT) test systems were compared with the kaolin partial thromboplastin time (KPTT) method to determine sensitivity in detecting minor coagulation defects. All reagent systems detected severe factor VIII-, IX-, and XI-deficient hemophilia. Homozygous states of factor XII deficiency, Fletcher factor deficiency, and high-molecular-weight kininogen deficiency (Fitzgerald trait) also showed abnormally long APTTs by all systems. Of 19 samples from patients with deficiencies of factors XII, VIII, IX, XI, and II ranging from 2.5 to 52%, eight had deficiencies that were not detected by reagent A (ellagic acid); two, by reagent B (ellagic acid); two, by reagent C (kaolin); one, by reagent D (silica); one, by the KPTT method. All deficiencies were detected by reagent E (celite). Heparin effect on plasma was less well detected by reagent A (ellagic acid) than with the other test systems. APTT test systems can vary greatly in their abilities to detect minor coagulation abnormalities.

  15. Bactericidal activity of partially oxidized nanodiamonds.

    PubMed

    Wehling, Julia; Dringen, Ralf; Zare, Richard N; Maas, Michael; Rezwan, Kurosch

    2014-06-24

    Nanodiamonds are a class of carbon-based nanoparticles that are rapidly gaining attention, particularly for biomedical applications, i.e., as drug carriers, for bioimaging, or as implant coatings. Nanodiamonds have generally been considered biocompatible with a broad variety of eukaryotic cells. We show that, depending on their surface composition, nanodiamonds kill Gram-positive and -negative bacteria rapidly and efficiently. We investigated six different types of nanodiamonds exhibiting diverse oxygen-containing surface groups that were created using standard pretreatment methods for forming nanodiamond dispersions. Our experiments suggest that the antibacterial activity of nanodiamond is linked to the presence of partially oxidized and negatively charged surfaces, specifically those containing acid anhydride groups. Furthermore, proteins were found to control the bactericidal properties of nanodiamonds by covering these surface groups, which explains the previously reported biocompatibility of nanodiamonds. Our findings describe the discovery of an exciting property of partially oxidized nanodiamonds as a potent antibacterial agent.

  16. The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

    PubMed Central

    Figueiredo, Ana C.; de Sanctis, Daniele; Pereira, Pedro José Barbosa

    2013-01-01

    The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin. PMID:23951260

  17. Cleavage of a specific bond in troponin C by thrombin.

    PubMed

    Leavis, P C; Rosenfeld, S; Lu, R C

    1978-08-21

    Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

  18. Concentration-Dependent Dual Role of Thrombin In Protection of Cultured Rat Cortical Neurons

    PubMed Central

    García, Paul S.; Ciavatta, Vincent T.; Fidler, Jonathan A.; Woodbury, Anna; Levy, Jerrold H.; Tyor, William R.

    2015-01-01

    Background Thrombin’s role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. Methods We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. Results We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor - 1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. Conclusions An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor

  19. Characterization of in vitro biotransformation of new, orally active, direct thrombin inhibitor ximelagatran, an amidoxime and ester prodrug.

    PubMed

    Clement, Bernd; Lopian, Katrin

    2003-05-01

    N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b(5), NADH-cytochrome b(5) reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b(5) and b(5) reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.

  20. Inhibition of thrombin generation in plasma by fibrin formation (Antithrombin I).

    PubMed

    de Bosch, N B; Mosesson, M W; Ruiz-Sáez, A; Echenagucia, M; Rodriguez-Lemoin, A

    2002-08-01

    The adsorption of thrombin to fibrin during clotting defines "Antithrombin I" activity. We confirmed that thrombin generation in afibrinogenemic or in Reptilase defibrinated normal plasma was higher than in normal plasma. Repletion of these fibrinogen-deficient plasmas with fibrinogen 1 (gamma A/gamma A), whose fibrin has two "low affinity" non-substrate thrombin binding sites, resulted in moderately reduced thrombin generation by 29-37%. Repletion with fibrinogen 2 (gamma'/gamma A), which in addition to low affinity thrombin-binding sites in fibrin, has a "high affinity" non-substrate thrombin binding site in the carboxy-terminal region of its gamma' chain, was even more effective and reduced thrombin generation by 57-67%. Adding peptides that compete for thrombin binding to fibrin [S-Hir53-64 (hirugen) or gamma'414-427] caused a transient delay in the onset of otherwise robust thrombin generation, indicating that fibrin formation is necessary for full expression of Antithrombin I activity. Considered together, 1) the increased thrombin generation in afibrinogenemic or fibrinogen-depleted normal plasma that is mitigated by fibrinogen replacement; 2) evidence that prothrombin activation is increased in afibrinogenemia and normalized by fibrinogen replacement; 3) the severe thrombophilia that is associated with defective thrombin-binding in dysfibrinogenemias Naples I and New York I, and 4) the association of afibrinogenemia or hypofibrinogenemia with venous or arterial thromboembolism, indicate that Antithrombin I (fibrin) modulates thromboembolic potential by inhibiting thrombin generation in blood.

  1. A thrombin receptor in resident rat peritoneal macrophages

    SciTech Connect

    Kudahl, K.; Fisker, S.; Sonne, O. )

    1991-03-01

    Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of {sup 125}I-labeled bovine thrombin is achieved after 1 min at 37{degrees}C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. {sup 125}I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.

  2. Partial pressure measurements with an active spectrometer

    SciTech Connect

    Brooks, N.H.; Jensen, T.H.; Colchin, R.J.; Maingi, R.; Wade, M.R.; Finkenthal, D.F.; Naumenko, N.; Tugarinov, S.

    1998-07-01

    Partial pressure neutral ga measurements have been made using a commercial Penning gauge in conjunction with an active spectrometer. In prior work utilizing bandpass filters and conventional spectrometers, trace concentrations of the hydrogen isotopes H, D, T and of the noble gases He, Ne and Ar were determined from characteristic spectral lines in the light emitted by the neutral species of these elements. For all the elements mentioned, the sensitivity was limited by spectral contamination from a pervasive background of molecular hydrogen radiation. The active spectrometer overcomes this limitations by means of a digital lock-in method and correlation with reference spectra. Preliminary measurements of an admixture containing a trace amount of neon in deuterium show better than a factor of 20 improvement in sensitivity over conventional techniques. This can be further improved by correlating the relative intensities of multiple lines to sets of reference spectra.

  3. Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-κB activation and endothelial cell inflammation.

    PubMed

    Leonard, Antony; Marando, Catherine; Rahman, Arshad; Fazal, Fabeha

    2013-11-01

    Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury and its more severe form acute respiratory distress syndrome. Alterations in actin cytoskeleton are shown to be crucial for NF-κB regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin in mediating cytoskeletal alterations essential for NF-κB activation and EC inflammation. The present study describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-κB activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion inhibited RelA/p65 phosphorylation at Ser(536), a critical event conferring transcriptional competency to the bound NF-κB. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 by blocking IKKβ-dependent phosphorylation/degradation of IκBα. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNF-α-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.

  4. Thrombin generation in clinical conditions.

    PubMed

    Ten Cate, Hugo

    2012-03-01

    Commercial assays for determining thrombin generation in plasma are being tested in clinical conditions associated with thrombosis or bleeding. While pre-analytical conditions remain a source of inter laboratory variation, demanding for further standardization, clinical research proceeds. In patients at risk of venous thrombosis thrombin generation (TG) analysis may be utilized to detect underlying thrombophilia and this has been achieved both with addition of thrombomodulin or activated protein C, to test the contribution of the protein C system. In patients with documented venous thromboembolism, increased TG values are seen in those patients at greatest risk for recurrence, although the data are not consistent yet. In patients with arterial vascular disease, effects on TG patterns are seen that both reflect atherosclerosis (and its risk factors) and link to risk of recurrent atherothrombosis (coronary or stroke), but the data are limited. In patients with a bleeding diathesis, like hemophilia, the main importance of TG assays lies in the application for monitoring replacement therapy, either with factor concentrate or rFVIIa. An interesting application is in conjunction with thromboelastography, for monitoring peri-operative transfusion policy. Finally, TG analysis may contribute to monitoring anticoagulant drug treatment, but these and other applications would greatly benefit from whole blood, point of care applications of TG testing.

  5. Disparate temporal expression of the prothrombin and thrombin receptor genes during mouse development.

    PubMed Central

    Soifer, S. J.; Peters, K. G.; O'Keefe, J.; Coughlin, S. R.

    1994-01-01

    The protease thrombin is a potent agonist for platelet aggregation, mesenchymal cell proliferation, and endothelial production of growth factors and adhesion molecules. Thrombin also modulates neurite outgrowth in neuronal cultures. These apparently disparate responses to thrombin appear to be largely mediated by the recently cloned thrombin receptor. In the adult, thrombin is generated from its zymogen prothrombin at sites of vascular injury when circulating coagulation factors meet extravascular tissue factor. In this context thrombin's varied actions may mediate responses to wounding. Whether thrombin's actions on cells may also play a role in development is unknown. We examined the expression of thrombin receptor, prothrombin, and tissue factor by in situ hybridization in mouse development. Thrombin receptor mRNA was expressed widely in mesenchymal cell populations during early organogenesis (E9.5) and was particularly abundant in developing heart and blood vessels. Robust receptor expression was also noted in the germinal epithelium of the hindbrain. Thrombin receptor expression became more restricted with time and by the fetal growth stage (E16.5) was most readily detected in certain neurons, endocardial and endothelial cells, and within lung and liver. In contrast to the thrombin receptor, prothrombin mRNA was limited to the embryonic liver and was not detected until E12.5, well after the onset of receptor expression. mRNA for tissue factor, one important trigger for thrombin generation in the adult, was detected in embryonic epithelia from E9.5-12.5. In several instances, tissue factor-expressing epithelia were surrounded by thrombin receptor-expressing mesenchyme. These data suggest a possible role for the thrombin receptor in development. The finding of robust thrombin receptor expression before prothrombin mRNA was detected raises the question of whether other proteases or peptide ligands can activate the thrombin receptor. Images Figure 1 Figure 2

  6. Addition of prothrombin to plasma can result in a paradoxical increase in activated partial thromboplastin time.

    PubMed

    Hansson, Kenny M; Björkqvist, Jenny; Deinum, Johanna

    2014-12-01

    In the activated partial thromboplastin time (APTT) assay, a variety of nonphysiological reagents is used to induce contact activation. The sensitivity of the APTT response for different thrombin inhibitors has previously been found to be dependent on the used reagent. Recently, infusion of prothrombin (FII) has been used in in-vivo coagulopathy models and its effect has been analyzed in different assays. Therefore, we investigated whether the FII plasma concentration might affect APTT using different commercial reagents, applying both turbidimetry and viscometry. We compared both plasma-derived human FII (pd-hFII) and recombinant human FII (r-hFII). Similar results were found for pd-hFII and r-hFII with different APTT reagents. As expected, no effect on APTT was found by increasing the plasma concentration of FII using APTT reagents consisting of ellagic acid (Actin FS or Actin). Although with Pathromtin SL, consisting of SiO2, only a slight increase was found, with most other commercial APTT reagents, consisting of SiO2 or kaolin, APTT dose-dependently increased by increasing concentration of FII. Therefore, both Pathromtin SL and Actin FS were used to compare r-hFII and pd-hFII by determining the KM at 37C using FII-depleted plasma, providing values of 6 ± 0.3 nmol/l FII for both. Thus, at normal plasma concentrations of FII, the maximal initial thrombin generation rate should be reached and no effect on the coagulation time is expected at higher FII concentrations. To completely avoid the paradoxical effect in the APTT assay at FII concentrations higher than normal, Actin or Actin FS is the preferable reagent.

  7. GpIbα interacts exclusively with exosite II of thrombin.

    PubMed

    Lechtenberg, Bernhard C; Freund, Stefan M V; Huntington, James A

    2014-02-20

    Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.

  8. The influence of Erythropoietin on platelet activation, thrombin generation and FVII/active FVII in patients with AMI

    PubMed Central

    2014-01-01

    Background Erythropoietin (Epo) has been shown to improve myocardial function in models of experimental myocardial infarction, but has also been associated with a rise in thromboembolic events. Thus, the aim of this study was to investigate the influence of Epo on platelet activation and coagulation in patients with acute myocardial infarction (AMI). Methods The study was designed as a substudy of the randomised, double-blind, placebo controlled REVIVAL-3 (REgeneration of VItal Myocardium in ST-Segment EleVation MyocardiAL Infarction by Erythropoietin) study that investigated the effects of recombinant human Epo in AMI. Serial venous blood samples were collected before and after study medication. Circulating prothrombin fragment F1 + 2, FVII, active FVII, beta thromboglobulin (TG) and P-Selectin were measured before and 60 hours after randomization by immunoassay (n = 94). In a randomly selected subgroup platelet aggregation was measured using whole blood aggregometry (Multiplate Analyzer, n = 45). Results After 5 days an increase in FVII was observed after Epo as compared to placebo (P = 0.02), yet active FVII and prothrombin fragment F1 + 2 remained unchanged. Moreover, no statistically significant differences in circulating TG or P-selectin were observed between the groups. As an expected response to peri-interventional therapy with clopidogrel and aspirin, platelet aggregation after stimulation with ADP, TRAP, ASPI or collagen decreased 12 hours and 2 days after PCI. However, no difference between the Epo and the placebo group was observed. Conclusion After treatment with Epo in patients with AMI a slight increase in circulating FVII after Epo was not associated with an increase in active FVII, prothrombin fragment F1 + 2, TG or P-selectin. Moreover, platelet aggregation was not altered after treatment with Epo as compared to placebo. Trial registration ClinicalTrials.gov Identifier: NCT01761435 PMID:25228850

  9. Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action.

    PubMed Central

    Bar-Shavit, R; Benezra, M; Eldor, A; Hy-Am, E; Fenton, J W; Wilner, G D; Vlodavsky, I

    1990-01-01

    Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation. Images PMID:1963793

  10. Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity.

    PubMed

    Bhakta, Varsha; Gierczak, Richard F; Sheffield, William P

    2013-12-01

    Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352-358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API

  11. Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1.

    PubMed

    Giacco, Ferdinando; Perruolo, Giuseppe; D'Agostino, Elio; Fratellanza, Giorgio; Perna, Enzo; Misso, Saverio; Saldalamacchia, Gennaro; Oriente, Francesco; Fiory, Francesca; Miele, Claudia; Formisano, Salvatore; Beguinot, Francesco; Formisano, Pietro

    2006-11-01

    Platelet components have found successful clinical utilization to initiate or to accelerate tissue-repair mechanisms. However, the molecular pathways by which platelet factors contribute to tissue regeneration have not been fully elucidated. We have studied the effect of thrombin-activated platelets (TAPs) on cell growth in vivo and in cultured cell systems. Application of TAPs to ulcerative skin lesions of diabetic patients induced local activation of ERK1/2 and Akt/PKB. Moreover, when applied to cultured human skin fibroblasts, TAPs promoted cell growth and DNA synthesis and activated platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 receptor tyrosine kinases. PDGF was released by TAPs and rapidly achieved a plateau. At variance, the release of IGF-1 was mainly provided by the TAPs-stimulated fibroblasts and progressively increased up to 48 h. The PDGF-R blocker Ag1296 reduced the activation of Akt/PKB and, at a lesser extent, of ERK1/2. Conversely, inhibition of IGF-1 signaling by Ag1024 and expression of a dominant-negative IGF-1R mutant selectively reduced the stimulation of ERK1/2 by TAPs and fibroblast-released factors, with minor changes of Akt/PKB activity. Thus, platelet factors promote fibroblast growth by acutely activating Akt/PKB and ERK1/2. Sustained activation of ERK1/2, however, requires autocrine production of IGF-1 by TAPs-stimulated fibroblasts.

  12. Effect of thrombin on maturing human megakaryocytes.

    PubMed Central

    Cramer, E. M.; Massé, J. M.; Caen, J. P.; Garcia, I.; Breton-Gorius, J.; Debili, N.; Vainchenker, W.

    1993-01-01

    Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis

  13. An inhibitor of thrombin activated fibrinolysis inhibitor (TAFI) can reduce extracellular matrix accumulation in an in vitro model of glucose induced ECM expansion.

    PubMed

    Atkinson, J M; Pullen, N; Johnson, T S

    2013-06-24

    Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is downregulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity and reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. The data presented here indicate that high glucose induces TAFI activity, inhibiting plasmin activation which results in elevated ECM levels in tubular epithelial cells. The results support the hypothesis that UK-396082 is able to reduce TAFI activity, normalising plasmin activity and preventing excess ECM accumulation suggesting that TAFI inhibition may have potential as an anti-scarring strategy in CKD.

  14. Thrombin generation in patients after acute deep-vein thrombosis.

    PubMed

    ten Cate-Hoek, Arina J; Dielis, Arne W J H; Spronk, Henri M H; van Oerle, René; Hamulyák, Karly; Prins, Martin H; ten Cate, Hugo

    2008-08-01

    Thrombin generation measurement may be of value for assessing the risk of venous thromboembolism, but its long term profile has not been assessed in patients. We evaluated thrombin generation by Calibrated Automated Thrombogram (CAT) in plasma during follow up of 104 consecutive patients after an acute episode of deep venous thrombosis. Blood was drawn three times over the course of 24 months. Thrombin generation was measured in absence and presence of thrombomodulin and compared to a reference range derived from thrombin generation curves in 137 healthy volunteers. Thrombin generation of patients showed significantly higher endogenous thrombin potential (ETP) and peak height compared to the reference population. Differences were more pronounced in assays triggered with 1 pM TF. Inhibition by thrombomodulin was attenuated in patients off anticoagulants as compared to the reference population (21% vs. 42.2%, p < 0.0001); inhibition in patients on anticoagulant treatment was less pronounced (9.7%, p < 0.0001). Protein C activity, protein S antigen as well as free protein S showed highly negative correlation with ETP in all patients. A significant negative relation was found between FVIII levels and thrombomodulin induced reduction of ETP and peak height. In conclusion, thrombin generation by CAT reflects changes in coagulation status in patients following a thromboembolic event and is most sensitive at CAT analysis triggered with 1 pM TF. A role for factor VIII as an important attributable cause of hypercoagulability is reflected by the reduced inhibitory effect of thrombomodulin at high factor VIII levels.

  15. Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes.

    PubMed

    Kranzhöfer, R; Clinton, S K; Ishii, K; Coughlin, S R; Fenton, J W; Libby, P

    1996-08-01

    Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

  16. Anticoagulant activity of native and partially degraded glycoglucuronomannan after chemical sulfation.

    PubMed

    de Oliveira Barddal, Helyn Priscila; Gracher, Ana Helena Pereira; Simas-Tosin, Fernanda Fogagnoli; Iacomini, Marcello; Cipriani, Thales Ricardo

    2015-09-01

    Heparin has great clinical importance as anticoagulant and antithrombotic agent. However, because of its risks of causing bleeding and contamination by animal pathogens, several studies aim to obtain alternatives to heparin. In the search for anticoagulant and antithrombotic agents from a non-animal source, a glycoglucuronomannan from the gum exudate of the plant Vochysia thyrsoidea was partially hydrolyzed, and both native and partially degraded polysaccharides were chemically sulfated, yielding VThS and Ph-VThS respectively. Methylation analysis indicated that sulfation occurred preferentially at the O-5 position of arabinose units in the VThS and at the O-6 position of mannose units in Ph-VThS. In vitro aPTT assay showed that VThS and Ph-VThS have anticoagulant activity, which could be controlled by protamine, and ex vivo aPTT assay demonstrated that Ph-VThS is absorbed by subcutaneous route. Like heparin, they were able to inhibit α-thrombin and factor Xa by a serpin-dependent mechanism. In vivo, VThS and Ph-VThS reduced thrombus formation by approximately 50% at a dose of 40 IU/kg, similarly to heparin. The results demonstrated that the chemically sulfated polysaccharides are promising anticoagulant and antithrombotic agents.

  17. Thrombin generation and low-molecular-weight heparin prophylaxis in pregnant women with thrombophilia.

    PubMed

    Selmeczi, Anna; Roach, Rachel E J; Móré, Csaba; Batta, Zoltán; Hársfalvi, Jolán; van der Bom, Johanna G; Boda, Zoltán; Oláh, Zsolt

    2015-02-01

    Pregnancy is associated with increased risk of venous thromboembolism, especially in the presence of thrombophilia. However, there is no consensus on the optimal approach for thromboprophylaxis in this population. Recent evidence suggests that thrombin generation correlates with the overall procoagulant state of the plasma. Our aim was to evaluate thrombin generation in a prospective cohort of thrombophilic pregnant women, and investigate the effectiveness of low-molecular-weight heparin (LMWH) prophylaxis in pregnancy. Women with severe (n=8), mild (n=47) and no (n=15) thrombophilia were followed throughout their pregnancies. Thrombin generation was evaluated in each trimester as well as five days and eight weeks postpartum (as a reference category). In women undergoing LMWH prophylaxis, thrombin generation and anti-Factor-Xa activity were measured just before and 4 hours after administration (peak effect). Thrombin generation was determined using Technothrombin TGA assay system. For the analysis, median peak thrombin and endogenous thrombin potential were used. Peak thrombin and endogenous thrombin potential were increased during pregnancy compared to the non-pregnant state with the highest results in the severe thrombophilia group. In women receiving LMWH prophylaxis a decrease was observed in thrombin generation at peak effect but over the progression of pregnancy the extent of this decrease reduced in a stepwise fashion. Our results show that thrombin generation demonstrates the hypercoagulable state in thrombophilic pregnancies. In addition, we found the effect of LMWH prophylaxis to progressively decrease with advancing stages of pregnancy.

  18. Activated partial thromboplastin time and anti-xa measurements in heparin monitoring: biochemical basis for discordance.

    PubMed

    Takemoto, Clifford M; Streiff, Michael B; Shermock, Kenneth M; Kraus, Peggy S; Chen, Junnan; Jani, Jayesh; Kickler, Thomas

    2013-04-01

    We examined the concordance of heparin levels measured by a chromogenic anti-Xa assay and the activated partial thromboplastin time (APTT) during unfractionated heparin therapy (UFH) and the biochemical basis for differences between these measures. We also investigated the endogenous thrombin potential (ETP) as a possible measure of anticoagulation. Paired measures of anti-Xa and APTT were performed on 569 samples from 149 patients on UFH. The anti-Xa values and the APTT were concordant in only 54% of measurements. One hundred twelve samples from 59 patients on UFH were assayed for APTT, anti-Xa, factor II, factor VIII, and ETP. Supratherapeutic APTT values but therapeutic anti-Xa results had decreased factor II activity. Subtherapeutic APTT but therapeutic anti-Xa values had high factor VIII activity. ETP correlated with anticoagulation status and UFH dose. In conclusion, factor II and VIII activity contributes to discordance between APTT and anti-Xa results. ETP measurements may provide an additional assessment of anticoagulation status.

  19. Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.

    PubMed

    Mogami, Haruta; Keller, Patrick W; Shi, Haolin; Word, R Ann

    2014-05-09

    Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.

  20. Thrombin Generation in the Glasgow Myocardial Infarction Study

    PubMed Central

    Smid, Machiel; Dielis, Arne W. J. H.; Spronk, Henri M. H.; Rumley, Ann; van Oerle, Rene; Woodward, Mark; ten Cate, Hugo; Lowe, Gordon

    2013-01-01

    Background Thrombin is a key protease in coagulation also implicated in complex pathology including atherosclerosis. To address the role of thrombin in relation to myocardial infarction (MI) we explored thrombin generation analysis in plasma from patients and controls that had participated in the Glasgow MI Study (GLAMIS). Methods Thrombin generation at 1 and 2 pM TF and with and without thrombomodulin (TM) was performed on plasmas from 356 subjects (171 cases, 185 age and sex matched controls) from GLAMIS collected between 3 and 9 months after the MI event. Results Although thrombin generation was slightly delayed in cases (lag time increased from 3.3 to 3.6 min) at the highest trigger, the overall potential to generate thrombin was increased by 7% for the ETP and by 15% for the peak height (both at the 1 pM TF trigger) in cases. Addition of TM did not reveal differences. Furthermore, an increased thrombin generation was associated with MI [normalized ETP: adjusted OR for the highest percentile = 2.4 (95% CI 1.3–4.5) and normalized peak height: adjusted OR = 2.6 (1.3–5.0)] at the lowest trigger; normalized ETP and peak height being 2.1 (1.1–3.8) and 2.0 (1.0–4.1) at the higher 2 pM trigger. Conclusion In GLAMIS, patients with a previous MI had an increased thrombin generation compared to controls. The absence of a clear difference in TM reduction suggests an unaltered anticoagulant activity in these patients. Further research is needed in order to unravel the underlying mechanisms of enhanced thrombin generation after MI. PMID:23826181

  1. Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

    PubMed Central

    Virgilio, Antonella; Petraccone, Luigi; Vellecco, Valentina; Bucci, Mariarosaria; Varra, Michela; Irace, Carlo; Santamaria, Rita; Pepe, Antonietta; Mayol, Luciano; Esposito, Veronica; Galeone, Aldo

    2015-01-01

    Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2′-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (ΔTm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications. PMID:26582916

  2. Evidence for a hyperglycaemia-dependent decrease of antithrombin III-thrombin complex formation in humans.

    PubMed

    Ceriello, A; Giugliano, D; Quatraro, A; Marchi, E; Barbanti, M; Lefèbvre, P

    1990-03-01

    In the presence of increased levels of fibrinopeptide A, decreased antithrombin III biological activity, and thrombin-antithrombin III complex levels are seen in diabetic patients. Induced-hyperglycaemia in diabetic and normal subjects decreased antithrombin III activity and thrombin-antithrombin III levels, and increased fibrinopeptide A plasma levels, while antithrombin III concentration did not change; heparin was shown to reduced these phenomena. In diabetic patients, euglycaemia induced by insulin infusion restored antithrombin III activity, thrombin-antithrombin III complex and fibrinopeptide A concentrations; heparin administration had the same effects. These data stress the role of a hyperglycaemia-dependent decrease of antithrombin III activity in precipitating thrombin hyperactivity in diabetes mellitus.

  3. Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

    PubMed Central

    Post, G R; Collins, L R; Kennedy, E D; Moskowitz, S A; Aragay, A M; Goldstein, D; Brown, J H

    1996-01-01

    In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells. Images PMID:8930892

  4. Antithrombotic effects of bromophenol, an alga-derived thrombin inhibitor

    NASA Astrophysics Data System (ADS)

    Shi, Dayong; Li, Xiaohong; Li, Jing; Guo, Shuju; Su, Hua; Fan, Xiao

    2010-01-01

    Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. A bromophenol derivative named (+)-3-(2,3-dibromo-4, 5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1, isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity. In this study, we investigated the inhibition of human thrombin in vitro with this bromophenol derivative, and its antithrombotic efficacy in vivo using the arteriovenous shunt model and the ferric chloride-induced arterial thrombosis model in rats. The results show that the bromophenol derivative is a potential inhibitor of thrombin (IC50=1.03 nmol/L). In antithrombotic experiments in vivo, the bromophenol derivative also shows good effect comparing with the control group. These data indicate that the bromophenol derivative is a potential drug for prophylaxis and the treatment of thrombotic diseases.

  5. Comparison of the effect of fondaparinux and enoxaparin on thrombin generation during in-vitro clotting of whole blood and platelet-rich plasma.

    PubMed

    Gerotziafas, Grigoris T; Depasse, François; Chakroun, Tahar; Van Dreden, Patrick; Samama, Meyer M; Elalamy, Ismail

    2004-03-01

    Fondaparinux, a selective antithrombin-dependent inhibitor of activated factor X (FXa), is effective in the prevention and treatment of deep vein thrombosis and seems to be superior to enoxaparin. However, the exact mechanism of fondaparinux antithrombotic action is still unclear. We compared the effect of clinically relevant concentrations of fondaparinux and enoxaparin on the initiation and propagation phase of prothrombin activation and on the endogenous thrombin potential (ETP). Coagulation was triggered either in whole blood or in platelet-rich plasma (PRP) by recalcification in the presence of diluted thromboplastin. Prothrombin activation in whole blood was assessed with an original method by measuring the kinetics of prothrombin F1+2 formation using an enzyme-linked immunosorbent assay. We also assessed the maximum concentration of thrombin (Cmax) and the ETP in PRP using the Thrombogram-Thrombinoscope assay. Concentrations of fondaparinux achieved in prophylaxis (0.11-0.28 anti-FXa IU/ml) prolonged the initiation phase and reduced the velocity of the propagation phase of F1+2 formation. Concentrations of enoxaparin achieved in prophylaxis (0.1-0.25 anti-FXa IU/ml) did not significantly modify these parameters. Concentrations of fondaparinux equal to or higher than 0.57 anti-FXa IU/ml significantly reduced the Cmax of F1+2 or thrombin as well as the ETP. At fondaparinux concentrations equal to or higher than 0.91 anti-FXa IU/ml, a maximum 60% inhibition of thrombin generation was observed. In the presence of enoxaparin concentrations equal to or higher than 0.8 anti-FXa IU/ml, the inhibition of thrombin generation was higher than 80%. Fondaparinux prolonged the initiation phase, decreased the velocity of the propagation phase of thrombin generation and partially reduced the total amount of generated thrombin. The inhibitory effect of fondaparinux on the initiation and propagation phase of thrombin generation seems to be responsible for its antithrombotic

  6. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils

    PubMed Central

    1991-01-01

    The adhesion of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC) is an early and fundamental event in acute inflammation. This process requires the regulated expression of molecules on both the EC and PMN. EC stimulated with histamine or thrombin coexpress two proadhesive molecules within minutes: granule membrane protein 140 (GMP-140), a member of the selectin family, and platelet-activating factor (PAF), a biologically active phospholipid. Coexpression of GMP-140 and PAF is required for maximal PMN adhesion and the two molecules act in a cooperative fashion. The component of adhesion mediated by EC-associated PAF requires activation of CD11/CD18 integrins on the PMN and binding of these heterodimers to counterreceptors on the EC. GMP-140 also binds to a receptor on the PMN; however, it tethers the PMN to the EC without requiring activation of CD11/CD18 integrins. This component of the adhesive interaction is blocked by antibodies to GMP-140 or by GMP-140 in the fluid phase. Experiments with purified GMP-140 indicate that binding to its receptor on the PMN does not directly induce PMN adhesiveness but that it potentiates the CD11/CD18-dependent adhesive response to PAF by a mechanism that involves events distal to the PAF receptor. Tethering of the PMN to the EC by GMP-140 may also be required for efficient interaction of PAF with its receptor on the PMN. These observations define a complex cell recognition system in which tethering of PMNs by a selectin, GMP-140, facilitates juxtacrine activation of the leukocytes by a signaling molecule, PAF. The latter event recruits the third component of the adhesive interaction, the CD11/CD18 integrins. PMID:1717478

  7. G-Quadruplex Aptamers to Human Thrombin Versus Other Direct Thrombin Inhibitors: The Focus on Mechanism of Action and Drug Efficiency as Anticoagulants.

    PubMed

    Zavyalova, Elena; Ustinov, Nikita; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2016-01-01

    Thrombin is a key enzyme of blood coagulation system which has multiple functions including pro- and anticoagulant, platelet aggregating and inflammatory activities. Unsurprisingly, this enzyme has been a target for anticoagulant drug development for decades. Among the most interesting direct thrombin inhibitors with intravenous administration route are the following ones: 1) hirudins, proteins with bivalent binding mode to the thrombin, 2) bivalirudin, the peptide with bivalent binding mode to the thrombin, 3) argatroban, the chemical that binds to the thrombin active site, and 4) G-quadruplex DNA aptamers, structured oligonucleotides with an affinity to protein-binding site of the thrombin. Efficiency of all these inhibitors has been studied in vivo in preclinical and clinical trials, as well as in vitro with various tests, allowing to compare them thoroughly. In the review three levels of comparison were used to highlight the features of each inhibitor: 1) thrombin inhibition constants as a characteristic of inhibitor potency in simple enzymatic system; 2) inhibition of fibrin fiber formation and thrombin generation in coagulation cascade as a characteristic of anticoagulant potency in human blood plasma; and 3) therapeutic doses used and therapeutic profiles obtained after intravenous administration into animals and humans. The data clearly demonstrate weak and strong aspects of thrombin binding aptamers providing a solid background for further novel anticoagulant development.

  8. Role of Krev Interaction Trapped-1 in Prostacyclin-Induced Protection against Lung Vascular Permeability Induced by Excessive Mechanical Forces and Thrombin Receptor Activating Peptide 6

    PubMed Central

    Meliton, Angelo; Meng, Fanyong; Tian, Yufeng; Shah, Alok A.; Birukova, Anna A.

    2015-01-01

    Mechanisms of vascular endothelial cell (EC) barrier regulation during acute lung injury (ALI) or other pathologies associated with increased vascular leakiness are an active area of research. Adaptor protein krev interaction trapped-1 (KRIT1) participates in angiogenesis, lumen formation, and stabilization of EC adherens junctions (AJs) in mature vasculature. We tested a role of KRIT1 in the regulation of Rho-GTPase signaling induced by mechanical stimulation and barrier dysfunction relevant to ventilator-induced lung injury and investigated KRIT1 involvement in EC barrier protection by prostacyclin (PC). PC stimulated Ras-related protein 1 (Rap1)–dependent association of KRIT1 with vascular endothelial cadherin at AJs, with KRIT1-dependent cortical cytoskeletal remodeling leading to EC barrier enhancement. KRIT1 knockdown exacerbated Rho-GTPase activation and EC barrier disruption induced by pathologic 18% cyclic stretch and thrombin receptor activating peptide (TRAP) 6 and attenuated the protective effects of PC. In the two-hit model of ALI caused by high tidal volume (HTV) mechanical ventilation and TRAP6 injection, KRIT1 functional deficiency in KRIT1+/− mice increased basal lung vascular leak and augmented vascular leak and lung injury caused by exposure to HTV and TRAP6. Down-regulation of KRIT1 also diminished the protective effects of PC against TRAP6/HTV-induced lung injury. These results demonstrate a KRIT1-dependent mechanism of vascular EC barrier control in basal conditions and in the two-hit model of ALI caused by excessive mechanical forces and TRAP6 via negative regulation of Rho activity and enhancement of cell junctions. We also conclude that the stimulation of the Rap1-KRIT1 signaling module is a major mechanism of vascular endothelial barrier protection by PC in the injured lung. PMID:25923142

  9. Thrombin Generation in Zebrafish Blood

    PubMed Central

    Hemker, Coenraad; Lindhout, Theo; Kelchtermans, Hilde; de Laat, Bas

    2016-01-01

    To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis. PMID:26872266

  10. Thrombin regulation of synaptic transmission and plasticity: implications for health and disease

    PubMed Central

    Ben Shimon, Marina; Lenz, Maximilian; Ikenberg, Benno; Becker, Denise; Shavit Stein, Efrat; Chapman, Joab; Tanne, David; Pick, Chaim G.; Blatt, Ilan; Neufeld, Miri; Vlachos, Andreas; Maggio, Nicola

    2015-01-01

    Thrombin, a serine protease involved in the blood coagulation cascade has been shown to affect neural function following blood-brain barrier breakdown. However, several lines of evidence exist that thrombin is also expressed in the brain under physiological conditions, suggesting an involvement of thrombin in the regulation of normal brain functions. Here, we review ours’ as well as others’ recent work on the role of thrombin in synaptic transmission and plasticity through direct or indirect activation of Protease-Activated Receptor-1 (PAR1). These studies propose a novel role of thrombin in synaptic plasticity, both in physiology as well as in neurological diseases associated with increased brain thrombin/PAR1 levels. PMID:25954157

  11. Argatroban-coupled Affi-Gel matrix for the purification of thrombin from plasma.

    PubMed

    Lefkowitz, Jerry B

    2005-10-01

    Sometimes it is necessary to obtain thrombin from limited amounts of human plasma for laboratory assay. None of the available purification methods easily deals with this subject. The procedure described in the present paper uses a readily available pharmaceutical agent, argatroban, to construct an affinity matrix. Argatroban has a high affinity for thrombin and its thrombin binding is reversible. Prothrombin derived from a Ba(2+) precipitate of human plasma is used as the starting material. The crude prothrombin can be bulk activated to thrombin using taipan-snake (Oxyuranus scutellatus) venom and bound to the argatroban-coupled matrix without further processing steps. The thrombin product eluted from the argatroban matrix is very pure as judged by high specific activity and by electrophoresis. This purification scheme is rapid, yielding purified thrombin within 2 days.

  12. Low Anticoagulant Heparin Blocks Thrombin-Induced Endothelial Permeability in a PAR-Dependent Manner

    PubMed Central

    Gonzales, Joyce N.; Kim, Kyung-mi; Zemskova, Marina A.; Rafikov, Ruslan; Heeke, Brenten; Varn, Matthew N.; Black, Stephen; Kennedy, Thomas P.; Verin, Alexander D.; Zemskov, Evgeny A.

    2014-01-01

    Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhances lung inflammation, activates endothelial cells and disrupts lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (EC). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin, were also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH, can interfere with thrombin-activated signaling. PMID:24469066

  13. Red blood cells and thrombin generation in sickle cell disease.

    PubMed

    Whelihan, Matthew F; Lim, Ming Y; Key, Nigel S

    2014-05-01

    The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platelet rich plasma and showed little difference in endogenous thrombin potential between the SCD cohort and healthy matched controls. In SCD, erythrocytes and monocytes have been demonstrated to exhibit procoagulant characteristics. Thus, the absence of these cellular components in standard thrombin generation assays may fail to reflect global hypercoagulability in the whole blood of patients with SCD. We were therefore surprised to see no difference in net thrombin generation in tissue factor-initiated initiated clotting of whole blood from patients with SCD. However, we are continuing to reconcile these seemingly disparate observations by slight modifications of the whole blood model that include alternative coagulation triggers and a re-examination of the net thrombin generation when the protein/protein S system is simultaneously interrogated.

  14. Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

    PubMed

    Brzoska, Tomasz; Suzuki, Yuko; Sano, Hideto; Suzuki, Seiichirou; Tomczyk, Martyna; Tanaka, Hiroki; Urano, Tetsumei

    2017-04-03

    Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

  15. Thrombin-induced regulation of CD95(Fas) expression in the N9 microglial cell line: evidence for involvement of proteinase-activated receptor(1) and extracellular signal-regulated kinase 1/2.

    PubMed

    Weinstein, Jonathan R; Zhang, Matthew; Kutlubaev, Mansur; Lee, Richard; Bishop, Caroline; Andersen, Henrik; Hanisch, Uwe-Karsten; Möller, Thomas

    2009-03-01

    Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase alpha-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human alpha-thrombin (rh-thr) induces a restricted set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose-response and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR(1) agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2). Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate the neuroinflammatory response in a variety of neurological disorders.

  16. The co-crystal structure of unliganded bovine alpha-thrombin and prethrombin-2: movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding.

    PubMed Central

    Malkowski, M. G.; Martin, P. D.; Guzik, J. C.; Edwards, B. F.

    1997-01-01

    Unliganded bovine alpha-thrombin and prethrombin-2 have been co-crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 A and 2.3 A, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine alpha-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen A alpha with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 A) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen. PMID:9232645

  17. Nanocomplexation of thrombin with cationic amylose derivative for improved stability and hemostatic efficacy

    PubMed Central

    Zhuang, Baoxiong; Li, Zhihua; Pang, Jiadong; Li, Wenbin; Huang, Pinbo; Wang, Jie; Zhou, Yu; Lin, Qing; Zhou, Quanbo; Ye, Xiao; Ye, Huilin; Liu, Yimin; Zhang, Li-Ming; Chen, Rufu

    2015-01-01

    As a topical hemostatic agent, thrombin has wide application for many surgical treatments. However, native thrombin always suffers from its physical and chemical instabilities. In this work, a nanocomplexation strategy was developed for modifying the stability and hemostatic efficacy of thrombin, in which a water-soluble cationic amylose derivative containing poly(l-lysine) dendrons was prepared by a click reaction and then used to complex thrombin in an aqueous system. For resultant thrombin nanocomplexes, their morphology and particle size distribution were investigated. Their stabilities were studied in terms of activity retention percentages under different storage time, pH values, and illumination time. In addition, their ability to achieve in vitro fibrinogen and blood coagulation were evaluated. Via a rat hepatic hemorrhage model and a rat iliac artery hemorrhage model, these thrombin nanocomplexes were confirmed to have good tissue biocompatibility and in vivo hemostatic effectiveness. PMID:25673989

  18. Activated partial thromboplastin time of owl monkey (Aotus trivirgatus) plasma.

    PubMed

    Mrema, J E; Johnson, G S; Kelley, S T; Green, T J

    1984-06-01

    Owl monkey plasma samples produced short, reproducible activated partial thromboplastin times, similar to those obtained with samples from many other mammalian species. This was an apparent contradiction to an earlier report of long irreproducible activated partial thromboplastin times from owl monkey samples. The discrepant data could not be explained by differences in anticoagulants (citrate or oxalate), assay reagents (partial thromboplastin with either diatomaceous earth or ellagic acid), or activation incubation times (2, 5, or 10 minutes); nor could they be explained by differences in the monkeys' sex, age or previous experimental exposure to Plasmodium falciparum malaria.

  19. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    PubMed

    Parker, William H; Qu, Zhi-chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism.

  20. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

    PubMed Central

    Parker, William H.; Qu, Zhi-chao; May, James M.

    2015-01-01

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  1. [Thrombin generation assays and their clinical application].

    PubMed

    Kern, Anita; Várnai, Katalin; Vásárhelyi, Barna

    2014-06-01

    Thrombin is a key enzyme of the coagulation system, having both pro- and anticoagulant functions. Thus, the generation of thrombin is one of the most important steps in coagulation. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Since thrombin generation is sensible for both pro- and anticoagulant processes it can be applied for the general characterisation of the risk of thrombosis and bleeding, too. Clinical studies confirmed augmented thrombin generation in patients with high risk of venous or arterial thrombosis. Anticoagulant therapy (also novel oral anticoagulant treatment) can be monitored by thrombin generation. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off values are required before its application in clinical practice.

  2. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  3. Crystal Structure of Thrombin Bound to the Uncleaved Extracellular Fragment of PAR1

    SciTech Connect

    Gandhi, Prafull S.; Chen, Zhiwei; Di Cera, Enrico

    2010-05-11

    Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 {angstrom} resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp{sup 39}, does not hinder binding to the active site and actually makes favorable interactions with Gly{sup 219} of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.

  4. A critical role for thrombin in vertebrate lens regeneration.

    PubMed Central

    Imokawa, Yutaka; Simon, András; Brockes, Jeremy P

    2004-01-01

    Lens regeneration in urodele amphibians such as the newt proceeds from the dorsal margin of the iris where pigment epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. A general problem in regeneration research is to understand how the events of tissue injury or removal are coupled to the activation of plasticity in residual differentiated cells or stem cells. Thrombin, a pivotal regulator of the injury response, has been implicated as a regulator of cell cycle re-entry in newt myotubes, and also in newt iris PEC. After removal of the lens, thrombin was activated on the dorsal margin for 5-7 days. Inactivation of thrombin by either of two different inhibitors essentially blocked S-phase re-entry by PEC at this location. The axolotl, a related species which can regenerate its limb but not its lens, can activate thrombin after amputation but not after lens removal. These data support the hypothesis that thrombin is a critical signal linking injury to regeneration, and offer a new perspective on the evolutionary and phylogenetic questions about regeneration. PMID:15293804

  5. Evaluation of Potential Thrombin Inhibitors from the White Mangrove (Laguncularia racemosa (L.) C.F. Gaertn.)

    PubMed Central

    Rodrigues, Caroline Fabri Bittencourt; Gaeta, Henrique Hessel; Belchor, Mariana Novo; Ferreira, Marcelo José Pena; Pinho, Marcus Vinícius Terashima; de Oliveira Toyama, Daniela; Toyama, Marcos Hikari

    2015-01-01

    The aim of this work was to verify the effects of methanol (MeOH) and hydroalcoholic (HA) extracts and their respective partition phases obtained from white mangrove (Laguncularia racemosa (L.) C.F. Gaertn.) leaves on human thrombin activity. Among the extracts and phases tested, only the ethyl acetate and butanolic partitions significantly inhibited human thrombin activity and the coagulation of plasma in the presence of this enzyme. Chromatographic analyses of the thrombin samples incubated with these phases revealed that different compounds were able to interact with thrombin. The butanolic phase of the MeOH extract had the most potent inhibitory effects, reducing enzymatic activity and thrombin-induced plasma coagulation. Two glycosylated flavonoids in this partition were identified as the most potent inhibitors of human thrombin activity, namely quercetin-3-O-arabinoside (QAra) and quercetin-3-O-rhamnoside (Qn). Chromatographic analyses of thrombin samples incubated with these flavonoids demonstrated the chemical modification of this enzyme, suggesting that the MeOH extract contained other compounds that both induced structural changes in thrombin and diminished its activity. In this article, we show that despite the near absence of the medical use of mangrove compounds, this plant contains natural compounds with potential therapeutic applications. PMID:26197325

  6. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  7. New cyanopeptide-derived low molecular weight thrombin inhibitors.

    PubMed

    Radau, Gregor; Gebel, Jana; Rauh, Daniel

    2003-08-01

    Thrombosis is the result of defective regulation of the hemostasis system. This cardiovascular disorder may lead to deep vein thrombosis, myocardial infarction, and stroke. The majority of current drug research is focused on finding inhibitors of thrombin - the global player in hemostasis. In our work, we emphasize investigation of the marine environment to yield new lead structures from marine organisms like blue-green algae (cyanobacteria). This article deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (2) as a lead structure, we have developed and synthesized new, selective acting inhibitors of thrombin (RA-1001 and RA-1002), which are suitable targets for further structure-activity studies.

  8. Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C.

    PubMed

    McKenzie, F R; Seuwen, K; Pouysségur, J

    1992-11-15

    In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity

  9. Dynamics of Thrombin Generation and Flux from Clots during Whole Human Blood Flow over Collagen/Tissue Factor Surfaces.

    PubMed

    Zhu, Shu; Lu, Yichen; Sinno, Talid; Diamond, Scott L

    2016-10-28

    Coagulation kinetics are well established for purified blood proteases or human plasma clotting isotropically. However, less is known about thrombin generation kinetics and transport within blood clots formed under hemodynamic flow. Using microfluidic perfusion (wall shear rate, 200 s(-1)) of corn trypsin inhibitor-treated whole blood over a 250-μm long patch of type I fibrillar collagen/lipidated tissue factor (TF; ∼1 TF molecule/μm(2)), we measured thrombin released from clots using thrombin-antithrombin immunoassay. The majority (>85%) of generated thrombin was captured by intrathrombus fibrin as thrombin-antithrombin was largely undetectable in the effluent unless Gly-Pro-Arg-Pro (GPRP) was added to block fibrin polymerization. With GPRP present, the flux of thrombin increased to ∼0.5 × 10(-12) nmol/μm(2)-s over the first 500 s of perfusion and then further increased by ∼2-3-fold over the next 300 s. The increased thrombin flux after 500 s was blocked by anti-FXIa antibody (O1A6), consistent with thrombin-feedback activation of FXI. Over the first 500 s, ∼92,000 molecules of thrombin were generated per surface TF molecule for the 250-μm-long coating. A single layer of platelets (obtained with αIIbβ3 antagonism preventing continued platelet deposition) was largely sufficient for thrombin production. Also, the overall thrombin-generating potential of a 1000-μm-long coating became less efficient on a per μm(2) basis, likely due to distal boundary layer depletion of platelets. Overall, thrombin is robustly generated within clots by the extrinsic pathway followed by late-stage FXIa contributions, with fibrin localizing thrombin via its antithrombin-I activity as a potentially self-limiting hemostatic mechanism.

  10. Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

    PubMed

    Brailoiu, Eugen; Shipsky, Megan M; Yan, Guang; Abood, Mary E; Brailoiu, G Cristina

    2017-02-15

    Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca(2+) concentration in RBMVEC via Ca(2+) release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca(2+) influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

  11. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies.

    PubMed

    Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang

    2017-03-09

    Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

  12. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

    NASA Astrophysics Data System (ADS)

    Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang

    2017-03-01

    Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

  13. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

    PubMed Central

    Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang

    2017-01-01

    Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs. PMID:28276481

  14. Planar Hall magnetoresistive aptasensor for thrombin detection.

    PubMed

    Sinha, B; Ramulu, T S; Kim, K W; Venu, R; Lee, J J; Kim, C G

    2014-09-15

    The use of aptamer-based assays is an emerging and attractive approach in disease research and clinical diagnostics. A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using a planar Hall magnetoresistive (PHR) sensor in cooperation with superparamagnetic labels. A PHR sensor has the great advantages of a high signal-to-noise ratio, a small offset voltage and linear response in the low-field region, allowing it to act as a high-resolution biosensor. In the system presented here, the sensor has an active area of 50 µm × 50 µm with a 10-nm gold layer deposited onto the sensor surface prior to the binding of thiolated DNA primary aptamer. A polydimethylsiloxane well of 600-µm radius and 1-mm height was prepared around the sensor surface to maintain the same specific area and volume for each sensor. The sensor response was traced in real time upon the addition of streptavidin-functionalized magnetic labels on the sensor. A linear response to the thrombin concentration in the range of 86 pM-8.6 µM and a lower detection limit down to 86 pM was achieved by the proposed present method with a sample volume consumption of 2 µl. The proposed aptasensor has a strong potential for application in clinical diagnosis.

  15. NMR reveals a dynamic allosteric pathway in thrombin

    PubMed Central

    Handley, Lindsey D.; Fuglestad, Brian; Stearns, Kyle; Tonelli, Marco; Fenwick, R. Bryn; Markwick, Phineus R. L.; Komives, Elizabeth A.

    2017-01-01

    Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R1, R2, 15N-{1H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with μs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na+-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain μs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form. PMID:28059082

  16. Thrombin and exercise similarly influence expression of cell cycle genes in cultured putative endothelial progenitor cells.

    PubMed

    Lockard, Michael M; Witkowski, Sarah; Jenkins, Nathan T; Spangenburg, Espen E; Obisesan, Thomas O; Hagberg, James M

    2010-06-01

    Acute exercise and exercise training may influence putative endothelial progenitor cell (EPC) number and colony forming units (CFU-ECs), although the mechanisms remain unclear. This study examined the effects of in vitro thrombin supplementation and acute exercise on CFU-EC gene expression, associated with cellular proliferation and differentiation. The effect of habitual physical activity was evaluated through analysis of EPCs from chronically high- and low-active men. Participants were healthy high- and low-active men (n=23), aged 55-80 yr. Circulating CD34+/VEGFR2+ number, CFU-ECs, plasma prothrombin fragment (F1+2), and thrombin-antithrombin III were measured at rest and after 30 min of exercise. Gene expression of cyclin A2, cyclin D1, p27, VE-cadherin, and VEGFR2 was assessed in postexercise CFU-ECs and resting CFU-ECs treated with 0, 1, 5, or 10 U/ml of thrombin. Outcomes were compared between high- and low-active participants. F1+2 and thrombin-antithrombin III, but not CD34+/VEGFR2+ number and CFU-ECs, increased with exercise. Exercise-induced changes in F1+2 correlated with changes in CD34+/VEGFR2+ number in both groups. Thrombin treatments and acute exercise increased cyclin A2 and cyclin D1 expression and decreased p27 expression. One unit per milliliter thrombin increased VEGFR2 and VE-cadherin expression, whereas 5 U/ml, 10 U/ml, and acute exercise did not elicit any changes. An exercise training effect was observed with greater decreases in p27 expression with 5 and 10 U/ml thrombin and greater increases in VEGFR2 and VE-cadherin expression with 1 U/ml thrombin in high-active men. Exercise-induced changes in putative EPC gene expression are associated with thrombin production and may be modulated by long-term exercise training.

  17. Thrombin and exercise similarly influence expression of cell cycle genes in cultured putative endothelial progenitor cells

    PubMed Central

    Lockard, Michael M.; Witkowski, Sarah; Jenkins, Nathan T.; Spangenburg, Espen E.; Obisesan, Thomas O.

    2010-01-01

    Acute exercise and exercise training may influence putative endothelial progenitor cell (EPC) number and colony forming units (CFU-ECs), although the mechanisms remain unclear. This study examined the effects of in vitro thrombin supplementation and acute exercise on CFU-EC gene expression, associated with cellular proliferation and differentiation. The effect of habitual physical activity was evaluated through analysis of EPCs from chronically high- and low-active men. Participants were healthy high- and low-active men (n = 23), aged 55–80 yr. Circulating CD34+/VEGFR2+ number, CFU-ECs, plasma prothrombin fragment (F1+2), and thrombin-antithrombin III were measured at rest and after 30 min of exercise. Gene expression of cyclin A2, cyclin D1, p27, VE-cadherin, and VEGFR2 was assessed in postexercise CFU-ECs and resting CFU-ECs treated with 0, 1, 5, or 10 U/ml of thrombin. Outcomes were compared between high- and low-active participants. F1+2 and thrombin-antithrombin III, but not CD34+/VEGFR2+ number and CFU-ECs, increased with exercise. Exercise-induced changes in F1+2 correlated with changes in CD34+/VEGFR2+ number in both groups. Thrombin treatments and acute exercise increased cyclin A2 and cyclin D1 expression and decreased p27 expression. One unit per milliliter thrombin increased VEGFR2 and VE-cadherin expression, whereas 5 U/ml, 10 U/ml, and acute exercise did not elicit any changes. An exercise training effect was observed with greater decreases in p27 expression with 5 and 10 U/ml thrombin and greater increases in VEGFR2 and VE-cadherin expression with 1 U/ml thrombin in high-active men. Exercise-induced changes in putative EPC gene expression are associated with thrombin production and may be modulated by long-term exercise training. PMID:20378705

  18. PACAP38 protects rat cortical neurons against the neurotoxicity evoked by sodium nitroprusside and thrombin

    PubMed Central

    Sanchez, Alma; Rao, Haripriya Vittal; Grammas, Paula

    2009-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 is a multifunctional anti-inflammatory and anti-apoptotic neuropeptide widely distributed in the nervous system. The objective of this study is to determine whether PACAP38 is neuroprotective against sodium nitroprusside (SNP) and thrombin, two mechanistically distinct neurotoxic agents. Treatment of primary cortical neuronal cultures with 1 mM SNP for 4 h causes neuronal cell death that is significantly reduced by 100 nM PACAP38. PACAP38 down-regulates SNP-induced cell cycle protein (cyclin E) expression and up-regulates p57KIP2, a cyclin-dependent kinase inhibitor as well as the anti-apoptotic protein Bcl-2. Similarly, neuronal death induced by 100 nM thrombin or the thrombin receptor activating peptide (TRAP 6) is reduced by PACAP38 treatment. Thrombin-stimulated cell cycle protein (cdk4) expression is decreased by PACAP38 while PACAP38 inhibits thrombin-mediated reduction of p57KIP2. However, the decrease in Bcl-2 evoked by thrombin is not affected by PACAP38. Finally, both SNP and thrombin (or TRAP) increase caspase 3 activity, an effect that is decreased by PACAP38. These data show that PACAP38 supports neuronal survival in vitro suppressing cell cycle progression and enhancing anti-apoptotic proteins. Our results support the possibility that PACAP could be a useful therapeutic agent for reducing neuronal cell death in neurodegenerative diseases. PMID:18682263

  19. Thrombin selectively induces transcription of genes in human monocytes involved in inflammation and wound healing.

    PubMed

    López, Mercedes L; Bruges, Gustavo; Crespo, Gustavo; Salazar, Victor; Deglesne, Pierre-Antoine; Schneider, Heike; Cabrera-Fuentes, Hector; Schmitz, M Lienhard; Preissner, Klaus T

    2014-11-01

    Thrombin is essential for blood coagulation but functions also as a mediator of cellular signalling. Gene expression microarray experiments in human monocytes revealed thrombin-induced upregulation of a limited subset of genes, which are almost exclusively involved in inflammation and wound healing. Among these, the expression of F3 gene encoding for tissue factor (TF) was enhanced indicating that this physiological initiator of coagulation cascade may create a feed-forward loop to enhance blood coagulation. Activation of protease-activated receptor type 1 (PAR1) was shown to play a main role in promoting TF expression. Moreover, thrombin induced phosphorylation of ERK1/2, an event that is required for expression of thrombin-regulated genes. Thrombin also increased the expression of TF at the protein level in monocytes as evidenced by Western blot and immunostaining. Furthermore, FXa generation induced by thrombin-stimulated monocytes was abolished by a TF blocking antibody and therefore it is entirely attributable to the expression of tissue factor. This cellular activity of thrombin provides a new molecular link between coagulation, inflammation and wound healing.

  20. [Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay].

    PubMed

    Martinuzzo, Marta E; Duboscq, Cristina; Viñuales, Estela S; Girardi, Beatriz; Penchasky, Diana; Ceresetto, José; Stemmelin, Germán; Otero, Victoria; Barrera, Luis H; López, Marina S; Otaso, Juan C; Hoyhamburu, José

    2017-01-01

    Dabigatran etexilate (direct thrombin inhibitor) is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc), correlate cc with classic coagulation assays, prothrombin time (PT) and activated partial thromboplastin time (APTT), and evaluate them according to the creatinine clearance (CLCr). Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation) receiving dabigatran at 150 (n = 19) or 110 (n = 21) mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL). PT and APTT (IL) were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL). DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients' cc: median 83 (4-945) ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min) showed significantly higher median cc: 308 (49-945), compared to the average 72 (12-190) and highest tertile, 60 (4-118) ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies.

  1. STIM1 and Orai1 mediate thrombin-induced Ca(2+) influx in rat cortical astrocytes.

    PubMed

    Moreno, Claudia; Sampieri, Alicia; Vivas, Oscar; Peña-Segura, Claudia; Vaca, Luis

    2012-12-01

    In astrocytes, thrombin leads to cytoplasmic Ca(2+) elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca(2+) increase generated by an interplay between ER-Ca(2+) release and store-operated Ca(2+) entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca(2+) depletion and binds Orai1 to activate Ca(2+) influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca(2+) response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca(2+) influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca(2+) influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca(2+) response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca(2+) release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca(2+) influx.

  2. Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates.

    PubMed Central

    Hosokawa, K; Ohnishi, T; Shima, M; Nagata, M; Koide, T

    2001-01-01

    Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes. PMID:11171108

  3. Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

    NASA Astrophysics Data System (ADS)

    Wheatley Myerson, Jacob; He, Li; Allen, John Stacy; Williams, Todd; Lanza, Gregory; Tollefsen, Douglas; Caruthers, Shelton; Wickline, Samuel

    2014-09-01

    Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (˜10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

  4. Thrombin Induces Inositol Trisphosphate-Mediated Spatially Extensive Responses in Lung Microvessels.

    PubMed

    Escue, Rachel; Kandasamy, Kathirvel; Parthasarathi, Kaushik

    2017-04-01

    Activation of plasma membrane receptors initiates compartmentalized second messenger signaling. Whether this compartmentalization facilitates the preferential intercellular diffusion of specific second messengers is unclear. Toward this, the receptor-mediated agonist, thrombin, was instilled into microvessels in a restricted region of isolated blood-perfused mouse lungs. Subsequently, the thrombin-induced increase in endothelial F-actin was determined using confocal fluorescence microscopy. Increased F-actin was evident in microvessels directly treated with thrombin and in those located in adjoining thrombin-free regions. This increase was abrogated by inhibiting inositol trisphosphate-mediated calcium release with Xestospongin C (XeC). XeC also inhibited the thrombin-induced increase in the amplitude of endothelial cytosolic Ca(2+) oscillations. Instillation of thrombin and XeC into adjacent restricted regions increased F-actin in microvessels in the thrombin-treated and adjacent regions but not in those in the XeC-treated region. Thus, inositol trisphosphate, and not calcium, diffused interendothelially to the spatially remote thrombin-free microvessels. Thus, activation of plasma membrane receptors increased the ambit of inflammatory responses via a second messenger different from that used by stimuli that induce cell-wide increases in second messengers. Thrombin however failed to induce the spatially extensive response in microvessels of mice lacking endothelial connexin43, suggesting a role for connexin43 gap junctions. Compartmental second messenger signaling and interendothelial communication define the specific second messenger involved in exacerbating proinflammatory responses to receptor-mediated agonists.

  5. Evaluation of Thrombin Generation Assay in Patients With Hemophilia.

    PubMed

    Haghpanah, Sezaneh; Bazrafshan, Asghar; Silavizadeh, Samir; Dehghani, Javad; Afrasiabi, Abdolreza; Karimi, Mehran

    2016-05-01

    We evaluated the correlation between thrombin generation (TG) parameters with bleeding symptoms and disease severity in patients with hemophilia. In this cross-sectional study, 59 patients with hemophilia without inhibitors and regardless of their severity were randomly selected from southern Iran and TG assays were conducted. Bleeding score (BS) was calculated by performing a clinical evaluation using Tosetto questionnaire. Only lag time showed a statistically significant correlation with BS (rs = .316,P= .016). All TG parameters except peak showed association with disease severity (P< .05). Endogenous thrombin potential showed a significant correlation with factor activity level (rs = .459,P< .001). Both lag time and start tail showed significant negative correlations with factor activity level (rs = -0.488,P< .001 andrs = - .289,P< .026, respectively). Although most of the TG parameters evaluated were not significantly correlated with the BS of patients with hemophilia, the majority of TG parameters were significantly associated with factor activity level and disease severity.

  6. Monitoring Heparin Therapy with the Activated Partial Thromboplastin Time

    PubMed Central

    Stuart, R. K.; Michel, A.

    1971-01-01

    Difficulties associated with the whole blood clotting time (W.B.C.T.) as a method of monitoring heparin therapy have led to the investigation of the activated partial thromboplastin time (A.P.T.T.) as an alternative. The conclusion is reached that the latter procedure possesses several advantages. Using the method described and a citrate-preserved blood sample collected just prior to the administration of the next serial dose of heparin, the suggested therapeutic duration of the A.P.T.T. is 70 seconds or twice the mean control value. A practical range for this method is 60 to 70 seconds. PMID:5557913

  7. PAF mediates neutrophil adhesion to thrombin or TNF-stimulated endothelial cells under shear stress.

    PubMed

    Macconi, D; Foppolo, M; Paris, S; Noris, M; Aiello, S; Remuzzi, G; Remuzzi, A

    1995-07-01

    Platelet-activating factor (PAF) is known to modulate polymorphonuclear leukocyte (PMN) adhesion to endothelial cells cultured under static conditions and activated by thrombin. In contrast, there are no data on the role of PAF in PMN adhesion to cells exposed to flow conditions and activated by stimuli other than thrombin. Here we used the PAF receptor antagonist L-659,989 to evaluate PMN adhesion to human umbilical vein endothelial cells (HUVEC) in basal conditions or upon challenge with thrombin or tumor necrosis factor-alpha (TNF-alpha). Experiments were performed under dynamic flow using a parallel-plate flow chamber and a computer-based image analysis system. Rolling and adhesion of PMNs to endothelial cells significantly increased upon stimulation with thrombin. Thrombin-stimulated HUVEC also synthesized higher amounts of PAF than untreated cells. Pretreatment of PMNs with L-659,989 significantly reduced their rolling and adhesion to thrombin-activated HUVEC. Stimulation of HUVEC with TNF-alpha significantly increased the number of rolling and adherent PMNs as compared with untreated cells. Adhesion of PMNs to and migration across TNF-alpha-stimulated HUVEC were reduced by L-659,989, whereas cell rolling was unchanged. We conclude that PAF mediates leukocyte interaction under flow conditions with HUVEC activated by inflammatory stimuli.

  8. Thrombin modulates persistent sodium current in CA1 pyramidal neurons of young and adult rat hippocampus.

    PubMed

    Lunko, O O; Isaev, D S; Krishtal, O O; Isaeva, E V

    2015-01-01

    Serine protease thrombin, a key factor of blood coagulation, participates in many neuronal processes important for normal brain functioning and during pathological conditions involving abnormal neuronal synchronization, neurodegeneration and inflammation. Our previous study on CA3 pyramidal neurons showed that application ofthrombin through the activation of specific protease-activated receptor 1 (PAR1) produces a significant hyperpolarizing shift of the activation of the TTX-sensitive persistent voltage-gated Na+ current (I(Nap)) thereby affecting membrane potential and seizure threshold at the network level. It was shown that PAR1 is also expressed in CA1 area of hippocampus and can be implicated in neuronal damage in this area after status epilepticus. The aim of the present study was to evaluate the effect of thrombin on I(NaP) in CA1 pyramidal neurons from adult and young rats. Using whole cell patch-clamp technique we demonstrate that thrombin application results in the hyperpolarization shift of I(NaP) activation as well as increase in the I(NaP) amplitude in both age groups. We have found that I(NaP) in pyramidal neurons of hippocampal CA 1 region is more vulnerable to the thrombin action than I(NaP) in pyramidal neurons of hippocampal CA3 region. We have also found that the immature hippocampus is more sensitive to thrombin action which emphasizes the contribution of thrombin-dependent pathway to the regulation of neuronal activity in immature brain.

  9. PAR1-dependent and independent increases in COX-2 and PGE2 in human colonic myofibroblasts stimulated by thrombin.

    PubMed

    Seymour, Michelle L; Zaidi, Nosheen F; Hollenberg, Morley D; MacNaughton, Wallace K

    2003-05-01

    Subepithelial myofibroblast-derived prostaglandin E(2) (PGE(2)) regulates epithelial chloride secretion in the intestine. Thrombin is elevated in inflammatory conditions of the bowel. Therefore, we sought to determine a role for thrombin in regulating PGE(2) synthesis by colonic myofibroblasts. Incubation of cultured CCD-18Co colonic myofibroblasts with thrombin, the proteinase-activated receptor 1 (PAR(1))-activating peptide (Cit-NH(2)), and peptides corresponding to 2 noncatalytic regions of thrombin (TP367 and TP508) for 18 h increased both cyclooxygenase (COX)-2 expression (immunocytochemistry) and PGE(2) synthesis (enzyme immunoassay). Inhibition of thrombin by D-Phe-Pro-Arg-chloromethylketone (PPACK) did not significantly reduce PGE(2) synthesis, which remained elevated compared with control. We also investigated the basic fibroblast growth factor (bFGF) dependence of thrombin-induced PGE(2) elevations. Recombinant human bFGF concentration dependently increased PGE(2) synthesis, and a bFGF neutralizing antibody inhibited PGE(2) synthesis induced by TP367 and TP508 (approximately 40%) and by thrombin (approximately 20%) (but not Cit-NH(2)). Thrombin, therefore, upregulates COX-2-derived PGE(2) synthesis by both catalytic cleavage of PAR(1) and bFGF-dependent noncatalytic activity. This presents a novel mechanism by which intestinal myofibroblasts might regulate epithelial chloride secretion.

  10. Kaolin-correctable prolongation of the activated partial thromboplastin time.

    PubMed

    Briselli, M F; Ellman, L

    1980-11-01

    Seven patients who had normal prothrombin times but prolonged activated partial thromboplastin times (aPTT) are described. The prolonged aPTT, obtained with micronized silica as the contact activating agent in a semi-automated optical end-point system, a nonautomated optical end-point system, and a conductivity end-point system, corrected to normal when kaolin was used as the contact activating agent. Abnormal results were also obtained with celite and ellagic acid as contact activating agents. The activities of various clotting factors were within normal limits in all cases where they were assayed. The thromboplastin dilution test was uniformly negative, and mixtures of one patient's plasma with that of another patient failed to correct the abnormal aPTT. No patients had a personal or family history of bleeding, and all underwent surgery without bleeding difficulties. This pattern of a prolonged aPTT that corrects to normal when kaolin is used as the contact activator appears to represent a previously unrecognized laboratory phenomenon.

  11. Reversal of trauma-induced amnesia in mice by a thrombin receptor antagonist.

    PubMed

    Itzekson, Zeev; Maggio, Nicola; Milman, Anat; Shavit, Efrat; Pick, Chaim G; Chapman, Joab

    2014-05-01

    Minimal traumatic brain injury (mTBI) is associated with the existence of retrograde amnesia and microscopic bleeds containing activated coagulation factors. In an mTBI model, we report that thrombin induces amnesia through its receptor protease-activated receptor 1 (PAR-1). Thrombin activity was significantly elevated (32 %, p < 0.05) 5 min following mTBI compared to controls. Amnesia was assessed by the novel object recognition test in mTBI animals and in animals injected intracerebroventricularly (ICV) with either thrombin or a PAR-1 agonist 1 h after the acquisition phase. Saline-injected controls had a preference index of over 0.3 while mTBI animals and those injected with thrombin or the PAR-1 agonist spent equal time with both objects indicating no recall of the object presented to them 24 h previously (p < 0.05). Co-injecting a PAR-1 antagonist (SCH79797) completely blocked the amnestic effects of mTBI, thrombin, and the PAR-1 agonist. Long-term potentiation, measured in hippocampal slices 24 h after mTBI, ICV thrombin or the PAR-1 agonist, was significantly impaired and this effect was completely reversed by the PAR-1 antagonist. The results support a crucial role for PAR-1 in the generation of amnesia following mTBI, revealing a novel therapeutic target for the cognitive effects of brain trauma.

  12. Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV.

    PubMed

    Karle, Michael; Knecht, Wolfgang; Xue, Yafeng

    2012-07-15

    In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.

  13. Treatment of accidental perianal injection of topical thrombin with intravenous antithrombin.

    PubMed

    Nielsen, Vance G; Paidy, Samata R; McLeod, Whitney; Fox, Alexandra; Nfonsam, Valentine N

    2017-04-01

    While topical thrombin application can markedly improve surgical hemostasis, rapid absorption of thrombin can result in pulmonary embolism and death. We report a case of accidental interstitial infiltration of topical thrombin after hemorrhoidectomy that was treated with administration of human antithrombin and heparin anticoagulation. Except for a marked decrease in antithrombin activity from super normal to normal values, the patient exhibited no laboratory or clinical signs of pulmonary embolism, thrombin mediated consumptive loss of procoagulants, or regional thrombosis. The patient had an uncomplicated recovery without sign of thrombotic morbidity. While it is hoped that such a medical misadventure should not occur, our case may serve as a reference to guide anticoagulant therapy if such a clinical scenario arises.

  14. Partial purification of benzene hydroxylase activity from rat liver mitoplasts

    SciTech Connect

    Karaszkiewicz, J.W.; Snyder, R.; Kalf, G.F.

    1986-05-01

    Benzene is a hemopoietic toxin and a carcinogen which causes aplastic anemia and leukemia in humans and leukemia and certain solid tumors in rodents. Bioactivation of benzene is required for toxicity and, presumably, carcinogenicity. The authors have demonstrated that rat liver mitochondria, stripped of outer membrane to avoid microsomal contamination (mitoplasts), metabolize benzene in an NADPH-dependent reaction to compounds capable of covalently binding to mitochondrial DNA. They report here on the partial purification of a benzene hydroxylase activity from rat liver mitoplasts which converts benzene to phenol and which appears to be a cytochrome P-450. The activity is solubilized with 0.4% sodium cholate and further purified by a 5-15% polyethylene glycol (PEG) fractionation. The production of (/sup 3/H)phenol from (/sup 3/H)benzene by the 5-15% PEG fraction requires an NADPH-generating system and is completely dependent on the addition of exogenous bovine adrenodoxin. Although benzene hydroxylase activity has been demonstrated in rat liver microsomes, adrenodoxin has no effect on the activity of microsomal cytochromes P-450, providing evidence that our activity is indeed of mitochondrial origin.

  15. Artificial neural networks from MATLAB in medicinal chemistry. Bayesian-regularized genetic neural networks (BRGNN): application to the prediction of the antagonistic activity against human platelet thrombin receptor (PAR-1).

    PubMed

    Caballero, Julio; Fernández, Michael

    2008-01-01

    Artificial neural networks (ANNs) have been widely used for medicinal chemistry modeling. In the last two decades, too many reports used MATLAB environment as an adequate platform for programming ANNs. Some of these reports comprise a variety of applications intended to quantitatively or qualitatively describe structure-activity relationships. A powerful tool is obtained when there are combined Bayesian-regularized neural networks (BRANNs) and genetic algorithm (GA): Bayesian-regularized genetic neural networks (BRGNNs). BRGNNs can model complicated relationships between explanatory variables and dependent variables. Thus, this methodology is regarded as useful tool for QSAR analysis. In order to demonstrate the use of BRGNNs, we developed a reliable method for predicting the antagonistic activity of 5-amino-3-arylisoxazole derivatives against Human Platelet Thrombin Receptor (PAR-1), using classical 3D-QSAR methodologies: Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA). In addition, 3D vectors generated from the molecular structures were correlated with antagonistic activities by multivariate linear regression (MLR) and Bayesian-regularized neural networks (BRGNNs). All models were trained with 34 compounds, after which they were evaluated for predictive ability with additional 6 compounds. CoMFA and CoMSIA were unable to describe this structure-activity relationship, while BRGNN methodology brings the best results according to validation statistics.

  16. Thrombin Generation in Acute Ischaemic Stroke

    PubMed Central

    Roberts, Lara N.; Patel, Raj; Pathansali, Rohan; Kalra, Lalit; Arya, Roopen

    2016-01-01

    Introduction. Stroke remains a global leading cause of death and disability. Traditional description of plasma biology in the aftermath of acute ischaemic stroke favours development of hypercoagulability, resulting from complex interplay between plasma and endothelial factors. However, no single assay measures the overall global coagulation process. We postulate that thrombin generation would assist in identifying coagulation abnormalities after acute stroke. Aim. To investigate the coagulation abnormalities after acute ischaemic stroke using thrombin generation. Methods. We evaluated thrombin generation, measured with calibrated automated thrombography in stroke of different aetiological types (n = 170) within 48 hours of symptoms onset (baseline) and in the second week (time 2) and in normal healthy volunteers (n = 71). Results. Two-point thrombin generation assays showed prolonged lag time and time to peak at baseline (3.3 (2.9, 4.0) versus 3.6 (3.2, 4.7); p = 0.005) and (3.3 (2.9, 4.0) versus 3.6 (3.2, 4.7); p = 0.002), respectively, and at time 2 (3.5 (2.9, 4.2) versus 4.0 (3.1, 4.9); p = 0.004) and (5.9 (5.3, 6.6) versus 6.8 (5.8, 7.7) p = 0.05), respectively, in cardioembolic stroke (n = 39), when compared to noncardioembolic stroke (n = 117). The result was reproduced in multiple comparisons between acute ischaemic stroke subgroups and normal healthy volunteers. Endogenous thrombin potential and peak thrombin did not indicate hypercoagulability after acute ischaemic stroke, and thrombolytic therapy did not affect thrombin generation assays. Conclusion. Our findings suggest that thrombin generation in platelet poor plasma is not useful in defining hypercoagulability in acute ischaemic stroke. This is similar to observed trend in coronary artery disease and contrary to other hypercoagulable states. PMID:28116215

  17. Tissue factor-driven thrombin generation and inflammation in atherosclerosis.

    PubMed

    ten Cate, Hugo

    2012-05-01

    The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

  18. Unimolecular, soluble semiconductor nanoparticle-based biosensors for thrombin using charge/electron transfer.

    PubMed

    Swain, Marla D; Octain, Jashain; Benson, David E

    2008-12-01

    Duplex DNA was attached to semiconductor nanoparticles providing selective detection of thrombin. Using the method reported here, semiconductor nanoparticles can have selective sensory functions for a host of additional analytes in the future. The system uses one DNA strand that selectively binds an analyte (thrombin), while the complementary DNA strand contains a redox-active metal complex. The accessibility of the metal complex to the nanoparticle surface is increased upon thrombin binding due to unravelling of the duplex DNA secondary structure. Increased interactions between the metal complex and the nanoparticle surface will decrease nanoparticle emission intensity, through charge transfer. Initially, water-soluble nanoparticles with carboxylate-terminated monolayers showed thrombin-specific responses in emission intensity (-30% for 1:1 nanoparticle to DNA, +50% for 1:5). Despite the selective responses, the thrombin binding isotherms indicated multiple binding equilibria and more than likely nanoparticle aggregation. The need for a nonaggregative system comes from the potential employment of these sensors in live cell or living system fluorescence assays. By changing the nanoparticle capping ligand to provide an ethylene glycol-terminated monolayer, the binding isotherms fit a two-state binding model with a thrombin dissociation constant of 3 nM in a physiologically relevant buffer. This article demonstrates the need to consider capping ligand effects in designing biosensors based on semiconductor nanoparticles and demonstrates an initial DNA-attached semiconductor nanoparticle system that uses DNA-analyte binding interactions (aptamers).

  19. Comparison of thrombin generation assay with conventional coagulation tests in evaluation of bleeding risk in patients with rare bleeding disorders.

    PubMed

    Zekavat, Omid R; Haghpanah, Sezaneh; Dehghani, Javad; Afrasiabi, Abdolreza; Peyvandi, Flora; Karimi, Mehran

    2014-09-01

    Based on the premise that the capacity of plasma to generate thrombin in vitro is a comprehensive and precise functional test of the clotting system, we designed a cross-sectional, single-center study involving 83 patients with rare bleeding disorders (RBDs) to compare the usefulness of the thrombin generation (TG) assay versus conventional tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT) in predicting bleeding risk in patients with RBD in southern Iran. The TG parameters consisted of endogenous thrombin potential, lag time, peak, time to peak (ttPeak), and start tail. The area under the receiver-operating characteristic (ROC) curve showed statistically significant associations between bleeding risk and lag time, ttPeak, and start tail. We determined cutoff values for these 3 TG parameters and obtained a negative predictive value of 86% to 90% in patients with RBD who had a bleeding score (BS) ≤13. The ROC curves for the association of PT and aPTT with BS did not indicate any significant association. Correlation analysis supported the results of ROC curve analysis, only lag time, ttPeak, and start tail showed significant positive correlations with BS (P < .05). Disease severity based on plasma factor activity was significantly associated with prolonged lag time and ttPeak and with prolonged PT (P <.05). We suggest that TG assay is a potentially more useful tool for predicting the bleeding risk in patients with RBD. However, the small sample size in different RBD subgroups precluded subgroup analysis. Prospective multicenter studies with larger numbers of patients are therefore advisable.

  20. Thrombin induces increased expression and secretion of VEGF from human FS4 fibroblasts, DU145 prostate cells and CHRF megakaryocytes.

    PubMed

    Huang, Y Q; Li, J J; Hu, L; Lee, M; Karpatkin, S

    2001-10-01

    Angiogenesis is required for tumor growth and metastasis. It has recently been suggested that thrombin is a potent promoter of angiogenesis. We therefore examined the possibility that thrombin could be inducing the expression of vascular endothelial growth factor (VEGF), which promotes endothelial growth. Primary human FS4 fibroblasts as well as tumor cell lines: prostate DU145 and megakaryocyte CHRF were incubated with thrombin (0.25-1 unit/ml) for 1-8 hrs and then examined for mRNA by Northern Analysis. Enhanced mRNA (approximately 3-4 fold over base line) was noted at 2-4 hrs, with 0.5 u/ml thrombin. The effect was specific for thrombin activity on its PAR-1 receptor, since equal units of hirudin completely inhibited the response and the thrombin effect could be mimicked with the 14 mer thrombin receptor activation peptide (TRAP). Upregulation of mRNA was associated with enhanced VEGF protein synthesis and secretion as assayed by immunoblot. Enhanced expression of VEGF mRNA was not secondary to enhanced transcription (nuclear run on experiments), but due to an >3 fold stabilization of mRNA (Actinomycin D chase experiment). Enhanced VEGF mRNA stabilization is promoted by the PI3Kinase and serine/threonine kinase pathways, since thrombin-induced mRNA expression is inhibited by Wortmanin and H7. No effect was noted with the MAPKinase inhibitor, PD98059. Thus, thrombin-induced tumorigenesis and metastasis is associated with enhanced VEGF protein synthesis and secretion via the stabilization of VEGF mRNA promoted by the PI3Kinase and serine/threonine kinase pathways. This could help explain how thrombin promotes angiogenesis.

  1. Thrombin inhibition with dabigatran protects against high-fat diet-induced fatty liver disease in mice.

    PubMed

    Kopec, Anna K; Joshi, Nikita; Towery, Keara L; Kassel, Karen M; Sullivan, Bradley P; Flick, Matthew J; Luyendyk, James P

    2014-11-01

    Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies.

  2. Haemophilus ducreyi partially activates human myeloid dendritic cells.

    PubMed

    Banks, Keith E; Humphreys, Tricia L; Li, Wei; Katz, Barry P; Wilkes, David S; Spinola, Stanley M

    2007-12-01

    Dendritic cells (DC) orchestrate innate and adaptive immune responses to bacteria. How Haemophilus ducreyi, which causes genital ulcers and regional lymphadenitis, interacts with DC is unknown. H. ducreyi evades uptake by polymorphonuclear leukocyte and macrophage-like cell lines by secreting LspA1 and LspA2. Many H. ducreyi strains express cytolethal distending toxin (CDT), and recombinant CDT causes apoptosis of DC in vitro. Here, we examined interactions between DC and H. ducreyi 35000HP, which produces LspA1, LspA2, and CDT. In human volunteers infected with 35000HP, the ratio of myeloid DC to plasmacytoid DC was 2.8:1 in lesions, compared to a ratio of 1:1 in peripheral blood. Using myeloid DC derived from monocytes as surrogates for lesional DC, we found that DC infected with 35000HP remained as viable as uninfected DC for up to 48 h. Gentamicin protection and confocal microscopy assays demonstrated that DC ingested and killed 35000HP, but killing was incomplete at 48 h. The expression of LspA1 and LspA2 did not inhibit the uptake of H. ducreyi, despite inactivating Src kinases. Infection of DC with live 35000HP caused less cell surface marker activation than infection with heat-killed 35000HP and lipopolysaccharide (LPS) and inhibited maturation by LPS. However, infection of DC with live bacteria caused the secretion of significantly higher levels of interleukin-6 and tumor necrosis factor alpha than infection with heat-killed bacteria and LPS. The survival of H. ducreyi in DC may provide a mechanism by which the organism traffics to lymph nodes. Partial activation of DC may abrogate the establishment of a full Th1 response and an environment that promotes phagocytosis.

  3. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

  4. Salmon-derived thrombin inhibits development of chronic pain through an endothelial barrier protective mechanism dependent on APC

    PubMed Central

    Smith, Jenell R; Galie, Peter A; Slochower, David R; Weisshaar, Christine L.; Janmey, Paul A; Winkelstein, Beth A

    2015-01-01

    Many neurological disorders are initiated by blood-brain barrier breakdown, which potentiates spinal neuroinflammation and neurodegeneration. Peripheral neuropathic injuries are known to disrupt the blood-spinal cord barrier (BSCB) and to potentiate inflammation. But, it is not known whether BSCB breakdown facilitates pain development. In this study, a neural compression model in the rat was used to evaluate relationships among BSCB permeability, inflammation and pain-related behaviors. BSCB permeability increases transiently only after injury that induces mechanical hyperalgesia, which correlates with serum concentrations of pro-inflammatory cytokines, IL-7, IL-12, IL-1α and TNF-α. Mammalian thrombin dually regulates vascular permeability through PAR1 and activated protein C (APC). Since thrombin protects vascular integrity through APC, directing its affinity towards protein C, while still promoting coagulation, might be an ideal treatment for BSCB-disrupting disorders. Salmon thrombin, which prevents the development of mechanical allodynia, also prevents BSCB breakdown after neural injury and actively inhibits TNF-α-induced endothelial permeability in vitro, which is not evident the case for human thrombin. Salmon thrombin’s production of APC faster than human thrombin is confirmed using a fluorogenic assay and APC is shown to inhibit BSCB breakdown and pain-related behaviors similar to salmon thrombin. Together, these studies highlight the impact of BSCB on pain and establish salmon thrombin as an effective blocker of BSCB, and resulting nociception, through its preferential affinity for protein C. PMID:26708087

  5. Effects of Aerobic Capacity on Thrombin-Induced Hydrocephalus and White Matter Injury.

    PubMed

    Ni, Wei; Gao, Feng; Zheng, Mingzhe; Koch, Lauren G; Britton, Steven L; Keep, Richard F; Xi, Guohua; Hua, Ya

    2016-01-01

    We have previously shown that intracerebral hemorrhage-induced brain injury is less in rats bred for high aerobic capacity (high capacity runners; HCR) compared with those bred for low aerobic capacity (low capacity runners; LCRs). Thrombin, an essential component in the coagulation cascade, is produced after cerebral hemorrhage. Intraventricular injection of thrombin causes significant hydrocephalus and white matter damage. In the present study, we examined the effect of exercise capacity on thrombin-induced hydrocephalus and white matter damage. Mid-aged (13-month-old) female LCRs (n = 13) and HCRs (n = 12) rats were used in this study. Rats received an intraventricular injection of thrombin (3 U, 50 μl). All rats underwent magnetic resonance imaging (MRI) at 24 h and were then euthanized for brain histology and Western blot. The mortalities were 20 % in LCRs and 33 % in HCRs after thrombin injection (p > 0.05). No rats died after saline injection. Intraventricular thrombin injection resulted in hydrocephalus and periventricular white matter damage as determined on MRI. In LCR rats, thrombin induced significant ventricle enlargement (23.0 ± 2.3 vs12.8 ± 1.9 mm(3) in LCR saline group; p < 0.01) and white matter lesion (9.3 ± 7.6 vs 0.6 ± 0.5 mm(3) in LCR saline group, p < 0.05). In comparison, in HCR rats thrombin induced less ventricular enlargement (17.3 ± 3.9 vs 23.0 ± 2.3 mm(3) in LCRs, p < 0.01) and smaller white matter lesions (2.6 ± 1.2 mm(3) vs 9.3 ± 7.6 mm(3) in LCRs, p < 0.05). In LCR rats, there was also upregulation of heat shock protein-32, a stress marker, and microglial activation in the periventricular white matter. These changes were significantly reduced in HCR rats. Intraventricular injection of thrombin caused more white matter damage and hydrocephalus in rats with low aerobic capacity. A differential effect of thrombin may contribute to differences in the effects of cerebral

  6. Presence of plasma proteins facilitates the uptake of /sup 125/I-thrombin by the rabbit thoracic aorta endothelium in vitro

    SciTech Connect

    Hatton, M.W.; Moar, S.L.

    1986-07-01

    Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of /sup 125/I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in /sup 125/I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with /sup 125/I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having /sup 125/I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed.

  7. Marine Diterpenes: Molecular Modeling of Thrombin Inhibitors with Potential Biotechnological Application as an Antithrombotic

    PubMed Central

    Pereira, Rebeca Cristina Costa; Lourenço, André Luiz; Terra, Luciana; Abreu, Paula Alvarez; Laneuville Teixeira, Valéria; Castro, Helena Carla

    2017-01-01

    Thrombosis related diseases are among the main causes of death and incapacity in the world. Despite the existence of antithrombotic agents available for therapy, they still present adverse effects like hemorrhagic risks which justify the search for new options. Recently, pachydictyol A, isopachydictyol A, and dichotomanol, three diterpenes isolated from Brazilian marine brown alga Dictyota menstrualis were identified as potent antithrombotic molecules through inhibition of thrombin, a key enzyme of coagulation cascade and a platelet agonist. Due to the biotechnological potential of these marine metabolites, in this work we evaluated their binding mode to thrombin in silico and identified structural features related to the activity in order to characterize their molecular mechanism. According to our theoretical studies including structure-activity relationship and molecular docking analysis, the highest dipole moment, polar surface area, and lowest electronic density of dichotomanol are probably involved in its higher inhibition percentage towards thrombin catalytic activity compared to pachydictyol A and isopachydictyol A. Interestingly, the molecular docking studies also revealed a good shape complementarity of pachydictyol A and isopachydictyol A and interactions with important residues and regions (e.g., H57, S195, W215, G216, and loop-60), which probably justify their thrombin inhibitor effects demonstrated in vitro. Finally, this study explored the structural features and binding mode of these three diterpenes in thrombin which reinforced their potential to be further explored and may help in the design of new antithrombotic agents. PMID:28335516

  8. Marine Diterpenes: Molecular Modeling of Thrombin Inhibitors with Potential Biotechnological Application as an Antithrombotic.

    PubMed

    Pereira, Rebeca Cristina Costa; Lourenço, André Luiz; Terra, Luciana; Abreu, Paula Alvarez; Laneuville Teixeira, Valéria; Castro, Helena Carla

    2017-03-20

    Thrombosis related diseases are among the main causes of death and incapacity in the world. Despite the existence of antithrombotic agents available for therapy, they still present adverse effects like hemorrhagic risks which justify the search for new options. Recently, pachydictyol A, isopachydictyol A, and dichotomanol, three diterpenes isolated from Brazilian marine brown alga Dictyota menstrualis were identified as potent antithrombotic molecules through inhibition of thrombin, a key enzyme of coagulation cascade and a platelet agonist. Due to the biotechnological potential of these marine metabolites, in this work we evaluated their binding mode to thrombin in silico and identified structural features related to the activity in order to characterize their molecular mechanism. According to our theoretical studies including structure-activity relationship and molecular docking analysis, the highest dipole moment, polar surface area, and lowest electronic density of dichotomanol are probably involved in its higher inhibition percentage towards thrombin catalytic activity compared to pachydictyol A and isopachydictyol A. Interestingly, the molecular docking studies also revealed a good shape complementarity of pachydictyol A and isopachydictyol A and interactions with important residues and regions (e.g., H57, S195, W215, G216, and loop-60), which probably justify their thrombin inhibitor effects demonstrated in vitro. Finally, this study explored the structural features and binding mode of these three diterpenes in thrombin which reinforced their potential to be further explored and may help in the design of new antithrombotic agents.

  9. Bufadienolides from Kalanchoe daigremontiana as thrombin inhibitors-In vitro and in silico study.

    PubMed

    Kolodziejczyk-Czepas, Joanna; Sieradzka, Malgorzata; Moniuszko-Szajwaj, Barbara; Pecio, Łukasz; Ponczek, Michal B; Nowak, Pawel; Stochmal, Anna

    2017-06-01

    Thrombin is an active plasma coagulation factor II, critical for the formation of fibrin clot during blood coagulation. For that reason, this protein is also a crucial target for different anti-thrombotic therapies. The work is based on in vitro evaluation of the inhibitory effect of bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (1-50μg/ml) on enzymatic properties of a serine proteinase - thrombin. The efficacy of the inhibition of amidolytic activity of thrombin (measured as a hydrolysis of the chromogenic substrate S-2238, Chromogenix) attained about 10 and 66%, respectively. The IC50, established for the examined bufadienolide fraction was 2.79μg/ml, while the IC50 calculated for argatroban (reference compound) was 0.78μg/ml. Linearization conducted using Lineweaver-Burk plot indicated that the K. daigremontiana fraction contains compounds that are uncompetitive inhibitors of thrombin. K. daigremontiana fraction was also able to reduce the proteolytic activity of thrombin towards its physiological substrate, i.e. fibrinogen. Additionally, this study is supported by in silico analysis of interactions of the most common compounds, identified in the examined in Kalanchoe extract to crystal structure of this enzyme.

  10. The influence of the sequence of nanoparticles injection to solution on the rate of fibrinogen-thrombin reaction

    NASA Astrophysics Data System (ADS)

    Kirichenko, M. N.; Krivokhiza, S. V.; Chaikov, L. L.; Bulychev, N. A.

    2017-01-01

    The influence of Fe2O3 nanoparticles on the rate of fibrinogen-thrombin reaction is studied. The nanoparticles were obtained in acoustoplasma discharge with cavitation. The sequence of nanoparticles injection appeared to change dramatically the rate and result of enzymatic reaction. In case of nanoparticles injection to fibrinogen before thrombin addition, enzymatic reaction practically stopped at the first stage. The mixing of nanoparticles with thrombin before its addition to fibrinogen leads to acceleration of gel formation in comparison with reaction without nanoparticles. We believe that Fe2O3 nanoparticles can modify the rate of enzymatic reaction, in one case acting as inhibitors of the reaction and as activators in other.

  11. A mathematical model of thrombin production in blood coagulation, Part I: The sparsely covered membrane case.

    PubMed

    Baldwin, S A; Basmadjian, D

    1994-01-01

    This paper presents the first attempt to model the blood coagulation reactions in flowing blood. The model focuses on the common pathway and includes activation of factor X and prothrombin, including feedback activation of cofactors VIII and V by thrombin, and plasma inhibition of factor Xa and thrombin. In this paper, the first of two, the sparsely covered membrane (SCM) case is presented. This considers the limiting situation where platelet membrane binding sites are in excess, such that no membrane saturation or binding competition occurs. Under these conditions, the model predicts that the two positive feedback loops lead to multiple steady-state behavior in the range of intermediate mass transfer rates. It will be shown that this results in three parameter regions exhibiting very different thrombin production patterns. The model predicts the effect of flow on steady-state and dynamic thrombin production and attempts to explain the difference between venous and arterial thrombi. The reliance of thrombin production on precursor procoagulant protein concentrations is also assessed.

  12. Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells.

    PubMed

    Smadja, David M; Basire, Agnès; Amelot, Aymeric; Conte, Aurélie; Bièche, Ivan; Le Bonniec, Bernard F; Aiach, Martine; Gaussem, Pascale

    2008-06-01

    Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.

  13. Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells

    PubMed Central

    Smadja, David M; Basire, Agnès; Amelot, Aymeric; Conte, Aurélie; Bièche, Ivan; Le Bonniec, Bernard F; Aiach, Martine; Gaussem, Pascale

    2008-01-01

    Abstract Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34+ cells. Fibrin networks generated in microplates by adding CaCl2 to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs. PMID:18494938

  14. The role of hyperglycaemia-induced alterations of antithrombin III and factor X activation in the thrombin hyperactivity of diabetes mellitus.

    PubMed

    Ceriello, A; Quatraro, A; Marchi, E; Barbanti, M; Dello Russo, P; Lefebvre, P; Giugliano, D

    1990-05-01

    Factor X concentration and factor X activation, antithrombin III anti-Xa activity and plasma concentration, and fibrinopeptide A were measured in 20 diabetic patients and 20 normal subjects. Although factor X activation (81.3 +/- 2.2 vs 97.3 +/- 2.1%, p less than 0.01; mean +/- SE) and antithrombin III activity (76.5 +/- 2.2 vs 96.3 +/- 1.8%, p less than 0.01) were reduced in the diabetic patients, fibrinopeptide A concentration was increased (3.7 +/- 0.4 vs 1.7 +/- 0.2 ng ml-1, p less than 0.01). The ratio of factor X activation to antithrombin III anti-factor Xa activity was increased in the diabetic patients (1.10 +/- 0.01 vs 1.01 +/- 0.02, p less than 0.01). Induced hyperglycaemia was able to mimic all these abnormalities, without changing factor X or antithrombin III concentration. The results suggest that in vivo hyperglycaemia produces a decrease of factor X activation, but at the same time increases fibrinopeptide A formation due to a greater decrease of antithrombin III anti-Xa activity.

  15. Thrombin induces ischemic LTP (iLTP): implications for synaptic plasticity in the acute phase of ischemic stroke

    PubMed Central

    Stein, Efrat Shavit; Itsekson-Hayosh, Zeev; Aronovich, Anna; Reisner, Yair; Bushi, Doron; Pick, Chaim G.; Tanne, David; Chapman, Joab; Vlachos, Andreas; Maggio, Nicola

    2015-01-01

    Acute brain ischemia modifies synaptic plasticity by inducing ischemic long-term potentiation (iLTP) of synaptic transmission through the activation of N-Methyl-D-aspartate receptors (NMDAR). Thrombin, a blood coagulation factor, affects synaptic plasticity in an NMDAR dependent manner. Since its activity and concentration is increased in brain tissue upon acute stroke, we sought to clarify whether thrombin could mediate iLTP through the activation of its receptor Protease-Activated receptor 1 (PAR1). Extracellular recordings were obtained in CA1 region of hippocampal slices from C57BL/6 mice. In vitro ischemia was induced by acute (3 minutes) oxygen and glucose deprivation (OGD). A specific ex vivo enzymatic assay was employed to assess thrombin activity in hippocampal slices, while OGD-induced changes in prothrombin mRNA levels were assessed by (RT)qPCR. Upon OGD, thrombin activity increased in hippocampal slices. A robust potentiation of excitatory synaptic strength was detected, which occluded the ability to induce further LTP. Inhibition of either thrombin or its receptor PAR1 blocked iLTP and restored the physiological, stimulus induced LTP. Our study provides important insights on the early changes occurring at excitatory synapses after ischemia and indicates the thrombin/PAR1 pathway as a novel target for developing therapeutic strategies to restore synaptic function in the acute phase of ischemic stroke. PMID:25604482

  16. Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme from Lachesis muta rhombeata Snake Venom

    PubMed Central

    Torres-Huaco, Frank Denis; Werneck, Cláudio C.; Vicente, Cristina Pontes; Vassequi-Silva, Talita; Nery-Diez, Ana Cláudia Coelho; Mendes, Camila B.; Antunes, Edson; Marangoni, Sérgio; Damico, Daniela C. S.

    2013-01-01

    We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC). PMID:24058917

  17. Comparison of the 'chemical' and 'structural' approaches to the optimization of the thrombin-binding aptamer.

    PubMed

    Tatarinova, Olga; Tsvetkov, Vladimir; Basmanov, Dmitry; Barinov, Nikolay; Smirnov, Igor; Timofeev, Edward; Kaluzhny, Dmitry; Chuvilin, Andrey; Klinov, Dmitry; Varizhuk, Anna; Pozmogova, Galina

    2014-01-01

    Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.

  18. The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.

    PubMed

    Siebenlist, K R; DiOrio, J P; Budzynski, A Z; Mosesson, M W

    1990-10-25

    Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21

  19. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

    PubMed Central

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-01

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed. PMID:27899589

  20. Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers.

    PubMed

    Pica, Andrea; Russo Krauss, Irene; Parente, Valeria; Tateishi-Karimata, Hisae; Nagatoishi, Satoru; Tsumoto, Kouhei; Sugimoto, Naoki; Sica, Filomena

    2017-01-09

    Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.

  1. Differential involvement of thrombin receptors in Ca2+ release from two different intracellular stores in human platelets

    PubMed Central

    Jardin, Isaac; Ben Amor, Nidhal; Bartegi, Ahgleb; Pariente, José A.; Salido, Ginés M.; Rosado, Juan A.

    2006-01-01

    Physiological agonists increase cytosolic free Ca2+ concentration to regulate a number of cellular processes. The platelet thrombin receptors, PAR (protease-activated receptor) 1 PAR-4 and GPIb-IX-V (glycoprotein Ib-IX-V) have been described as potential contributors of thrombin-induced platelet aggregation. Platelets present two separate Ca2+ stores, the DTS (dense tubular system) and acidic organelles, differentiated by the distinct sensitivity of their respective SERCAs (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPases) to TG (thapsigargin) and TBHQ [2,5-di-(tert-butyl)-1,4-hydroquinone]. However, the involvement of the thrombin receptors in Ca2+ release from each Ca2+ store remains unknown. Depletion of the DTS using ADP, which releases Ca2+ solely from the DTS, in combination with 10 nM TG, to selectively inhibit SERCA2 located on the DTS reduced Ca2+ release evoked by the PAR-1 agonist, SFLLRN, and the PAR-4 agonist, AYPGKF, by 80 and 50% respectively. Desensitization of PAR-1 and PAR-4 or pre-treatment with the PAR-1 and PAR-4 antagonists SCH 79797 and tcY-NH2 reduced Ca2+ mobilization induced by thrombin, and depletion of the DTS after desensitization or blockade of PAR-1 and PAR-4 had no significant effect on Ca2+ release stimulated by thrombin through the GPIb-IX-V receptor. Converse experiments showed that depletion of the acidic stores using TBHQ reduced Ca2+ release evoked by SFLLRN or AYPGKF, by 20 and 50% respectively, and abolished thrombin-stimulated Ca2+ release through the GPIb-IX-V receptor when PAR-1 and PAR-4 had been desensitized or blocked. Our results indicate that thrombin-induced activation of PAR-1 and PAR-4 evokes Ca2+ release from both Ca2+ stores, while activation of GPIb-IX-V by thrombin releases Ca2+ solely from the acidic compartments in human platelets. PMID:16939417

  2. TMJ function after partial condylectomy in active mandibular condylar hyperplasia

    PubMed Central

    Olate, Sergio; Martinez, Felipe; Uribe, Francisca; Pozzer, Leandro; Cavalieri-Pereira, Lucas; de Moraes, Marcio

    2014-01-01

    Condylar hyperplasia is a frequent pathology that causes severe facial asymmetries. The partial condylectomy generally halts the disease. The aim of this research was to examine post-condylectomy TMJ function; 14 patients were included in this study, 6 male and 8 female. The average age was 21 years old. In all, the partial condylectomy was performed with preauricular or endaural access and the osteotomy were performed with drills, saw or an ultrasonic system. The patients were assessed with 3 or more time after surgery and were considering maximum mouth opening, right and left lateralities, presence of pain, noises, alterations in the facial nerve (VII) and esthetic alteration from the scar. The analysis was performed with the visual analog scale (VAS) and with a 7 cm metallic rule. Data analysis was descriptive plus chi-square test considering p value < 0.05 for statistical differences. With an average of 11 month after surgery, the results showed that the open mouth (over 35 mm) and lateralities (average 9 mm for the both right and left side) were normal and without statistical differences between the right or left side. Noise was observed in 3 patients and pain was observed in two patients with level 2 and 1 (VAS score). Scar was not related to problem with patient and the temporal branch of facial nerve was observed with limitations but without problem for patients. It can be concluded that the condylectomy is a safe and effective procedure with low morbidity for patients. PMID:24753777

  3. Thrombin as important factor for cutaneous wound healing: comparison of fibrin biomatrices in vitro and in a rat excisional wound healing model.

    PubMed

    Gugerell, Alfred; Pasteiner, Waltraud; Nürnberger, Sylvia; Kober, Johanna; Meinl, Alexandra; Pfeifer, Sabine; Hartinger, Joachim; Wolbank, Susanne; Goppelt, Andreas; Redl, Heinz; Mittermayr, Rainer

    2014-01-01

    Fibrin biomatrices have been used for many years for hemostasis and sealing and are a well-established surgical tool. The objective of the present study was to compare two commercially available fibrin biomatrices regarding the effect of their thrombin concentration on keratinocytes and wound healing in vitro and in vivo. Keratinocytes showed significant differences in adhesion, viability, and morphology in the presence of the fibrin matrices in vitro. A high thrombin concentration (800-1,200 IU/mL) caused deteriorated cell compatibility. By using a thrombin inhibitor, those differences could be reversed. In a rat excisional wound healing model, we observed more rapid wound closure and less wound severity in wounds treated with a fibrin matrix containing a lower concentration of thrombin (4 IU/mL). Furthermore, fewer new functional vessels and a lower level of vascular endothelial growth factor were measured in wounds after 7 days treated with the matrix with higher thrombin concentration. These in vivo results may be partially explained by the in vitro biocompatibility data. Additionally, results show that low thrombin biomatrices were degraded faster than the high thrombin material. Hence, we conclude that the composition of fibrin biomatrices influences keratinocytes and therefore has an impact on wound healing.

  4. Electrostatic interactions in hirudin-thrombin binding.

    PubMed

    Sharp, K A

    1996-08-30

    Hirudin is a good anticoagulant owing to potent inhibition of the serine protease thrombin. An aspartate- and glutamate-rich portion of hirudin plays an important part in its tight binding to thrombin through a ladder of salt bridges, and these residues have previously been mutated to asparagine or glutamine. Detailed calculations of the electrostatic contribution to changes in binding from these mutations have been performed using the finite-difference Poisson-Boltzmann method which include charge--charge interactions, solvation interactions, the residual electrostatic interaction of mutant residues, pKa shifts, and ionic strength. Single mutant effects on binding energy were close to experimental values, except for the D55N mutant whose effect is overestimated, perhaps because of displacement of a bound chloride ion from the site where it binds. Multiple mutation values were generally overestimated. The effect of pKa shifts upon the binding is significant for one hirudin residue E58, but this appears to be due to a poor salt bridge with thrombin caused by crystal contacts. Electrostatic interaction between the acidic residues is unfavorable. However, analysis of experimental multiple mutation/single mutation data shows apparently negative interactions between these residues, from which it is concluded that structural changes can occur in the complex to relieve an unfavorable interaction when more than one acidic residue is mutated. In all cases, there is a loss in stability of the complex from mutations due to loss of favorable charge--charge interactions with thrombin, but this is largely compensated for by reduced unfavorable desolvation interactions, and by residual polar interactions in the Asn/Gln mutants.

  5. Rigidification of the autolysis loop enhances Na[superscript +] binding to thrombin

    SciTech Connect

    Pozzi, Nicola; Chen, Raymond; Chen, Zhiwei; Bah, Alaji; Di Cera, Enrico

    2011-09-20

    Binding of Na{sup +} to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na{sup +} is weak due to large heat capacity and enthalpy changes associated with binding, and the K{sub d} = 80 mM ensures only 64% saturation of the site at the concentration of Na{sup +} in the blood (140 mM). Residues controlling Na{sup +} binding and activation have been identified. Yet, attempts to improve the interaction of Na{sup +} with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na{sup +} affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na{sup +} binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.

  6. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

    PubMed Central

    Xie, Lishi; Chiang, Eddie T.; Kelly, Gabriel T.; Kanteti, Prasad; Singleton, Patrick A.; Camp, Sara M.; Zhou, Tingting; Dudek, Steven M.; Natarajan, Viswanathan; Wang, Ting; Black, Steven M.; Garcia, Joe G. N.; Jacobson, Jeffrey R.

    2016-01-01

    Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis. PMID:27442243

  7. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

    PubMed Central

    2015-01-01

    Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin. PMID:24635452

  8. A comparison between two commercially available activators for determining the partial thromboplastin time.

    PubMed

    Smith, L G; Kitchens, C S

    1985-03-01

    Activators that are used to determine the activated partial thromboplastin time should be sensitive enough to detect mild deficiencies of coagulation factors in a reproducible fashion. We compared two commercially available activators--a newly modified preparation of ellagic acid activator and a silica activator. Both reagents produced results that were reproducible over a broad range of concentrations of factors involved in the intrinsic coagulation pathway. The ellagic acid activator was more sensitive in detecting a partial deficiency of nearly all of these factors.

  9. Flow and delta-P dictate where thrombin, fibrin, and von Willebrand Factor will be found.

    PubMed

    Diamond, Scott L

    2016-05-01

    Hemostasis occurs in two different topological scenarios: complete severing of a vessel or disruption of the vessel wall. Either to meet the daily rigors of active life or during an acute trauma, hemostasis involves the regulated and self-limiting production of thrombin to stop bleeding. In contrast, arterial and venous thrombosis typically involves the unregulated, intraluminal growth of a clot, in the absence of bleeding. For either hemostasis or thrombosis, the presence of flow and pressure gradients (delta-P, ΔP) dictates when and where thrombin and fibrin are located and in what quantity. For hemostatic clots, fibrin formation helped limit clot growth. We found that γ'-fibrinogen had a role in limiting clot growth via anti-thrombin activity at venous, but not arterial conditions. For hemophilic blood, severe factor deficiency (<1% healthy) led to a defect in both platelet and fibrin deposition under flow. However, moderate deficiency, which is associated with a less severe bleeding phenotype, had normalized platelet function but still lacked fibrin production. We conclude signaling levels of thrombin can be generated during moderate hemophilia to sufficiently activate platelets to achieve primary hemostasis, even if fibrin formation remains defective. Finally, as a clot grows, shear stresses can become sufficiently extreme in diseased arteries to drive von Willebrand Factor self-association into massive fibers, potentially the final burst of clot growth towards full thrombotic occlusion.

  10. Hypersensitivity to thrombin of platelets from hypercholesterolemic rats

    SciTech Connect

    Winocour, P.D.; Rand, M.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-03-01

    Hypersensitivity of platelets to thrombin has been associated with hypercholesterolemia. The authors have examined the mechanisms involved in this hypersensitivity. Rats were given diets rich in milk fat and containing added cholesterol and taurocholate to produce hypercholesterolemia (HC) (262 +/- 25 mg%) or added sitosterol as a normocholesterolemic control (NC) (89 +/- 6 mg%). Washed platelets were prelabelled with /sup 14/C-serotonin. In the presence of acetylsalicyclic acid (ASA) (to inhibit thromboxane A/sub 2/ (TXA/sub 2/) formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (26 +/- 1%) and released more /sup 14/C (9.1 +/- 2.0%) than NC platelets (aggregation: 0%, p < 0.001; /sup 14/C release: 1.5 +/- 0.5%, p < 0.002) in response to thrombin (0.075 U/ml). Thus, a pathway independent of released ADP or TXA/sub 2/ formation is involved in the hypersensitivity of HC platelets to thrombin. Total binding of /sup 125/I-thrombin to HC platelets was less than that to NC platelets but HC platelets were smaller and had less protein than NC platelets; the thrombin binding per mg platelet protein was the same for HC and NC platelets, indicating that hypersensitivity to thrombin of HC platelets does not result from increased thrombin binding. Thus, hypersensitivity of HC platelets to thrombin is not due to TXA/sub 2/ formation, the action of released ADP or increased thrombin binding.

  11. The role of topical thrombin in skin grafting.

    PubMed Central

    Ofodile, F. A.; Sadana, M. K.

    1991-01-01

    A prospective study to evaluate the efficacy of thrombin as a hemostatic agent in burn patients was conducted on 24 patients undergoing debridement and skin grafting. All patients also acted as their own control. Results showed a 43.5% reduction in bleeding on the thrombin-treated sites compared with the control sites. There was no adverse effect on the rate of wound healing from the thrombin, and no difference in the nature of the scar seen at the thrombin-treated site compared with the control site. Images Figure 1 PMID:1875421

  12. Partial characterization of Acanthamoeba castellanii (T4 genotype) DNase activity.

    PubMed

    Iqbal, Junaid; Panjwani, Shamvil; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2015-02-01

    The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba.

  13. Thrombin Cleavage of Inter-α-inhibitor Heavy Chain 1 Regulates Leukocyte Binding to an Inflammatory Hyaluronan Matrix.

    PubMed

    Petrey, Aaron C; de la Motte, Carol A

    2016-11-18

    Dynamic alterations of the extracellular matrix in response to injury directly modulate inflammation and consequently the promotion and resolution of disease. During inflammation, hyaluronan (HA) is increased at sites of inflammation where it may be covalently modified with the heavy chains (HC) of inter-α-trypsin inhibitor. Deposition of this unique, pathological form of HA (HC-HA) leads to the formation of cable-like structures that promote adhesion of leukocytes. Naive mononuclear leukocytes bind specifically to inflammation-associated HA matrices but do not adhere to HA constitutively expressed under homeostatic conditions. In this study, we have directly investigated a role for the blood-coagulation protease thrombin in regulating the adhesion of monocytic cells to smooth muscle cells producing an inflammatory matrix. Our data demonstrate that the proteolytic activity of thrombin negatively regulates the adhesion of monocytes to an inflammatory HC-HA complex. This effect is independent of protease-activated receptor activation but requires proteolytic activity toward a novel substrate. Components of HC-HA complexes were predicted to contain conserved thrombin-susceptible cleavage sites based on sequence analysis, and heavy chain 1 (HC1) was confirmed to be a substrate of thrombin. Thrombin treatment is sufficient to cleave HC1 associated with either cell-surface HA or serum inter-α-trypsin inhibitor. Furthermore, thrombin treatment of the inflammatory matrix leads to dissolution of HC-HA cable structures and abolishes leukocyte adhesion. These data establish a novel mechanism whereby thrombin cleavage of HC1 regulates the adhesive properties of an inflammatory HA matrix.

  14. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.

    PubMed

    Gu, J-M; Ramsey, P; Evans, V; Tang, L; Apeler, H; Leong, L; Murphy, J E; Laux, V; Myles, T

    2014-07-01

    Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

  15. Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

    PubMed

    Russo Krauss, Irene; Spiridonova, Vera; Pica, Andrea; Napolitano, Valeria; Sica, Filomena

    2016-01-29

    Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

  16. Chemical characteristic and anticoagulant activity of the sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta).

    PubMed

    Mao, Wenjun; Li, Hongyan; Li, Yi; Zhang, Huijuan; Qi, Xiaohui; Sun, Haihong; Chen, Yin; Guo, Shoudong

    2009-01-01

    A polysaccharide was isolated from marine green algae Monostroma latissimum, and its chemical characteristic and anticoagulant activity were investigated. The results demonstrated that the polysaccharide was high rhamnose-containing sulfated polysaccharide, and was mainly composed of 1,2-linked l-rhamnose residues with sulfate groups substituted at positions C-3 and/or C-4. The sulfated polysaccharide exhibited high anticoagulant activities by assays of the activated partial thromboplastin time (APTT) and thrombin time (TT). The anticoagulant property of the sulfated polysaccharide was mainly attributed to powerful potentiation thrombin by heparin cofactor II.

  17. RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents

    PubMed Central

    Chen, I-Shan; Furutani, Kazuharu; Inanobe, Atsushi; Kurachi, Yoshihisa

    2014-01-01

    Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist–receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K+ inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist–receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine–M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses. PMID:24421355

  18. Development of a photometric assay for activated partial thromboplastin time and its application to the Cobas Bio centrifugal analyzer.

    PubMed

    Becker, U; Bartl, K; Lill, H; Wahlefeld, A W

    1985-12-15

    We describe a two-step procedure for APTT that can be performed on photometric devices. It includes preincubation of diluted plasma with ellagic acid and phospholipids and a starting reagent that contains calcium and a chromogenic peptide substrate for thrombin, Tos-Gly-Pro-Arg-pNA. Reaction time is recorded from addition of the starting reagent until thrombin formation occurs, and a prefixed amount of substrate is cleaved. The pattern of sensitivity to clotting factors and heparin was similar to clotting assays and the substrate used did not interfere with the activity of factor Xa. An application of the method was made for the Cobas(R) Bio centrifugal analyzer. Absorbance readings were sent to an external computer and were transformed into reaction times by a computer program. Although the results are independent on fibrinogen concentrations, from kinetic data of the reaction curve fibrinogen concentrations can be estimated. Correlation studies showed good correspondence to clotting methods (r = 0.92, n = 53) as well as an excellent precision (CV 3% for inter-assays, n = 15) and high throughput of samples (greater than 100/h) in the automated assay.

  19. Phospholipase C-beta3 mediates the thrombin-induced Ca2+ response in glial cells.

    PubMed

    Hwang, Jong-Ik; Shin, Kum-Joo; Oh, Yong-Seok; Choi, Jung-Woong; Lee, Zee-Won; Kim, Daesoo; Ha, Kwon-Soo; Shin, Hee-Sup; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-06-30

    Phospholipase C-beta (PLC-beta) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-beta1 [PLC-beta1 (-/-)] or PLC-beta3 [PLC-beta3 (-/-)], we examined which isotype of PLC-beta participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-beta1 (-/-) cells, but was negligible in PLC-beta3 (-/-) cells. Expression of PLC-beta3 in PLC-beta3 (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-beta1 in PLC-beta1 (-/-) cells did not have any effect on IP generation. The thrombin-induced [Ca2+]i increase was delayed and attenuated in PLC-beta3 (-/-) cells, but normal in PLC-beta1 (-/-) cells. Pertussis toxin evoked a delayed [Ca2+]i increase in PLC-beta3 (-/-) cells as well as in PLC-beta1 (-/-) cells. These results suggest that activation of PLC-beta3 by pertussis toxin-sensitive G proteins is responsible for the transient [Ca2+]i increase in response to thrombin, whereas the delayed [Ca2+]i increase may be due to activation of some other PLC, such as PLC-beta4, acting via PTx-insensitive G proteins.

  20. Lupus anticoagulants form immune complexes with prothrombin and phospholipid that can augment thrombin production in flow.

    PubMed

    Field, S L; Hogg, P J; Daly, E B; Dai, Y P; Murray, B; Owens, D; Chesterman, C N

    1999-11-15

    Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid binding proteins, including prothrombin. We found that a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a phospholipid-coated flow reactor. The flow reactor was incubated with prothrombin, calcium ions, and the IgGs and then perfused with prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 minutes. In summary, LA IgGs concentrate prothrombin on a phospholipid surface that can augment thrombin production by prothrombinase in flow. These observations suggest that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall.

  1. Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure.

    PubMed

    Clare, B W; Scozzafava, A; Supuran, C T

    2001-01-01

    A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.

  2. Immunomodulatory activity and partial characterisation of polysaccharides from Momordica charantia.

    PubMed

    Deng, Yuan-Yuan; Yi, Yang; Zhang, Li-Fang; Zhang, Rui-Fen; Zhang, Yan; Wei, Zhen-Cheng; Tang, Xiao-Jun; Zhang, Ming-Wei

    2014-08-29

    Momordica charantia Linn. is used as an edible and medicinal vegetable in sub-tropical areas. Until now, studies on its composition and related activities have been confined to compounds of low molecular mass, and no data have been reported concerning the plant's polysaccharides. In this work, a crude polysaccharide of M. charantia (MCP) fruit was isolated by hot water extraction and then purified using DEAE-52 cellulose anion-exchange chromatography to produce two main fractions MCP1 and MCP2. The immunomodulatory effects and physicochemical characteristics of these fractions were investigated in vitro and in vivo. The results showed that intragastric administration of 150 or 300 mg·kg-·d⁻¹ of MCP significantly increased the carbolic particle clearance index, serum haemolysin production, spleen index, thymus index and NK cell cytotoxicity to normal control levels in cyclophosphamide (Cy)-induced immunosuppressed mice. Both MCP1 and MCP2 effectively stimulated normal and concanavalin A-induced splenic lymphocyte proliferation in vitro at various doses. The average molecular weights of MCP1 and MCP2, which were measured using high-performance gel permeation chromatography, were 8.55×10⁴ Da and 4.41×10⁵ Da, respectively. Both fractions exhibited characteristic polysaccharide bands in their Fourier transform infrared spectrum. MCP1 is mainly composed of glucose and galactose, and MCP2 is mainly composed of glucose, mannose and galactose. The results indicate that MCP and its fractions have good potential as immunotherapeutic adjuvants.

  3. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  4. Acidosis, magnesium and acetylsalicylic acid: Effects on thrombin

    NASA Astrophysics Data System (ADS)

    Borisevich, Nikolaj; Loznikova, Svetlana; Sukhodola, Aleksandr; Halets, Inessa; Bryszewska, Maria; Shcharbin, Dzmitry

    2013-03-01

    Thrombin, an enzyme from the hydrolase family, is the main component of the blood coagulation system. In ischemic stroke it acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin forming blood clots in the brain. It has been found to phosphoresce at room temperature in the millisecond and microsecond ranges. The phosphorescence of thrombin was studied under physiological conditions, in acidosis (decrease of pH from 8.0 to 5.0) and on the addition of salts (magnesium sulfate and sodium chloride) and of acetylsalicylic acid, and its connection with thrombin function is discussed. Acidosis significantly increased the internal dynamics of thrombin. We propose that lactate-acidosis plays a protective role in stroke, preventing the formation of clots. The addition of NaCl and MgSO4 in different concentrations increased the internal dynamics of thrombin. Also, the addition of MgSO4 decreased thrombin-induced platelet aggregation. However, magnesium sulfate and acetylsalicylic acid in the therapeutic concentrations used for treatment of ischemic stroke had no effect on thrombin internal dynamics. The data obtained will help to elucidate the conformational stability of thrombin under conditions modulating lactate-acidosis and in the presence of magnesium sulfate.

  5. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  6. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  7. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7875 Thrombin time test....

  8. Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay

    PubMed Central

    Schenk, B.; Würtinger, P.; Streif, W.; Sturm, W.; Fries, D.; Bachler, M.

    2016-01-01

    Background In major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). Methods Blood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. Results When added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV−t), and with all measured thrombin generation parameters. In vivo, CT, MaxV−t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). Conclusions Thromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. Clinical trial registration EudracCT trial no. 213-00474-30. PMID:27623677

  9. Muscle Activation Differs Between Partial And Full Back Squat Exercise With External Load Equated.

    PubMed

    Jarbas da Silva, Josinaldo; Schoenfeld, Brad Jon; Marchetti, Priscyla Nardi; Pecoraro, Silvio Luis; D'Andréa Greve, Julia Maria; Marchetti, Paulo Henrique

    2017-02-13

    Changes in range of motion affect the magnitude of the load during the squat exercise and, consequently may influence muscle activation. The purpose of this study was to evaluate muscle activation between the partial and full back squat exercise with external load equated on a relative basis between conditions. Fifteen young, healthy, resistance trained men (age: 26±5 years, height: 173±6 cm) performed a back squat at their 10 repetition maximum using two different ranges of motion (partial and full) in a randomized, counterbalanced fashion. Surface electromyography was used to measure muscle activation of the vastus lateralis (VL), vastus medialis (VM), rectus femoris (RF), biceps femoris (BF), semitendinosus (ST), erector spinae (ES), soleus (SL), and gluteus maximus (GM). In general, muscle activity was highest during the partial back squat for GM (P=0.004), BF (P=0.009), and SL (P=0.031) when compared to full. There was no significant difference for RPE between partial and full back squat exercise at 10RM (8±1 and 9±1, respectively). In conclusion, the range of motion in the back squat alters muscle activation of the prime mover (gluteus maximus), and stabilizers (soleus and biceps femoris) when performed with the load equated on a relative basis. Thus, the partial back squat maximizes the level of muscle activation of the gluteus maximus and associated stabilizer muscles.

  10. Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation

    PubMed Central

    Mitrophanov, Alexander Y.; Rosendaal, Frits R.

    2015-01-01

    BACKGROUND: Acidosis, a frequent complication of trauma and complex surgery, results from tissue hypoperfusion and IV resuscitation with acidic fluids. While acidosis is known to inhibit the function of distinct enzymatic reactions, its cumulative effect on the blood coagulation system is not fully understood. Here, we use computational modeling to test the hypothesis that acidosis delays and reduces the amount of thrombin generation in human blood plasma. Moreover, we investigate the sensitivity of different thrombin generation parameters to acidosis, both at the individual and population level. METHODS: We used a kinetic model to simulate and analyze the generation of thrombin and thrombin–antithrombin complexes (TAT), which were the end points of this study. Large groups of temporal thrombin and TAT trajectories were simulated and used to calculate quantitative parameters, such as clotting time (CT), thrombin peak time, maximum slope of the thrombin curve, thrombin peak height, area under the thrombin trajectory (AUC), and prothrombin time. The resulting samples of parameter values at different pH levels were compared to assess the acidosis-induced effects. To investigate intersubject variability, we parameterized the computational model using the data on clotting factor composition for 472 subjects from the Leiden Thrombophilia Study. To compare acidosis-induced relative parameter changes in individual (“virtual”) subjects, we estimated the probabilities of relative change patterns by counting the pattern occurrences in our virtual subjects. Distribution overlaps for thrombin generation parameters at distinct pH levels were quantified using the Bhattacharyya coefficient. RESULTS: Acidosis in the range of pH 6.9 to 7.3 progressively increased CT, thrombin peak time, AUC, and prothrombin time, while decreasing maximum slope of the thrombin curve and thrombin peak height (P < 10–5). Acidosis delayed the onset and decreased the amount of TAT generation (P

  11. Significantly different coagulation factor activities underlying the variability of 'normal' activated partial thromboplastin time.

    PubMed

    Park, Kyoung-Jin; Kwon, Eui-Hoon; Ma, Youngeun; Park, In-Ae; Kim, Seon-Woo; Kim, Sun-Hee; Kim, Hee-Jin

    2012-01-01

    The activated partial thromboplastin time (aPTT) is a widely used coagulation screening test in routine laboratories. The aPTT level in the control population varies and is reflected by the reference interval. However, there have been no studies to investigate the coagulation status determining the variability of the aPTT. The aim of this study was to investigate the coagulation factor activities underlying the variability of aPTT in the population. The study participants were reference individuals with prothrombin time and aPTT within reference intervals. The aPTT was determined using STA-PTT Automate (Diagnostica Stago, Asnieres, France; local reference interval, 29.1-41.9 s). Those with aPTT within the marginal ranges of reference interval were selected for factor assays. We defined the lower marginal group as the lowest 10 percentile of reference interval (29.1-30.9 s) and the upper marginal group as the highest 10 percentile (38.0-41.9 s). Activities of factor II, V, VIII, IX, X, XI, and XII were determined in both groups. The lower marginal and upper marginal groups consisted of 220 and 209 individuals, respectively. All coagulation factors were significantly higher in the lower marginal than in the upper marginal group (P = 0.0127 for factor II and P < 0.0001 for the others). Multiple logistic regression analyses revealed factor XII and VIII were two strongest contributors determining the aPTT level, whereas factor XI was not significantly different between the groups (P = 0.095). This study firstly demonstrated significantly different coagulation factor activities underlying the variability of aPTT in reference individuals. The results suggested the possibility of disease association or phenotypic contribution of variable coagulation activities in the population.

  12. APTAMER-BASED SERRS SENSOR FOR THROMBIN DETECTION

    SciTech Connect

    Cho, H; Baker, B R; Wachsmann-Hogiu, S; Pagba, C V; Laurence, T A; Lane, S M; Lee, L P; Tok, J B

    2008-07-02

    We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human a-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5{prime}-capped, 3{prime}-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes.

  13. [Laboratory assessment of haemostatic parameters in patients taking a direct thrombin inhibitor].

    PubMed

    Beliavskaia, O O; Vavilova, T V

    2014-01-01

    The problem of prevention and treatment of thromboembolic complications has a significant place in clinical practice for many years. The gold-standard agents in long-term protection from embologenic strokes, secondary prevention of venous thromboses and embolisms still remain vitamin K antagonists (in Russia - warfarin). However, despite high efficacy, administration of warfarin is fraught with dangers and associated with a series of inconveniences. A direct thrombin inhibitor, dabigatran etexilate (hereinafter referred to as dabigatran) was approved in the Russian Federation for prevention of thromboembolic complications in orthopaedic practice (2009), for prevention of ischaemic embologenic stroke in atrial fibrillation (2011) and for treatment of recurrent thrombosis of deep veins and pulmonary artery thromboembolism (2014). A characteristic feature of a therapeutic agent possessing an anticoagulation effect is correlation between intensity of hypocoagulation and haemorrhage. The effect of dabigatran on the laboratory parameters of haemostasis has been studied insufficiently, with no practical guidelines on assessing these alterations for prediction of the risk for haemorrhagic and thromboembolic complications. The present study included a total of 65 patients with non-valvular aetiology atrial fibrillation, taking dabigatran during from 6 to 18 months. All patients underwent laboratory assessment of the coagulation level and measuring blood coagulation activation markers in dynamics 10-14 days, 1, 6, 12 and 18 months after taking the agent. Thromboembolic and haemorrhagic risks were also assessed. It was revealed that administration of dabigatran leads to alterations in the main parameters of coagulogram. Determination of prothrombin (in % according to Quick's method) and activated partial thromboplastin time may be used for qualitative assessment of hypocoagulation. During the follow up period no statistically significant changes in the coagulation activation

  14. Bacithrocins A, B and C, novel thrombin inhibitors.

    PubMed

    Kamiyama, T; Umino, T; Nakamura, Y; Itezono, Y; Sawairi, S; Satoh, T; Yokose, K

    1994-09-01

    Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988. The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis. Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C. Bacithrocins prolong the clotting time induced by thrombin and factor Xa.

  15. Thrombin/Matrix Metalloproteinase-9-Dependent SK-N-SH Cell Migration is Mediated Through a PLC/PKC/MAPKs/NF-κB Cascade.

    PubMed

    Yang, Chien-Chung; Lin, Chih-Chung; Chien, Peter Tzu-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-11-01

    Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH3), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH3, Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

  16. Human activities recognition by head movement using partial recurrent neural network

    NASA Astrophysics Data System (ADS)

    Tan, Henry C. C.; Jia, Kui; De Silva, Liyanage C.

    2003-06-01

    Traditionally, human activities recognition has been achieved mainly by the statistical pattern recognition methods or the Hidden Markov Model (HMM). In this paper, we propose a novel use of the connectionist approach for the recognition of ten simple human activities: walking, sitting down, getting up, squatting down and standing up, in both lateral and frontal views, in an office environment. By means of tracking the head movement of the subjects over consecutive frames from a database of different color image sequences, and incorporating the Elman model of the partial recurrent neural network (RNN) that learns the sequential patterns of relative change of the head location in the images, the proposed system is able to robustly classify all the ten activities performed by unseen subjects from both sexes, of different race and physique, with a recognition rate as high as 92.5%. This demonstrates the potential of employing partial RNN to recognize complex activities in the increasingly popular human-activities-based applications.

  17. Free Fatty Acids Modulate Thrombin Mediated Fibrin Generation Resulting in Less Stable Clots

    PubMed Central

    Tanka-Salamon, Anna; Komorowicz, Erzsébet; Szabó, László; Tenekedjiev, Kiril

    2016-01-01

    Upon platelet activation, free fatty acids are released at the stage of thrombus formation, but their effects on fibrin formation are largely unexplored. Our objective was to characterize the kinetic effects of fatty acids on thrombin activity, as well as the structural and mechanical properties of the resultant fibrin clots. Thrombin activity on fibrinogen was followed by turbidimetry and detailed kinetic characterization was performed using a fluorogenic short peptide substrate. The viscoelastic properties of fibrin were measured with rotatory oscillation rheometer, whereas its structure was analyzed with scanning electron microscopy (SEM). In turbidimetric assays of fibrin generation, oleate and stearate at physiologically relevant concentrations (60–600 μM) produced a bell-shaped inhibitory dose response, increasing 10- to 30-fold the time to half-maximal clotting. Oleate inhibited thrombin activity on a short peptide substrate according to a mixed-type inhibitor pattern (a 9-fold increase of the Michaelis constant, Km and a 20% decrease of the catalytic constant), whereas stearate resulted in only a minor (15%) drop in the catalytic constant without any change in the Km. Morphometric analysis of SEM images showed a 73% increase in the median fiber diameter in the presence of stearate and a 20% decrease in the presence of oleate. Concerning the viscoelastic parameters of the clots, storage and loss moduli, maximal viscosity and critical shear stress decreased by 32–65% in the presence of oleate or stearate, but loss tangent did not change indicating decreased rigidity, higher deformability and decreased internal resistance to shear stress. Our study provides evidence that free fatty acids (at concentrations comparable to those reported in thrombi) reduce the mechanical stability of fibrin through modulation of thrombin activity and the pattern of fibrin assembly. PMID:27942000

  18. On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

    PubMed Central

    Henriques, Elsa S.; Fonseca, Nelson; Ramos, Maria João

    2004-01-01

    Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors α-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors. PMID:15322279

  19. Polyvinylpyrrolidone induced artefactual prolongation of activated partial thromboplastin times in intravenous drug users with renal failure.

    PubMed

    Kristoffersen, A H; Bjånes, T K; Jordal, S; Leh, S; Leh, F; Svarstad, E

    2016-05-01

    Essentials Prolonged activated partial thromboplastin times (APTT) were found in drug users with renal failure. An oral methadone solution containing polyvinylpyrrolidone (PVP) had been injected intravenously. Spiking normal plasma with increasing concentrations of PVP resulted in artifically prolonged APTT. APTT prolongation may indicate PVP deposits as underlying cause in patients with renal failure.

  20. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  1. Comparison of the ‘Chemical’ and ‘Structural’ Approaches to the Optimization of the Thrombin-Binding Aptamer

    PubMed Central

    Tatarinova, Olga; Tsvetkov, Vladimir; Basmanov, Dmitry; Barinov, Nikolay; Smirnov, Igor; Timofeev, Edward; Kaluzhny, Dmitry; Chuvilin, Andrey; Klinov, Dmitry; Varizhuk, Anna; Pozmogova, Galina

    2014-01-01

    Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies. PMID:24586736

  2. Discovery of non-competitive thrombin inhibitor derived from competitive tryptase inhibitor skeleton: Shift in molecular recognition resulted from skeletal conversion of carboxylate into phosphonate.

    PubMed

    Aoyama, Hiroshi; Ijuin, Ryosuke; Kato, Jun-ya; Urushiyama, Sarasa; Tetsuhashi, Masashi; Hashimoto, Yuichi; Yokomatsu, Tsutomu

    2015-09-01

    A novel series of terminal and internal phosphonate esters based on our previously developed aryl carboxylate-type tryptase selective inhibitor 1 was synthesized. The potency of these synthesized compounds was assessed in vitro with an enzyme inhibition assay using three available serine proteases, that is, tryptase, trypsin, and thrombin. The internal phosphonate derivative 6 showed potent thrombin inhibitory activity with an IC50 value of 1.0 μM, whereas it exhibited no or only weak tryptase and trypsin inhibition at 10 μM. The Lineweaver-Burk plot analysis indicates that the inhibition pattern of thrombin with 6 is non-competitive in spite of the fact that the lead carboxylate compound 1 is competitive inhibitor. Therefore, the skeletal conversion of the carboxylate into a phosphonate alters the mode of molecular recognition of these inhibitors by thrombin.

  3. Thrombin mediates migration of rat brain astrocytes via PLC, Ca²⁺, CaMKII, PKCα, and AP-1-dependent matrix metalloproteinase-9 expression.

    PubMed

    Lin, Chih-Chung; Lee, I-Ta; Wu, Wen-Bin; Liu, Chiung-Ju; Hsieh, Hsi-Lung; Hsiao, Li-Der; Yang, Chien-Chung; Yang, Chuen-Mao

    2013-12-01

    Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca(2+)-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca(2+) concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca(2+)/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.

  4. Exponential stability of delayed and impulsive cellular neural networks with partially Lipschitz continuous activation functions.

    PubMed

    Song, Xueli; Xin, Xing; Huang, Wenpo

    2012-05-01

    The paper discusses exponential stability of distributed delayed and impulsive cellular neural networks with partially Lipschitz continuous activation functions. By relative nonlinear measure method, some novel criteria are obtained for the uniqueness and exponential stability of the equilibrium point. Our method abandons usual assumptions on global Lipschitz continuity, boundedness and monotonicity of activation functions. Our results are generalization and improvement of some existing ones. Finally, two examples and their simulations are presented to illustrate the correctness of our analysis.

  5. Thrombin generation test for evaluation of antiplatelet treatment in patients with coronary artery disease after percutaneous coronary intervention.

    PubMed

    Berezovskaya, Gelena; Smirnova, Olga; Malev, Eduard; Khromov-Borisov, Nikita; Klokova, Elena; Karpenko, Mikhail; Papayan, Lyudmila; Petrishchev, Nikolay

    2017-04-04

    To study the possibility of using thrombin generation tests in platelet-rich and platelet-poor plasma for evaluation of dual antiplatelet therapy efficacy in patients with coronary artery disease (CAD), following percutaneous coronary intervention. Venous blood was analyzed from CAD patients aged 53-75 years who had undergone percutaneous coronary intervention with stenting within one year and had been receiving standard doses of clopidogrel and aspirin (75 and 75-100 mg per day, respectively). The control group comprised age- and sex-matched subjects without clinical signs of CAD who were not receiving these drugs. Thrombin generation tests were performed in platelet-rich and platelet-poor plasma. Intravascular platelet activation, induced platelet aggregation, and routine coagulation were evaluated. Antiplatelet treatment did not influence results of routine coagulation tests or intravascular platelet activation. The dual antiplatelet therapy affects collagen-induced platelet aggregation (44 ± 2.5 vs. 7.9 ± 2.6%, р = 10(-7)) and leads to decreases in endogenous thrombin potential (1900 ± 85 vs. 1740 ± 95 nM∙min, p = 0.0045), maximum thrombin concentration (134 ± 9.5 vs. 106 ± 6.5 nM, p = 4∙10(-6)), and increases in time to peak thrombin (27 ± 1.5 vs. 31 ± 2 min, p = 0.0012). Decreases in thrombin generation rate showed the highest statistical significance (13 ± 2 vs. 7.9 ± 0.8 nM/min, p = 10(-8)). Antiplatelet treatment did not alter thrombogram parameters for platelet-poor plasma.

  6. A partially active mutant aldolase B from a patient with hereditary fructose intolerance.

    PubMed

    Brooks, C C; Tolan, D R

    1994-01-01

    Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients are deficient in aldolase B, the isozyme expressed in fructose-metabolizing tissues. The eight protein coding exons, including splicing signals, of the aldolase B gene from one American HFI patient were amplified by the polymerase chain reaction (PCR). Single-strand conformational polymorphism (SSCP) analysis and direct sequence determination were applied to the amplified fragments. The mutations in the patient's alleles were identified as a nonsense mutation (R59op) in exon 3 and a missense mutation (C134R) in exon 5. These mutations were confirmed by sequence determination of cloned PCR-amplified exons 3 and 5 from the patient. Allele specific oligonucleotide (ASO) hybridizations of amplified exons 3 and 5 showed the Mendelian inheritance of both mutations. Site-directed mutagenesis was used to generate an expression plasmid for the C134R mutation, and the mutant enzyme was expressed in bacteria. Assays of partially purified enzyme preparations showed that this missense mutation results in an apparently unstable enzyme that retains partial activity. This is the first evidence for a partially active aldolase B from an HFI individual with an identified mutation, and supports the hypothesis that adequate gluconeogenesis/glycolysis is maintained in HFI patients by the presence of partially active enzymes.

  7. Exposure of Mice to Topical Bovine Thrombin Induces Systemic Autoimmunity

    PubMed Central

    Schoenecker, Jonathan G.; Johnson, Rachel K.; Lesher, Aaron P.; Day, Jarrod D.; Love, Stephanie D.; Hoffman, Maureane R.; Ortel, Thomas L.; Parker, William; Lawson, Jeffrey H.

    2001-01-01

    Bovine thrombin is used as an aid to hemostasis in medical and surgical procedures. At least 500,000 Americans are exposed to this therapeutic annually and reports suggest that exposure is associated with the development of autoreactive antibodies. To determine whether bovine thrombin can induce pathological autoimmunity we exposed nonautoimmune-prone galactose-α1-3-galactose-deficient mice to the two bovine thrombin preparations currently approved for use in the United States. We found that, like humans exposed to bovine thrombin, mice developed an immune response against the therapeutic and the xenogeneic carbohydrate galactose-α1-3-galactose, and some mice developed autoantibodies against clotting factors. Further, unexpectedly, a single exposure to this therapeutic also induced autoimmunity with features characteristic of systemic lupus erythematosus including antibodies against nuclear antigens, native DNA, double-stranded DNA, and cardiolipin. High levels of these autoantibodies correlated with glomerulonephritis in all mice evaluated. This autoimmune syndrome was detected in mice 15 weeks after a secondary exposure to bovine thrombin and female mice were found to develop the syndrome at a significantly greater frequency than males. Thus, these studies indicate that exposure to bovine thrombin preparations can induce a pathological systemic autoimmune syndrome with lupus-like serology. PMID:11696457

  8. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

  9. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.« less

  10. Quadratic partial eigenvalue assignment problem with time delay for active vibration control

    NASA Astrophysics Data System (ADS)

    Pratt, J. M.; Singh, K. V.; Datta, B. N.

    2009-08-01

    Partial pole assignment in active vibration control refers to reassigning a small set of unwanted eigenvalues of the quadratic eigenvalue problem (QEP) associated with the second order system of a vibrating structure, by using feedback control force, to suitably chosen location without altering the remaining large number of eigenvalues and eigenvectors. There are several challenges of solving this quadratic partial eigenvalue assignment problem (QPEVAP) in a computational setting which the traditional pole-placement problems for first-order control systems do not have to deal with. In order to these challenges, there has been some work in recent years to solve QPEVAP in a computationally viable way. However, these works do not take into account of the practical phenomenon of the time-delay effect in the system. In this paper, a new "direct and partial modal" approach of the quadratic partial eigenvalue assignment problem with time-delay is proposed. The approach works directly in the quadratic system without requiring transformation to a standard state-space system and requires the knowledge of only a small number of eigenvalues and eigenvectors that can be computed or measured in practice. Two illustrative examples are presented in the context of active vibration control with constant time-delay to illustrate the success of our proposed approach. Future work includes generalization of this approach to a more practical complex time-delay system and extension of this work to the multi-input problem.

  11. An electrochemical aptasensor based on TiO2/MWCNT and a novel synthesized Schiff base nanocomposite for the ultrasensitive detection of thrombin.

    PubMed

    Heydari-Bafrooei, Esmaeil; Amini, Maryam; Ardakani, Mehdi Hatefi

    2016-11-15

    A sensitive aptasensor based on a robust nanocomposite of titanium dioxide nanoparticles, multiwalled carbon nanotubes (MWCNT), chitosan and a novel synthesized Schiff base (SB) (TiO2/MWCNT/CHIT/SB) on the surface of a glassy carbon electrode (GCE) was developed for thrombin detection. The resultant nanocomposite can provide a large surface area, excellent electrocatalytic activity, and high stability, which would improve immobilization sites for biological molecules, allow remarkable amplification of the electrochemical signal and contribute to improved sensitivity. Thrombin aptamers were simply immobilized onto the TiO2-MWCNT/CHIT-SB nanocomposite matrix through simple π - π stacking and electrostatic interactions between CHIT/SB and aptamer strands. The electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to analyze the surface characterization of unmodified GCE and TiO2-MWCNT/CHIT-SB modified GCE, and also the interaction between aptamer and thrombin. In the presence of thrombin, the aptamer on the adsorbent layer captures the target on the electrode interface, which makes a barrier for electrons and inhibits electron transfer, thereby resulting in decreased DPV and increased impedance signals of the TiO2-MWCNT/CHIT-SB modified GCE. Furthermore, the proposed aptasensor has a very low LOD of 1.0fmolL(-1) thrombin within the detection range of 0.00005-10nmolL(-1). The aptasensor also presents high specificity and reproducibility for thrombin, which is unaffected by the coexistence of other proteins. Clinical application was performed with analysis of the thrombin levels in blood and CSF samples obtained from patients with MS, Parkinson, Epilepsy and Polyneuropathy using both the aptasensor and commercial ELISA kit. The results revealed the proposed system to be a promising candidate for clinical analysis of thrombin.

  12. Dilute Russell's viper venom and activated partial thromboplastin time in lupus anticoagulant diagnosis: is mixing essential?

    PubMed

    Chandrashekar, Vani

    2016-06-01

    Dilute Russell's viper venom (DRVV) testing and activated partial thromboplastin time (APTT) have been effectively used in combination for lupus anticoagulant testing. The purpose of our study was to evaluate the role of mixing in activated partial thromboplastin and dilute Russell's viper venom testing for evaluation of lupus anticoagulants. Citrated blood from patients who were not on oral anticoagulant therapy was studied. Mixing study with 1 : 1 normal plasma for elevated APTT and also few samples with elevated screen time was carried out. Elevated APTT was seen in only 48.1% of patients with lupus anticoagulant. Correction of APTT was seen in 27.8% of lupus anticoagulant-positive patients. DRVV test on mixing resulted in 83.8% false-negative values. Integrated DRVV test could be a standalone test for testing lupus anticoagulant. Mixing study may be restricted for patients on oral anticoagulants or patients with strong lupus anticoagulant.

  13. Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

    PubMed Central

    Ganesh, V.; Lee, A. Y.; Clardy, J.; Tulinsky, A.

    1996-01-01

    Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174

  14. Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide.

    PubMed

    Jensen, Baard Olav; Selheim, Frode; Døskeland, Stein Ove; Gear, Adrian R L; Holmsen, Holm

    2004-11-01

    The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistive-particle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PET-cGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-Br-cAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPT-cGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change.

  15. Treatment of partial seizures and seizure-like activity with felbamate in six dogs.

    PubMed

    Ruehlmann, D; Podell, M; March, P

    2001-08-01

    Six dogs with partial seizures or partial seizure-like activity were treated with the antiepileptic drug felbamate between 1993 and 1998. All dogs had a history and results of diagnostic testing suggestive of either primary (idiopathic) or occult secondary epilepsy. Dogs ranged between four months and eight years of age at the onset of seizure activity. The median time period between onset of the first seizure and the start of felbamate therapy was 3.8 months (range 0.75 to 36 months). Median duration of therapy was nine months (range two to 22 months). All dogs experienced a reduction in seizure frequency after felbamate administration. Median total number of seizures post-treatment was two (range 0 to 9). Two dogs had an immediate and prolonged cessation of seizure activity. Steady-state trough serum felbamate concentrations measured at two weeks, and one, 12 and 22 months after the commencement of therapy in four dogs ranged between 13 and 55 mg/litre (median 35 mg/litre). Reversible haematological adverse effects were detected in two dogs, with one dog developing concurrent keratoconjunctivitis sicca. These results suggest that felbamate can be an effective antiepileptic drug without life-threatening complications when used as monotherapy for partial seizures in the dog.

  16. Core-shell nanostructures for ultrasensitive detection of α-thrombin

    NASA Astrophysics Data System (ADS)

    Chen, Xia; Liu, Hongli; Zhou, Xiaodong; Hu, Jiming

    2010-12-01

    We have synthesized a stable, sensitive and specific surface-enhanced Raman tag, and demonstrated its application in human α-thrombin detection. The tag consists of aptamer-modified core-shell nanoparticles with hydrophobic Au@Ag as core and silica as shell encapsulating Raman active molecules. By taking advantage of the Raman signal enhancement effect by metallic nanostructures, high stability and robustness of glass-coated core-shell nanostructures and the recognition capabilities of aptamers, we designed a sandwich detection for protein identification with high selectivity and sensitivity. In this way, we realized the ultrasensitive detection of α-thrombin. GDNs (glass-coated, dye-tagged nanoparticles), which were conjugated with oligonucleotides or antibodies, were extremely soluble in water, and had mechanical and chemical stability, easily controllable-size distribution and friendly biocompatibility. Specifically, the glass coating renders the particles amenable to use in many solvents without altering the Raman spectral response and makes agglomeration a nonfactor. All these merits open the door of the real applications in diagnostics or medical investigations in complex biofluids, such as human plasma and serum. Using the aptamer-modified GDNs as Raman tags, we successfully performed the detection of α-thrombin in human plasma. Furthermore, the overall method have been proved effective and selective, and may be implemented for multiplex target analysis simultaneously.

  17. Efficient thrombin generation requires molecular phosphatidylserine, not a membrane surface.

    PubMed

    Majumder, Rinku; Weinreb, Gabriel; Lentz, Barry R

    2005-12-27

    Activation of prothrombin to thrombin is catalyzed by a "prothrombinase" complex, traditionally viewed as factor X(a) (FX(a)) in complex with factor V(a) (FV(a)) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through "channeling" (activation without the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human "prothrombinase" complex in solution. Both FX(a) and FV(a) bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX(a) and FV(a2) (light isoform of FV(a)), these proteins form a tight (Kd = 0.6 +/- 0.09 nM at 37 degrees C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX(a) and one FV(a2), further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 x 10(8) M(-1) s(-1)) and via the same reaction pathway (50-60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 x 10(8) M(-1) s(-1)). These results question the accepted role of PS membranes as providing "dimensionality reduction" and favor a regulatory role for platelet-membrane-exposed PS.

  18. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    PubMed Central

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  19. Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

    PubMed

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A; Gordon, Dalia; Gurevitz, Michael

    2010-10-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward

  20. Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat

    SciTech Connect

    Asami, K.; Kobayashi, H.; Fujiwara, A.; Yasumasu, I. )

    1989-09-01

    X rays (4.8 Gy) inhibit both DNA synthesis and phosphorylation of histone H1 in the regenerating liver of the rat. To determine the cause of the inhibition of histone H1 phosphorylation, changes in the nuclear protein kinase activities during the prereplicative phase of regeneration were measured. The cAMP-dependent protein kinase activity was low during regeneration, and the changes in the activity were not statistically significant. The cAMP-independent protein kinase activity increased at 15 h, decreased at 18 h, and increased again at 24 h after partial hepatectomy. X irradiation prior to partial hepatectomy did not inhibit the increase at 15 h, but it did inhibit the increase at 24 h. The activity was not inhibited by isoquinolinesulfonamide inhibitors such as H-7, and it was activated by a commercial preparation of an inhibitor protein of the cAMP-dependent kinase. It was also inhibited by quercetin. The possibility that the radiation-sensitive nuclear protein kinase is a nuclear cAMP-independent protein kinase specific for histone H1 is considered.

  1. Partially Oxidized Sub-10 nm MnO Nanocrystals with High Activity for Water Oxidation Catalysis

    PubMed Central

    Jin, Kyoungsuk; Chu, Arim; Park, Jimin; Jeong, Donghyuk; Jerng, Sung Eun; Sim, Uk; Jeong, Hui-Yun; Lee, Chan Woo; Park, Yong-Sun; Yang, Ki Dong; Kumar Pradhan, Gajendra; Kim, Donghun; Sung, Nark-Eon; Hee Kim, Sun; Nam, Ki Tae

    2015-01-01

    The oxygen evolution reaction (OER) is considered a major bottleneck in the overall water electrolysis process. In this work, highly active manganese oxide nano-catalysts were synthesized via hot injection. Facile surface treatment generated Mn(III) species on monodisperse 10 nm MnO nanocrystals (NCs). Size dependency of MnO NCs on OER activity was also investigated. Surprisingly, the partially oxidized MnO NCs only required 530 mV @ 5 mA cm−2 under near neutral conditions. PMID:25998696

  2. [Test for detection of activated partial thromboplastin time using ellagic acid].

    PubMed

    Berkovskiĭ, A L; Sergeeva, E V; Kachalova, N D; Prostakova, T M; Kozlov, A A

    1999-06-01

    A simple and sensitive method for estimation of activated partial thromboplastin time (APTT) is developed, making use a complex reagent containing the activator (plant phospholipids) and contact factor (ellagic acid). The test requires additionally only 0.025 M CaCl2. The test is more sensitive to the presence of heparin in the blood and to insufficiency of blood clotting factors VIII and IX than the reagents containing insoluble substances (kaolin and animal phosphatides). Addition of soluble ellagic acid into reagent for APTT estimation allows studies on optic coagulometers.

  3. The interaction of alpha-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin.

    PubMed Central

    Klausner, Y S; Rigbi, M; Ticho, T; De Jong, P J; Neginsky, E J; Rinott, Y

    1978-01-01

    The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin. Images PLATE 1 PMID:629742

  4. Thrombin-activatable fibrinolysis inhibitor in hypothyroidism and hyperthyroxinaemia.

    PubMed

    Verkleij, Chantal J N; Stuijver, Danka J F; van Zaane, Bregje; Squizzato, Alessandro; Brandjes, Dees P M; Büller, Harry R; Meijers, Joost C M; Gerdes, Victor E A

    2013-02-01

    Endocrine disorders affect both the coagulation and fibrinolytic systems, and have been associated with the development of cardiovascular diseases. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a link between coagulation and the fibrinolytic system. The aim of this study was to determine the effect of thyroid hormone excess and deficiency on TAFI levels and function. The effect of hyperthyroxinemia on TAFI was studied in healthy volunteers who were randomised to receive levothyroxine or no medication for 14 days in a crossover design. The effect of hypothyroidism on TAFI was studied in a multicentre observational cohort study. Blood was drawn before treatment of patients with newly diagnosed hypothyroidism and when euthyroidism was achieved. Plasma clot-lysis times, activated TAFI (TAFIa)-dependent prolongation of clot-lysis and TAFI levels were measured. Thyroid hormone excess resulted in a hypofibrinolytic condition and in an enhanced TAFIa-dependent prolongation of clot lysis. A trend towards decreased plasma TAFI levels was observed in healthy volunteers who used levothyroxine. Hypothyroidism resulted in hyperfibrinolysis and a reduced TAFIa-dependent prolongation of clot lysis. In conclusion, alterations of TAFIa-dependent prolongation of clot lysis in patients with thyroid disorders may cause an impaired haemostatic balance. The disturbed haemostatic balance in patients with hyperthyroidism might make them prone to thrombosis, while the risk for bleeding may increase in patients with hypothyroidism.

  5. Thrombin Increases Expression of Fibronectin Antigen on the Platelet Surface

    NASA Astrophysics Data System (ADS)

    Ginsberg, Mark H.; Painter, Richard G.; Forsyth, Jane; Birdwell, Charles; Plow, Edward F.

    1980-02-01

    Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

  6. A Novel Photoelectrochemical Biosensor for Tyrosinase and Thrombin Detection

    PubMed Central

    Chen, Jiexia; Liu, Yifan; Zhao, Guang-Chao

    2016-01-01

    A novel photoelectrochemical biosensor for step-by-step assay of tyrosinase and thrombin was fabricated based on the specific interactions between the designed peptide and the target enzymes. A peptide chain with a special sequence which contains a positively charged lysine-labeled terminal, tyrosine at the other end and a cleavage site recognized by thrombin between them was designed. The designed peptide can be fixed on surface of the CdTe quantum dots (QDs)-modified indium-tin oxide (ITO) electrode through electrostatic attraction to construct the photoelectrochemical biosensor. The tyrosinase target can catalyze the oxidization of tyrosine by oxygen into ortho-benzoquinone residues, which results in a decrease in the sensor photocurrent. Subsequently, the cleavage site could be recognized and cut off by another thrombin target, restoring the sensor photocurrent. The decrease or increase of photocurrent in the sensor enables us to assay tyrosinase and thrombin. Thus, the detection of tyrosinase and thrombin can be achieved in the linear range from 2.6 to 32 μg/mL and from 4.5 to 100 μg/mL with detection limits of 1.5 μg/mL and 1.9 μg/mL, respectively. Most importantly, this strategy shall allow us to detect different classes of enzymes simultaneously by designing various enzyme-specific peptide substrates. PMID:26805846

  7. Sensitivity of three activated partial thromboplastin time reagents to coagulation factor deficiencies.

    PubMed

    Turi, D C; Peerschke, E I

    1986-01-01

    Three activated partial thromboplastin time (APTT) reagent test systems, General Diagnostics Automated APTT, American Dade Actin FS, and Pacific Hemostasis (Thromboscreen KAPTT) reagent, containing different activators for the APTT assay, were evaluated for their precision and sensitivity to factor deficiencies in the intrinsic coagulation system. The data suggest that micronized silica and ellagic acid reagent systems were similar in sensitivity to Factor VIII, X, and XII deficiencies, whereas, the micronized kaolin reagent was significantly less sensitive to these deficiencies. Factor XI deficiency was detected equally well with the use of all three reagent systems. The ellagic acid reagent was somewhat more sensitive to Factor IX deficiency than the micronized silica reagent, and the micronized kaolin reagent was again least sensitive. Both the micronized silica and ellagic acid based reagents were insensitive to all but severe deficiencies in prekallikrein, whereas the micronized kaolin reagent was unable to detect this deficiency. All three reagents were insensitive to all but severe deficiencies in high-molecular-weight kininogen. The authors conclude that the reagent systems tested, containing micronized silica or ellagic acid as activators, are similar in sensitivity when used in a routine activated partial thromboplastin time to screen for factor deficiencies, whereas the reagent system containing micronized kaolin as an activator is less sensitive.

  8. [Preparation and evaluation of antibacterial activity of partial fractions of Vallesia antillana Wood].

    PubMed

    Rojas Hernández, N M; Cuellar Cuellar, A

    1978-01-01

    A study on the antibacterial properties of alkaloidal fractions of Vallesia antillana Wood, a Cuban plant belonging to the Apocynaceae family, is performed for the first time. Four partial fractions from bark of stems were prepared according to a classical extraction of total alkaloids and a partial fractionation of these, with the use of tartaric acid and ethyl ether and by a pH gradient with values of 2.5, 5.0, 7.0 and alkaline one. Data from thin layer chromatography on the alkaloidal content of different fractions are added and an assessment of antibacterial activity was made; for this, a method of sections in agar medium with bacterial species of human clinical interest was used; inhibitory halos found within 24 hours of incubation at 37 degrees C are reported in millimeters.

  9. A study on partially biodegradable microparticles as carriers of active glycolipids.

    PubMed

    López-Donaire, M L; Fernández-Gutiérrez, M; Parra-Cáceres, J; Vázquez-Lasa, B; García-Alvarez, I; Fernández-Mayoralas, A; Román, J San

    2010-04-01

    This paper describes a study on the preparation and characterisation of partially biodegradable microparticles of poly(epsilon-caprolactone)/poly(ethyl methacrylate) (PCL/PEMA) as carriers of synthetic glycolipids with antimitotic activity against brain tumour cells. Microparticles prepared by suspension polymerisation of methacrylate in the presence of already polymerised PCL showed a predominantly spherical but complex morphology, with segregation of PCL micro/nano-domains towards the surface. Small diameter discs were prepared by compression moulding of blends of microparticles and the active principle under mild conditions. The in vitro behaviour of the discs and release of the glycolipid were studied in different simulated fluid models. Ingress of fluids increased with increasing hydrophobicity of the medium. Release of the glycolipid was sustained in all fluids, the most prolonged profile being in human synovial fluid and phosphate-buffered saline modified with 20 vol.% dioxane. Slow disintegration of the discs and partial degradation of the microparticles was evident in accelerated studies. The antimitotic activity of glycolipid released from the discs was proved against a human glioblastoma line. This activity, along with selectivity against human fibroblasts, could be controlled by the amount of drug charged in the disc.

  10. 75 FR 51754 - Certain Activated Carbon from the People's Republic of China: Notice of Partial Rescission of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-23

    ... Activated Carbon from the People's Republic of China: Notice of Partial Rescission of Antidumping Duty... of initiation of an administrative review of the antidumping duty order on certain activated carbon... Activated Carbon Plant; Datong Forward Activated Carbon Co., Ltd.; Datong Guanghua Activated Carbon Co.,...

  11. Aptamer-based fiber sensor for thrombin detection

    NASA Astrophysics Data System (ADS)

    Coelho, Luís; Marques Martins de Almeida, José Manuel; Santos, José Luís; da Silva Jorge, Pedro Alberto; Martins, Maria Cristina L.; Viegas, Diana; Queirós, Raquel B.

    2016-08-01

    The detection of thrombin based on aptamer binding is studied using two different optical fiber-based configurations: long period gratings coated with a thin layer of titanium dioxide and surface plasmon resonance devices in optical fibers coated with a multilayer of gold and titanium dioxide. These structures are functionalized and the performance to detect thrombin in the range 10 to 100 nM is compared in transmission mode. The sensitivity to the surrounding refractive index (RI) of the plasmonic device is higher than 3100 nm RIU-1 in the RI range 1.335 to 1.355, a factor of 20 greater than the sensitivity of the coated grating. The detection of 10 nM of thrombin was accomplished with a wavelength shift of 3.5 nm and a resolution of 0.54 nM.

  12. Mechanism of poly(acrylic acid) acceleration of antithrombin inhibition of thrombin: implications for the design of novel heparin mimics.

    PubMed

    Monien, Bernhard H; Cheang, Kai I; Desai, Umesh R

    2005-08-11

    The bridging mechanism of antithrombin inhibition of thrombin is a dominant mechanism contributing a massive approximately 2500-fold acceleration in the reaction rate and is also a key reason for the clinical usage of heparin. Our recent study of the antithrombin-activating properties of a carboxylic acid-based polymer, poly(acrylic acid) (PAA), demonstrated a surprisingly high acceleration in thrombin inhibition (Monien, B. H.; Desai, U. R. J. Med. Chem. 2005, 48, 1269). To better understand this interesting phenomenon, we have studied the mechanism of PAA-dependent acceleration in antithrombin inhibition of thrombin. Competitive binding studies with low-affinity heparin and a heparin tetrasaccharide suggest that PAA binds antithrombin in both the pentasaccharide- and the extended heparin-binding sites, and these results are corroborated by molecular modeling. The salt-dependence of the K(D) of the PAA-antithrombin interaction shows the formation of five ionic interactions. In contrast, the contribution of nonionic forces is miniscule, resulting in an interaction that is significantly weaker than that observed for heparins. A bell-shaped profile of the observed rate constant for antithrombin inhibition of thrombin as a function of PAA concentration was observed, suggesting that inhibition proceeds through the "bridging" mechanism. The knowledge gained in this mechanistic study highlights important rules for the rational design of orally available heparin mimics.

  13. An anti-fouling aptasensor for detection of thrombin by dual polarization interferometry.

    PubMed

    Zheng, Yu; Hu, Tao; Chen, Chuanxia; Yang, Fan; Yang, Xiurong

    2015-04-04

    An anti-fouling surface was designed to effectively resist nonspecific protein adsorption using dual polarization interferometry, based on which the aptasensor for detection of thrombin was fabricated according to the specific interaction between thrombin and its 15-mer aptamer.

  14. Targeting thrombin: an inflammatory neurotoxin in Alzheimer's disease.

    PubMed

    Grammas, Paula; Martinez, Joseph M

    2014-01-01

    The Alzheimer's disease (AD) epidemic proceeds unabated. Estimates suggest 5.4 million Americans and 36 million people worldwide have AD. No single mechanism or pathologic mediator can account for AD progression. Currently no disease modifying therapies are available. There is a large literature documenting an association among cardiovascular risk factors (CVRFs), especially diabetes and hypoxia, with increased AD incidence. CVRFs directly impair vascular function and could mediate cerebrovascular dysfunction in AD. This is important as cerebrovascular dysfunction precedes cognitive decline and onset of neurodegenerative changes in AD and AD animal models. In this review we present evidence that thrombin may be a heretofore unexplored target for AD therapy. This idea is based on the following observations. Thrombin is elevated in the brain and cerebral microvasculature in AD, is directly neurotoxic, and causes pro-inflammatory effects in endothelial cells, microglia, and astrocytes. Diabetes- and hypoxia-induced cerebrovascular effects are mediated by thrombin. Thrombin inhibitors block the effects of hypoxia on brain endothelial cells and reduce vascular inflammation in transgenic AD mice. Based on reports that reducing cerebrovascular expression of inflammatory proteins in AD mice is associated with improved cognition, we propose thrombin inhibitors could prove useful for improving cognition in AD patients. The next generation of AD therapeutics should not focus on single target drugs but rather employ a multi-component cocktail approach. We propose thrombin inhibitors be considered as potential contributors to the dementia therapy pharmacopeia. The urgent need for disease-modifying drugs in AD demands new thinking about disease pathogenesis and exploration of novel drug targets.

  15. [Pancreatic tail pseudoaneurysm: percutaneous treatment by thrombin injection].

    PubMed

    Pacheco Jiménez, M; Moreno Sánchez, T; Moreno Rodríguez, F; Guillén Rico, M

    2014-01-01

    Visceral artery pseudoaneurysms secondary to acute and/or chronic pancreatitis are a relatively common and potentially serious complication. Endovascular techniques are the most currently accepted techniques, given the higher morbidity-mortality of surgery. The thrombosis of the pseudoaneurysm using an ultrasound-guided percutaneous thrombin injection is emerging as a useful option in those cases in which endovascular embolisation is not possible. We present the case of a patient with a pseudoaneurysm of the transverse pancreatic artery secondary to chronic pancreatitis, and successfully treated by administering percutaneous thrombin.

  16. Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress

    NASA Technical Reports Server (NTRS)

    Kudryashov, B. A.; Uljanov, A. M.; Shapiro, F. B.; Bazazyan, G. G.

    1981-01-01

    Thrombin marked with I-131 resulted in a considerable increase of the thrombined clearance rate in healthy male rats during stress caused by an immobilization lasting 30 minutes, and in an increase of thrombin clearance occurred by a combination of immobilization and administration of adrenocorticotropin (ACTH). Contrary to ACTH, the thrombin clearance is not stimulated in healthy animals by hydrocortisone. The results of the examination are presented.

  17. Increased 99mTc-MDP Activity in a Partially Calcified Malignant Mediastinal Teratoma.

    PubMed

    Li, Wei; Zhang, Linqi; Zhang, Rusen

    2016-02-01

    A 41-year-old woman presented with cough and shortness of breath for 3 weeks. Chest x-ray and CT showed a large, partially calcified soft tissue mass adjacent to the right side of the heart. Whole-body bone was acquired to evaluate possible metastases, which showed abnormal accumulation of Tc-MDP in the right chest. Further SPECT/CT imaging that demonstrated intense Tc-MDP activity was mainly in the calcification portion of mass. Histopathological examination from biopsy specimen of the lesion was consistent with malignant teratoma.

  18. Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

    PubMed Central

    Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

  19. Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists.

    PubMed

    Liberato, Marcelo Vizoná; Nascimento, Alessandro S; Ayers, Steven D; Lin, Jean Z; Cvoro, Aleksandra; Silveira, Rodrigo L; Martínez, Leandro; Souza, Paulo C T; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A R; Skaf, Munir S; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

  20. Anticoagulant activities of persicarin and isorhamnetin.

    PubMed

    Ku, Sae-Kwang; Kim, Tae Hoon; Bae, Jong-Sup

    2013-04-01

    Persicarin and isorhamnetin were isolated from Oenanthe javanica and their anticoagulant activities were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). In addition, the effects of persicarin and isorhamnetin on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). The data obtained showed that persicarin and isorhamnetin both prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. In addition, they both inhibited the generations of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, persicarin and isorhamnetin prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by persicarin. Interestingly, the anticoagulant and profibrinolytic effects of persicarin were greater than those of isorhamnetin, which suggest that the sulfonate group of persicarin positively regulates its anticoagulatory function. Accordingly, our results suggest that persicarin and isorhamnetin possess antithrombotic activities and that they could provide bases for the development of new anticoagulant agents.

  1. Factor XIa and Thrombin Generation Are Elevated in Patients with Acute Coronary Syndrome and Predict Recurrent Cardiovascular Events

    PubMed Central

    Loeffen, Rinske; van Oerle, René; Leers, Mathie P. G.; Kragten, Johannes A.; Crijns, Harry; Spronk, Henri M. H.; ten Cate, Hugo

    2016-01-01

    Objective In acute coronary syndrome (ACS) cardiac cell damage is preceded by thrombosis. Therefore, plasma coagulation markers may have additional diagnostic relevance in ACS. By using novel coagulation assays this study aims to gain more insight into the relationship between the coagulation system and ACS. Methods We measured plasma thrombin generation, factor XIa and D-dimer levels in plasma from ACS (n = 104) and non-ACS patients (n = 42). Follow-up measurements (n = 73) were performed at 1 and 6 months. Associations between coagulation markers and recurrent cardiovascular events were calculated by logistic regression analysis. Results Thrombin generation was significantly enhanced in ACS compared to non-ACS patients: peak height 148±53 vs. 122±42 nM. There was a significantly diminished ETP reduction (32 vs. 41%) and increased intrinsic coagulation activation (25 vs. 7%) in ACS compared to non-ACS patients. Furthermore, compared to non-ACS patients factor XIa and D-dimer levels were significantly elevated in ACS patients: 1.9±1.1 vs. 1.4±0.7 pM and 495(310–885) vs. 380(235–540) μg/L. Within the ACS spectrum, ST-elevated myocardial infarction patients had the highest prothrombotic profile. During the acute event, thrombin generation was significantly increased compared to 1 and 6 months afterwards: peak height 145±52 vs. 100±44 vs. 98±33 nM. Both peak height and factor XIa levels on admission predicted recurrent cardiovascular events (OR: 4.9 [95%CI 1.2–20.9] and 4.5 [1.1–18.9]). Conclusion ACS patients had an enhanced prothrombotic profile, demonstrated by an increased thrombin generation potential, factor XIa and D-dimer levels. This study is the first to demonstrate the positive association between factor XIa, thrombin generation and recurrent cardiovascular events. PMID:27419389

  2. Prolonged Activated Partial Thromboplastin Time: Difficulties in Discriminating Coexistent Factor VIII Inhibitor and Lupus Anticoagulant.

    PubMed

    Ames, Paul R J; Graf, Maria; Archer, Jeremy; Scarpato, Nicola; Iannaccone, Luigi

    2015-03-01

    To review the diagnostic difficulties of a prolonged activated partial thromboplastin time (aPTT) when 2 inhibitors with opposite clinical presentations coexist, we searched MEDLINE from January 1970 to November 2013 using acquired, factor VIII (FVIII), factor IX, hemophilia A and B, inhibitor, lupus anticoagulant (LA), antiphospholipid, anticardiolipin, anti-β2-glycoprotein I, antibodies, syndrome, bleeding, and thrombosis. We identified 13 articles for a total of 15 cases of possible coexistence of FVIII inhibitor and LA. The presenting clinical manifestation was thrombosis in 6 cases and bleeding in 9 cases. Activated partial thromboplastin time was the presenting laboratory abnormality in all cases, and first-line investigations suggested the coexistence of LA and acquired FVIII inhibitor. None of the articles addressed the diagnostic accuracy of the screening tests by performing "second line" assays. We reviewed the diagnostic pitfalls of the cases under study and provide some guidance for alternative tests when facing a prolonged aPTT that may have a double meaning.

  3. Deformation of partially pumped active mirrors for high average-power diode-pumped solid-state lasers.

    PubMed

    Albach, Daniel; LeTouzé, Geoffroy; Chanteloup, Jean-Christophe

    2011-04-25

    We discuss the deformation of a partially pumped active mirror amplifier as a free standing disk, as implemented in several laser systems. We rely on the Lucia laser project to experimentally evaluate the analytical and numerical deformation models.

  4. Analysis of the activated partial thromboplastin time test using mathematical modeling.

    PubMed

    Kogan, A E; Kardakov, D V; Khanin, M A

    2001-02-15

    Activated partial thromboplastin time (APTT) is a laboratory test for the diagnosis of blood coagulation disorders. The test consists of two stages: The first one is the preincubation of a plasma sample with negatively charged materials (kaolin, ellagic acid etc.) to activate factors XII and XI; the second stage begins after the addition of calcium ions that triggers a chain of calcium-dependent enzymatic reactions resulting in fibrinogen clotting. Mathematical modeling was used for the analysis of the APTT test. The process of coagulation was described by a set of coupled differential equations that were solved by the numerical method. It was found that as little as 2.3 x 10(-9) microM of factor XIIa (1/10000 of its plasma concentration) is enough to cause the complete activation of factor XII and prekallikrein (PK) during the first 20 s of the preincubation phase. By the end of this phase, kallikrein (K) is completely inhibited, residual activity of factor XIIa is 54%, and factor XI is activated by 26%. Once a clot is formed, factor II is activated by 4%, factor X by 5%, factor IX by 90%, and factor XI by 39%. Calculated clotting time using protein concentrations found in the blood of healthy people was 40.5 s. The most pronounced prolongation of APTT is caused by a decrease in factor X concentration.

  5. 21 CFR 864.7875 - Thrombin time test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Thrombin time test. 864.7875 Section 864.7875 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL... fibrinogen split products for the evaluation of bleeding disorders. (b) Classification. Class II...

  6. Oral direct thrombin inhibition: a double-edged sword?

    PubMed

    Fragasso, Gabriele; Corti, Angelo; Loiacono, Ferdinando; Margonato, Alberto; D'Angelo, Armando

    2015-01-01

    New oral anticoagulants have been shown to be not inferior to vitamin K antagonists in reducing thrombo-embolic events in patients with non-valvular atrial fibrillation and venous thrombo-embolism. However, those among them whichdirectly inhibit thrombin have been associated with greater risk of myocardial infarction. In this article we review the pleiotropic physiological effects of thrombin and their potential link with the observed greater incidence of myocardial infarction during therapy with oral direct thrombin inhibitors. On this basis, we believe that further studies are necessary to clear out doubts on the use of these drugs in the general population and, more specifically, in patients with coronary artery disease. For these reasons, in our opinion at present it may be prudent to especially caution high risk patients initiating therapy with a direct thrombin inhibitor (or those who are already taking it) about this possible risk. For patients with established coronary artery disease an alternative oral anticoagulant may be at present a better choice.

  7. Antitcoagulant and antiplatelet activities of scolymoside

    PubMed Central

    Yoon, Eun-Kyung; Ku, Sae-Kwang; Lee, Wonhwa; Kwak, Soyoung; Kang, Hyejin; Jung, Byeongjin; Bae, Jong-Sup

    2015-01-01

    Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain. Here, the anticoagulant effects of scolymoside, an active compound in C. subternata, were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin and activated factor X (FXa). The effects of scolymoside on plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) expression were evaluated in tumor necrosis factor (TNF)-α-activated human endothelial cells. Treatment with scolymoside resulted in prolonged aPTT and PT and the inhibition of thrombin and FXa activities and production. In addition, scolymoside inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Scolymoside also elicited anticoagulant effects in mice, including a significant reduction in the PAI-1 to t-PA ratio. Collectively, these findings indicate that scolymoside possesses anticoagulant activities and could be developed as a novel anticoagulant. [BMB Reports 2015; 48(10): 577-582] PMID:25887749

  8. Antithrombotic actions of the thrombin inhibitor, argatroban, in a canine model of coronary cyclic flow: comparison with heparin.

    PubMed Central

    Duval, N.; Lunven, C.; O'Brien, D. P.; Grosset, A.; O'Connor, S. E.; Berry, C. N.

    1996-01-01

    1. The antithrombotic action of argatroban, a synthetic thrombin inhibitor, was studied in a canine model of coronary cyclic flow having some of the characteristics of acute unstable angina. Heparin was studied as a reference anticoagulant. 2. Localized endothelial damage was induced in the circumflex coronary artery of anaesthetized open-chest foxhounds and a critical stenosis was applied by use of a Lexan constrictor placed around the artery at the site of endothelial damage. An electro-magnetic flow probe was placed distal to the lesion, and cyclic flow variations (CFVs) were observed, as thrombi formed at the site of the arterial lesion and were dislodged. Test compounds were administered by i.v. infusion commencing 1 h after the appearance of CFVs, and maintained for 1 h. On termination of the treatments, coronary flow was observed for a further 60 min. A series of blood samples were taken at predetermined times throughout each experiment in order to determine the coagulation parameters, thrombin time (TT) activated partial thromboplastin time (aPTT) and for the determination of fibrinopeptide A (FpA) levels before, during and post-treatment. 3. Argatroban and heparin showed antithrombotic effects in this model. Argatroban dose-dependently increased the minimum coronary flow at the nadir of the CFVs from 5.4 +/- 1.7 to 9.1 +/- 2.1 ml min-1 (30 micrograms kg-1 min-1, P = 0.041) and from 2.9 +/- 0.9 to 16.3 +/- 4.5 ml min-1 (100 micrograms kg-1 min-1, P = 0.023, n = 8 dogs at each dose level). Heparin (5 and 15 iu kg-1 min-1) also increased minimum flow, but the increase was not statistically significant at the 5% level, although the P value in animals treated with 15 iu kg-1 min-1 (P = 0.0521, n = 6 dogs) fell just outside this limit. Although neither compound significantly decreased the overall CFV frequency, argatroban (100 micrograms kg-1 min-1) significantly (P < 0.01) decreased the number of large amplitude CFVs (minimum coronary flow < 10 ml min-1) by 63

  9. Immobilization of the direct thrombin inhibitor-bivalirudin on 316L stainless steel via polydopamine and the resulting effects on hemocompatibility in vitro.

    PubMed

    Lu, Lei; Li, Quan-Li; Maitz, Manfred F; Chen, Jia-Long; Huang, Nan

    2012-09-01

    Bivalirudin (BV), a peptidic direct thrombin inhibitor, derived from hirudin, has gained increasing interest in clinical anticoagulant therapy in the recent years. In this work, a hemocompatible surface was prepared by immobilization of BV on 316L stainless steel (SS) using a bonding layer of polydopamine (DA). X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces to characterize polydopamine intermediate layer and the immobilized BV. The quantity of bound BV was measured by quartz crystal microbalance (QCM). The hemocompatibility in vitro was evaluated by coagulating time of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assay, platelet adhesion and activation, fibrinogen adsorption, and activation and whole blood test. The effect of sterilizing method on the bioactivity of immobilized BV was also evaluated. The results showed that BVs were successfully immobilized on SS surface with the DA interlayer at a density of 98 ng/cm(2) . BV coating surface prolonged aPTT and PT, inhibited the activation of platelet and fibrinogen significantly. Sterilization by ultraviolet radiation was possible with only marginal loss of activity. Thus, the approach described here may provide a basis for the preparation of 316L SS surface modification for use in cardiovascular implants.

  10. Bioregulatory Functions of Thrombin (Annals of the New York Academy of Sciences, Volume 485)

    DTIC Science & Technology

    1986-12-29

    procedure was repeated and 0.2 ml of the substrate p- nitrophenyl phosphate (Sigma), I mg/ml in 10% diethanolamine !b,.Wer, pH 9.8, waQ added. The color...were added at two different dilutions, 1/400 and 1/800. After an 18-h incubation at 4 C and washing, the substrate p- nitrophenyl phosphate was added...Platelet-Activation Factor Synthesis . By G. A. ZIMMERMAN, T. M. MCINTYRE, and S. M. PRESCOTT .......... 349 The Role of Thrombin in Transcellular

  11. Acquired factor V inhibitor after exposure to topical human thrombin related to an otorhinolaryngological procedure.

    PubMed

    Donohoe, K; Levine, R

    2015-10-01

    Acquired factor V (FV) inhibitors occur rarely and classically develop after exposure to bovine thrombin. The clinical presentation is variable, ranging from asymptomatic with incidental laboratory abnormalities to significant bleeding. With the development of human-derived thrombin agents, bovine thrombin is less frequently used. We report a case of an acquired FV inhibitor that developed in a patient after exposure to human thrombin used as a hemostatic agent during an otorhinolaryngology surgical procedure. Our review of the literature revealed only one prior reported case of FV inhibitor after exposure to human thrombin. Hematologists and surgeons should be aware of this rare, but potentially life-threatening, complication in the postprocedural setting.

  12. Direct thrombin inhibitors: a case indicating benefit from 'plasmapheresis' in toxicity: a call for establishing "guidelines" in overdose and to find an "antidote"!

    PubMed

    Kamboj, Jasmine; Kottalgi, Manjunath; Cirra, Vidyasagar Reddy; Shah, Nainesh; Kamboj, Rahul

    2012-11-01

    Patient presented with passage of fresh blood mixed with clots per rectum. In the ER, she was found to have bright red blood per rectum with clots, with frank blood on nasogastric tube. She was on dabigatran for atrial fibrillation and aspirin, with intermittent intake of ibuprofen. Vitals were positive for orthostatic hypotension. The pertinent findings in the physical examination were altered mental status with orientation*1, weak peripheral pulses, irregularly irregular heart rate, and bilateral pitting edema 2+ in bilateral lower extremities. Patient was intubated and put on mechanical ventilation. A massive transfusion protocol was followed. Laboratories and imaging: hemoglobin/hematocrit, 7.2/22.1; white blood cells, 7.7, platelet, 210; international normalized ratio, 2.5; prothrombin time, 19.2; activated partial thromboplastin time, 88.2; CMP was WNL; BNP, 621; fibrinogen, 500 mg/dL. Electrocardiogram showed atrial fibrillation with inferolateral ischemia. Ultrasonography of the liver and gallbladder showed no acute pathology. Echocardiogram showed an EF of 70% with hyperdynamic LV. Patient was transferred to the intensive care unit. Dabigatran, aspirin, and nonsteroidal anti-inflammatory drugs were discontinued, and antihypertensives were held. She was given blood and FFPs. Hemoglobin, hematocrit, and coagulation profile was monitored every 6 hours. Gastroenterology, general surgery, interventional radiology, and hematology services were called stat. IR placed a double-lumen, power central venous catheter. In gastroenterology, EGD and colonoscopy was performed, which showed active bleed at distal esophagus, stopped with local epinephrine. No active bleed seen on colonoscopy. The patient was put on Nexium drip. Hematology service recommended thrombin time (>200) and factors 2, 5, 7, 9, 10-41(l), 80, 68, 48(l), 61. Prothrombin time and activated partial thromboplastin time mixing studies were done, which indicated the presence of thrombin inhibition

  13. Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple's anticancer activity.

    PubMed

    He, Xiangjiu; Liu, Rui Hai

    2007-05-30

    Bioactivity-guided fractionation of apple peels was used to determine the chemical identity of bioactive constituents. Thirteen triterpenoids were isolated, and their chemical structures were identified. Antiproliferative activities of the triterpenoids against human HepG2 liver cancer cells, MCF-7 breast cancer cells, and Caco-2 colon cancer cells were evaluated. Most of the triterpenoids showed high potential anticancer activities against the three human cancer cell lines. Among the compounds isolated, 2alpha-hydroxyursolic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid showed higher antiproliferative activity toward HepG2 cancer cells. Ursolic acid, 2alpha-hydroxyursolic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid exhibited higher antiproliferative activity against MCF-7 cancer cells. All triterpenoids tested showed antiproliferative activity against Caco-2 cancer cells, especially 2alpha-hydroxyursolic acid, maslinic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid, which displayed much higher antiproliferative activities. These results showed the triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for the anticancer activities of whole apples.

  14. Cerebral blood flow during paroxysmal EEG activation induced by sleep in patients with complex partial seizures

    SciTech Connect

    Gozukirmizi, E.; Meyer, J.S.; Okabe, T.; Amano, T.; Mortel, K.; Karacan, I.

    1982-01-01

    Cerebral blood flow (CBF) measurements were combined with sleep polysomnography in nine patients with complex partial seizures. Two methods were used: the 133Xe method for measuring regional (rCBF) and the stable xenon CT method for local (LCBF). Compared to nonepileptic subjects, who show diffuse CBF decreases during stages I-II, non-REM sleep onset, patients with complex partial seizures show statistically significant increases in CBF which are maximal in regions where the EEG focus is localized and are predominantly seen in one temporal region but are also propagated to other cerebral areas. Both CBF methods gave comparable results, but greater statistical significance was achieved by stable xenon CT methodology. CBF increases are more diffuse than predicted by EEG paroxysmal activity recorded from scalp electrodes. An advantage of the 133Xe inhalation method was achievement of reliable data despite movement of the head. This was attributed to the use of a helmet which maintained the probes approximated to the scalp. Disadvantages were poor resolution (7 cm3) and two-dimensional information. The advantage of stable xenon CT method is excellent resolution (80 mm3) in three dimensions, but a disadvantage is that movement of the head in patients with seizure disorders may limit satisfactory measurements.

  15. Degeneration of dopaminergic neurons induced by thrombin injection in the substantia nigra of the rat is enhanced by dexamethasone: role of monoamine oxidase enzyme.

    PubMed

    Argüelles, Sandro; Herrera, Antonio J; Carreño-Müller, Eloisa; de Pablos, Rocío M; Villarán, Ruth F; Espinosa-Oliva, Ana M; Machado, Alberto; Cano, Josefina

    2010-01-01

    Anti-inflammatory strategies receive growing attention for their potential to prevent pathological deterioration in disorders such as Parkinson's disease, which is accompanied by inflammatory reactions that might play a critical role in the degeneration of nigral dopaminergic neurons. We investigated the influence of dexamethasone - a potent synthetic member of the glucocorticoids class of steroid hormones that acts as an anti-inflammatory - on the degeneration of the dopaminergic neurons of rats observed after intranigral injection of thrombin, a serine protease that induces inflammation through microglia proliferation and activation. We evaluated tyrosine hydroxylase (TH)-positive neurons as well as astroglial and microglial populations; dexamethasone prevented the loss of astrocytes but was unable to stop microglial proliferation induced by thrombin. Moreover, dexamethasone produced alterations in the levels of nexin and the thrombin receptor PAR-1, and facilitated accumulation of alpha-synuclein induced by thrombin in dopaminergic neurons. Dexamethasone increased oxidative stress and expression of monoamine oxidase A and B, along with changes on different MAP kinases related to degenerative processes, resulting in a bigger loss of dopaminergic neurons after intranigral injection of thrombin in dexamethasone-treated animals. It is interesting to ascertain that inhibition of monoamine oxidase by tranylcypromine prevented neurodegeneration of dopaminergic neurons, thus suggesting that the deleterious effects of dexamethasone might be mediated by monoamine oxidase.

  16. [Effect of composition of reagents for activated partial thromboplastin time on their sensitivity during analysis of blood coagulation factors].

    PubMed

    Berkovskiĭ, A L; Vasil'ev, S A; Sergeeva, E V; Kozlov, A A

    2000-04-01

    Brain cephaline-based reagents for evaluating activated partial thromboplastin time (APTT) and soybean phosphatides with ellagic acid complex activator with intermediate metal ions have been studied. The sensitivity of these reagents to internal clotting factors (VIII and IX) and heparin is determined by phospholipid nature and type of metal. The results help obtain highly active and sensitive APTT reagents.

  17. Effects of thrombin on the integrity of monolayers of cultured human endothelial cells

    SciTech Connect

    Galdal, K.S.; Evensen, S.A.; Brosstad, F.

    1982-09-01

    /sup 51/Cr-prelabelled endothelial cells (EC) in confluent monolayers were incubated in RPMI 1640 + foetal calf serum 20% (v/v) to which purified thrombin was added. Thrombin (greater than or equal to 0.1 NIH U/ml) significantly accelerated /sup 51/Cr-release and caused extensive but reversible cell contraction. Thrombin-exposed EC reacted to a new dose of thrombin with no appreciable shape change, but /sup 51/Cr-efflux was again accelerated. EC exposed to thrombin pretreated with N-bromosuccinimide (modifying the macromolecular site) or phenylmethylsulfonyl fluoride (blocking the serine site) retained normal morphology and did not leak excess amounts of /sup 51/Cr. Antithrombin III also inhibited the effect of thrombin. Pretreatment of EC with either indomethacin, aspirin, sulfinpyrazone, pronase or neuraminidase did not influence the effect of subsequent thrombin exposure.

  18. Active vibration control of thin-plate structures with partial SCLD treatment

    NASA Astrophysics Data System (ADS)

    Lu, Jun; Wang, Pan; Zhan, Zhenfei

    2017-02-01

    To effectively suppress the low-frequency vibration of a thin-plate, the strategy adopted is to develop a model-based approach to the investigation on the active vibration control of a clamped-clamped plate with partial SCLD treatment. Firstly, a finite element model is developed based on the constitutive equations of elastic, piezoelectric and viscoelastic materials. The characteristics of viscoelastic materials varying with temperature and frequency are described by GHM damping model. A low-dimensional real modal control model which can be used as the basis for active vibration control is then obtained from the combined reduction. The emphasis is placed on the feedback control system to attenuate the vibration of plates with SCLD treatments. A modal controller in conjunction with modal state estimator is designed to solve the problem of full state feedback, making it much more feasible to real-time control. Finally, the theoretical model is verified by modal test, and an active vibration control is validated by hardware-in-the-loop experiment under different external excitations. The numerical and experimental study demonstrate how the piezoelectric actuators actively control the lower modes (first bending and torsional modes) using modal controller, while the higher frequency vibration attenuated by viscoelastic passive damping layer.

  19. Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

    PubMed Central

    Yu, Yiqun; de March, Claire A.; Ni, Mengjue J.; Adipietro, Kaylin A.; Golebiowski, Jérôme; Matsunami, Hiroaki; Ma, Minghong

    2015-01-01

    Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood. PMID:26627247

  20. Activated Partial Thromboplastin Time Monitoring of Unfractionated Heparin Therapy: Issues and Recommendations.

    PubMed

    Marlar, Richard A; Clement, Bernadette; Gausman, Jana

    2016-06-06

    When administering unfractionated heparin (UFH), therapeutic levels of anticoagulation must be achieved rapidly and maintained consistently in the therapeutic range. The basic assays for monitoring UFH therapy are the activated partial thromboplastin time (APTT) and/or the chromogenic antifactor Xa or antithrombin assays. For many laboratories, the APTT is the preferred standard of practice; however, the APTT is a surrogate marker that only estimates the heparin concentration. Many factors, including patient variation, reagents of the APTT, UFH composition, and concentration can influence the APTT result. This article reviews various methods to determine the heparin therapeutic range and presents recommendations for the laboratory to establish an APTT heparin therapeutic range for all sizes of hospitals.

  1. A patient with a prolonged activated partial thromboplastin time and a deep intracerebral haemorrhage.

    PubMed

    Schindhelm, Roger K; Wondergem, Mariëlle J; Admiraal, Joke; Nap, Gert; Boekel, Edwin Ten; Hani, Lahcen

    2012-05-01

    We report on a 57-year-old woman with a pontine haemorrhage and an extremely prolonged activated partial thromboplastin time (aPTT) of more than 240 s, suggestive of a coagulation disorder. Given the location of the haemorrhage, which is associated with a high mortality rate, recombinant factor VIIa was administered, although not all necessary laboratory analyses could be performed at that time. In our case, a deficiency of factor XII was found, which is not associated with an increased bleeding risk. In an acute setting, evaluation of a prolonged aPTT may cause diagnostic and therapeutic challenges, in particular in situations where additional laboratory investigations may not be readily available.

  2. Benzyloxycarbonyl-D-Phe-Pro-methoxypropylboroglycine: a novel inhibitor of thrombin with high selectivity containing a neutral side chain at the P1 position.

    PubMed Central

    Claeson, G; Philipp, M; Agner, E; Scully, M F; Metternich, R; Kakkar, V V; DeSoyza, T; Niu, L H

    1993-01-01

    Thrombin, the blood-clotting enzyme, is a serine proteinase with trypsin-like specificity and is able to cleave Arg-Xaa peptide bonds but only in a very limited number of substrates (and sites therein). For the prevention and treatment of thrombosis the control of thrombin activity is a key target, and a variety of synthetic inhibitors have been introduced recently, all of which have a positive charge at the P1 site. We report the synthesis of the first example of a new class of inhibitor containing a neutral side chain at the P1 site, the peptide benzyloxycarbonyl-D-Phe-Pro- methoxypropylboroglycine. The peptide is a potent inhibitor of thrombin [Ki (limiting) = 7 nM] and is highly selective for its target enzyme in respect of other serine proteinases. This may be expected to confer considerable advantage in terms of specificity of action and reduced toxicity over conventional, positively charged, inhibitors. PMID:8452516

  3. Artifactual prolongation of activated partial thromboplastin time with PEGylated compounds in silica-based assays.

    PubMed

    Murphy, Bethanne; Parrula, Maria C M; Perigard, Christopher J; Li, Jinze

    2014-12-01

    Conjugation of polyethylene glycol (PEG) to another molecule prolongs its half-life in the body, but has a potential to artifactually increase the activated partial thromboplastin time (aPTT) as measured with certain assays. Studies conducted in-house at Bristol-Myers Squibb using the STA-PTT Automate 5 assay, the routine assay used to measure aPTT, demonstrated prolongation of aPTT in plasma samples spiked in vitro with 40-kDa branched PEG (PEG40) conjugated compounds or PEG40 alone, but not in samples spiked with vehicle or non-PEGylated compound, suggesting that the interference is because of the PEG40 moiety. To investigate the cause of this phenomenon, cynomolgus monkey and rat plasma samples were spiked with different concentrations of PEG40 and the aPTT was measured using different proprietary assays. With one exception, prolongation of aPTT was observed with all assays containing silica as the contact activator. No changes in aPTT were noted in assays using kaolin as a contact activator. The findings indicated that the observed prolongation of aPTT is largely because of interference of PEG40 with the silica, but other features of the reagent mixture may also influence aPTT times.

  4. Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora.

    PubMed

    Silva, T M A; Alves, L G; de Queiroz, K C S; Santos, M G L; Marques, C T; Chavante, S F; Rocha, H A O; Leite, E L

    2005-04-01

    The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-beta-D-glucuronic acid 1-> or 4-beta-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or beta-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

  5. Thrombin generation in Cushing's Syndrome: do the conventional clotting indices tell the whole truth?

    PubMed

    Koutroumpi, S; Spiezia, L; Albiger, N; Barbot, M; Bon, M; Maggiolo, S; Gavasso, S; Simioni, P; Frigo, A; Mantero, F; Scaroni, C

    2014-02-01

    Cushing's Syndrome (CS) is associated with an increased mortality, where hypercoagulability seems to have a crucial role in both arterial and venous thrombosis. Parameters of in vitro thrombin generation (TG) such as lag time, peak thrombin and endogenous thrombin potential (ETP), that describe the time until thrombin burst, the peak amount of TG and the total amount of thrombin generated, respectively as well as classical clotting markers were evaluated in 33 CS patients compared to both a group of 28 patients matched for the features of Metabolic Syndrome (MetS) and 31 healthy individuals. CS and MetS patients had shorter lag time (p < 0.0001), higher peak and ETP (p < 0.0001) than healthy controls, though lag time was less shortened in CS (p < 0.0001) respect to MetS group. Prothrombin time (PT) was increased (p < 0.0001) in both CS and MetS patients, while partial thromboplastin time (PTT) was shorter (p < 0.0001) in CS compared to both MetS and healthy group (p < 0.0001). Factor VIII (FVIII), Antithrombin (AT), protein C and S were increased only in CS patients (p < 0.0001). lag time, AT and FVIII correlated to night salivary cortisol (r = + 0.59; p = 0.0005, r = + 0.40; p = 0.003, r = + 0.40; p = 0.04, respectively); PTT correlated inversely to urinary free cortisol (r = -0.45; p = 0.009). BMI correlated negatively to lag time (r = -0.40; p = 0.0001) and positively to peak and ETP (r = + 0.34; p = 0.001, r = + 0.28; p = 0.008, respectively). Obese and diabetic patients had shorter lag time (p = 0.0005; p = 0.0002, respectively), higher ETP (p = 0.0006; p = 0.007, respectively) and peak (p = 0.0003; p = 0.0005, respectively) as well as a more prolonged PT (p = 0.04; p = 0.009, respectively). Hypertensive individuals had higher ETP (p = 0.004), peak (p = 0.0008) and FVIII (p = 0.001). Our findings confirm a prothrombotic state in both CS and MetS patients, though lag time was less shortened in

  6. Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity.

    PubMed

    Gierczak, Richard F; Bhakta, Varsha; Xie, Michael; Sheffield, William P

    2015-08-20

    Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352-357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering.

  7. Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy

    PubMed Central

    Enko, Dietmar; Mangge, Harald; Münch, Andreas; Niedrist, Tobias; Mahla, Elisabeth; Metzler, Helfried; Prüller, Florian

    2017-01-01

    Introduction The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%. Conclusions Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced. PMID:28392742

  8. THROMBIN GENERATION AND PROCOAGULANT MICROPARTICLE (MP) PROFILES AFTER ACUTE TRAUMA: A PROSPECTIVE COHORT STUDY

    PubMed Central

    Park, Myung S.; Xue, Ailing; Spears, Grant M.; Halling, Timothy M.; Ferrara, Michael J.; Kuntz, Melissa M.; Dhillon, Sabtir K.; Jenkins, Donald H.; Harmsen, William S.; Ballman, Karla V.; Heit, John A.

    2015-01-01

    Objective The two sides of Trauma Induced Coagulopathy (TIC), the hypo- and the hyper- coagulable states, are poorly understood. To identify potential mechanisms for venous thromboembolism and bleeding after acute trauma, we estimated changes in circulating procoagulant MPs and thrombin activity during hospitalization for trauma. Methods Whole blood was collected by venipuncture into 3.2% trisodium citrate at 0, 6, 12, 24 and 72 hours after injury, and discharge. Platelet poor plasma was harvested and stored at −80°C until analysis. Thrombin generation was determined using the calibrated automated thrombogram (CAT), reported as lagtime (minutes), peak height (nM thrombin) and time to reach peak height (ttPeak - minutes). The concentration of total procoagulant MPs (number/uL]) was measured by flow cytometry. Data are presented as median [interquartile range]. Results Among 443 trauma patients (1734 samples; ISS=13.0 [6.0, 22.0], hospital LOS=4.0 [2.0, 10.0] days, age=48 [28, 65] years, 70.7% male, 95% with blunt mechanism, mortality 3.2%), no discernable patterns in thrombin generation or MP concentration were observed over time. The peak height and MPs were significantly different from healthy volunteers and were 337 [285, 395] nM and 400 [211, 772] per uL plasma, respectively. Extreme (defined as highest or lowest 5%) values reflecting a possible “hypercoagulable state” (lagtime ≤ 1.98, peak height ≥ 486.2, ttPeak ≤ 3.61, and total procoagulant MP ≥ 2278) were reached within 12 hours after acute trauma, while extreme values representing a possible “hypocoagulable state” (lagtime ≥ 18.6, peak height ≤ 17.8 and ttPeak ≥ 29.45) were not reached until 1-3 days. Conclusion Although there was no predictable pattern of coagulopathy observed in each patient after trauma, those who reached extreme values did so relatively early after injury. These findings should be taken into account when designing risk model tools involving coagulation

  9. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  10. Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

    NASA Astrophysics Data System (ADS)

    Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

    Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

  11. Thrombin-induced translocation of GLUT3 glucose transporters in human platelets.

    PubMed Central

    Sorbara, L R; Davies-Hill, T M; Koehler-Stec, E M; Vannucci, S J; Horne, M K; Simpson, I A

    1997-01-01

    Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155+/-18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 degrees C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3, -bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands

  12. How to report results of prothrombin and activated partial thromboplastin times.

    PubMed

    Tripodi, Armando; Lippi, Giuseppe; Plebani, Mario

    2016-02-01

    Prothrombin time (PT) and activated partial thromboplastin time (APTT) are the most widely used tests to investigate coagulation abnormalities. Varied result reporting have been introduced over the years for the two tests, thus making their interpretation rather confusing in different clinical settings. PT results have been reported as clotting time, percentage activity, PT-ratio (patient-to-normal clotting time) and as international normalized ratio (INR). The INR scale has been devised to harmonize results stemming from different thromboplastins from patients on treatment with vitamin K antagonists. Therefore, there are some theoretical and evidence-based considerations that make the INR formally invalid when the test is used to analyze patients in other clinical settings. Unfortunately, this limitation has been frequently overlooked, and the INR has been (and is currently) used as a universal system of results harmonization. The APTT has been historically reported as clotting time or as ratio (patient-to-normal clotting time). In this opinion paper we review the current state-of-the-art for result reporting and attempt to give practical guidance on how PT and APTT should be reported in different clinical conditions for which the tests are requested.

  13. Mnemonic strategy training partially restores hippocampal activity in patients with mild cognitive impairment

    PubMed Central

    Hampstead, Benjamin M.; Stringer, Anthony Y.; Stilla, Randall F.; Giddens, Michelle; Sathian, K.

    2012-01-01

    Learning and memory deficits typify patients with mild cognitive impairment (MCI) and are generally attributed to medial temporal lobe dysfunction. Although the hippocampus is perhaps the most commonly studied neuroanatomical structure in these patients, there have been few attempts to identify rehabilitative interventions that facilitate its functioning. Here, we present results from a randomized, controlled, single-blind study in which patients with MCI and healthy elderly controls (HEC) were randomized to either 3 sessions of mnemonic strategy training (MS) or a matched-exposure control group (XP). All participants underwent pre- and post-training fMRI scanning as they encoded and retrieved object-location associations. For the current report, fMRI analyses were restricted to the hippocampus, as defined anatomically. Before training, MCI patients showed reduced hippocampal activity during both encoding and retrieval, relative to HEC. Following training, the MCI MS group demonstrated increased activity during both encoding and retrieval. There were significant differences between the MCI MS and MCI XP groups during retrieval, especially within the right hippocampus. Thus, MS facilitated hippocampal functioning in a partially restorative manner. We conclude that cognitive rehabilitation techniques may help mitigate hippocampal dysfunction in MCI patients. PMID:22368035

  14. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed Central

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate. PMID:11536662

  15. Active learning of plans for safety and reachability goals with partial observability.

    PubMed

    Nam, Wonhong; Alur, Rajeev

    2010-04-01

    Traditional planning assumes reachability goals and/or full observability. In this paper, we propose a novel solution for safety and reachability planning with partial observability. Given a planning domain, a safety property, and a reachability goal, we automatically learn a safe permissive plan to guide the planning domain so that the safety property is not violated and that can force the planning domain to eventually reach states that satisfy the reachability goal, regardless of how the planning domain behaves. Our technique is based on the active learning of regular languages and symbolic model checking. The planning method first learns a safe plan using the L (*) algorithm, which is an efficient active learning algorithm for regular languages. We then check whether the safe plan learned is also permissive by Alternating-time Temporal Logic (ATL) model checking. If the plan is permissive, it is indeed a safe permissive plan. Otherwise, we identify and add a safe string to converge a safe permissive plan. We describe an implementation of the proposed technique and demonstrate that our tool can efficiently construct safe permissive plans for four sets of examples.

  16. Preparation of Partially Poisoned Alkanethiolate-Capped Platinum Nanoparticles for Hydrogenation of Activated Terminal Alkynes.

    PubMed

    San, Khin Aye; Chen, Vivian; Shon, Young-Seok

    2017-03-22

    Stable and isolable alkanethiolate-stabilized Pt nanoparticles (PtNP) were synthesized using the two-phase thiosulfate method with sodium S-alkylthiosulfate as ligand precursor. The mechanistic formation of octanethiolate-capped PtNP (Pt-SC8) from both sodium S-octylthiosulfate and 1-octanethiol ligands was investigated by using (1)H NMR and UV-vis spectroscopies, which revealed the formation of different Pt complexes as the reaction intermediates. The synthesis using S-octylthiosulfate ligand precursor produced Pt-SC8 in higher yields than that using 1-octanethiol ligand. The obtained nanoparticles were characterized by (1)H NMR, UV-vis spectroscopy, infrared spectroscopy (IR), thermogravimetric analysis, and transmission electron microscopy (TEM). The results obtained from (1)H NMR, IR, and UV-vis spectroscopy were consistent with the formation of stable and pure alkanethiolate-capped PtNP. TEM images of PtNP confirmed their small average core size (∼1.5 nm) and high monodispersity. The partially poisoned PtNP with thiolate monolayer ligands were further investigated for the hydrogenation of various alkynes to understand the organic ligands-induced geometric and electronic surface properties of colloidal Pt nanoparticle catalysts. The high catalytic activity of activated terminal alkynes, but the significantly low activity of internal alkynes and unactivated terminal alkynes, were observed under the mild reaction conditions (room temperature and atmospheric pressure). These results indicated that the presence of alkanethiolate ligands could decrease the coordination activity of PtNP surface especially for the bulkier and unactivated substrates.

  17. Thrombin aptasensing with inherently electroactive graphene oxide nanoplatelets as labels

    NASA Astrophysics Data System (ADS)

    Loo, Adeline Huiling; Bonanni, Alessandra; Pumera, Martin

    2013-05-01

    Graphene and its associated materials are commonly used as the transducing platform in biosensing. We propose a different approach for the application of graphene in biosensing. Here, we utilized graphene oxide nanoplatelets as the inherently electroactive labels for the aptasensing of thrombin. The basis of detection lies in the ability of graphene oxide to be electrochemically reduced, thereby providing a well-defined reduction wave; one graphene oxide nanoplatelet of dimension 50 × 50 nm can provide a reduction signal by accepting ~22 000 electrons. We demonstrate that by using graphene oxide nanoplatelets as an inherently electroactive label, we can detect thrombin in the concentration range of 3 pM-0.3 μM, with good selectivity of the aptamer towards interferences by bovine serum albumin, immunoglobulin G and avidin. Therefore, the inherently electroactive graphene oxide nanoplatelets are a material which can serve as an electroactive label, in a manner similar to metallic nanoparticles.

  18. Antiplatelet, anticoagulant, and profibrinolytic activities of cudratricusxanthone A.

    PubMed

    Yoo, Hayoung; Ku, Sae-Kwang; Lee, Wonhwa; Kwak, Soyoung; Baek, Young-Doo; Min, Byung-Woon; Jeong, Gil-Saeng; Bae, Jong-Sup

    2014-08-01

    Cudratricusxanthone A (CTXA), a natural bioactive compound extracted from the roots of Cudrania tricuspidata Bureau, is known to possess hepatoprotective, antiproliferative and anti-inflammatory activities. However, antiplatelet, anticoagulant, and profibrinolytic properties have not been studied. The anticoagulant activities of CTXA were measured by monitoring activated partial thromboplastin-time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). The effects of CTXA on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells. Our data showed that CTXA inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation, prolonged aPTT and PT significantly and inhibited the activities and production of thrombin and FXa. CTXA prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by CTXA. Collectively, these results indicate that CTXA possesses antithrombotic activities and suggest that the current study could provide bases for the development of new anticoagulant agents.

  19. Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

    SciTech Connect

    Thompson, Scott K.; Washburn, David G.; Frazee, James S.; Madauss, Kevin P.; Hoang, Tram H.; Lapinski, Leahann; Grygielko, Eugene T.; Glace, Lindsay E.; Trizna, Walter; Williams, Shawn P.; Duraiswami, Chaya; Bray, Jeffrey D.; Laping, Nicholas J.

    2010-09-03

    Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

  20. Thrombin, kallikrein and complement C5b-9 adsorption on hydrophilic and hydrophobic titanium and glass after short time exposure to whole blood.

    PubMed

    Yahyapour, Noushin; Eriksson, Cecilia; Malmberg, Per; Nygren, Håkan

    2004-07-01

    Hydrophilic and hydrophobic titanium and glass were exposed to capillary whole blood between 5s and 24h. The time-sequence for adsorption of thrombin, kallikrein and complement C5b-9, and their relationship with adherent platelets and polymorphonuclear granulocyte (PMN) activation were investigated. Adsorbed thrombin and kallikrein were measured by cleavage of specific chromogenic substances, S-2238 and S-2303, respectively. Complement C5b-9 and expression of CD11b, CD66b, CD62P and Pan-platelets were measured by immunofluorescence. Thrombin and kallikrein were present on the surfaces during the whole investigated periods. Platelet adhesion and PMN cell adhesion and activation on all surfaces and activation of platelets on hydrophobic surfaces showed a similar pattern to thrombin adsorption. Kallikrein adsorption had a different pattern on each surface. C5b-9 was detected between 32min and 24h of blood exposure and a varying pattern of C5b-9 coverage was observed on each surface. In conclusion, our results indicate that the interaction between material and blood coagulation and kinin-activating proteins regulate the adhesion and activation of blood cells, whereas after longer time the coagulation and kallikrein-kinin system play minor roles and the complement system is decisive for mediating and elongating the inflammatory process.

  1. Yersinia pestis YopM: thrombin binding and overexpression.

    PubMed Central

    Reisner, B S; Straley, S C

    1992-01-01

    In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression. Images PMID:1452357

  2. Thrombin-Based Hemostatic Agent in Primary Total Knee Arthroplasty.

    PubMed

    Fu, Xin; Tian, Peng; Xu, Gui-Jun; Sun, Xiao-Lei; Ma, Xin-Long

    2017-02-01

    The present meta-analysis pooled the results from randomized controlled trials (RCTs) to identify and assess the efficacy and safety of thrombin-based hemostatic agent in primary total knee arthroplasty (TKA). Potential academic articles were identified from the Cochrane Library, Medline (1966-2015.5), PubMed (1966-2015.5), Embase (1980-2015.5), and ScienceDirect (1966-2015.5). Relevant journals and the recommendations of expert panels were also searched by using Google search engine. RCTs assessing the efficacy and safety of thrombin-based hemostatic agent in primary TKA were included. Pooling of data was analyzed by RevMan 5.1 (The Cochrane Collaboration, Oxford, UK). A total of four RCTs met the inclusion criteria. The meta-analysis revealed significant differences in postoperative hemoglobin decline (p < 0.00001), total blood loss (p < 0.00001), drainage volume (p = 0.01), and allogenic blood transfusion (p = 0.01) between the treatment group and the control group. No significant differences were found regarding incidence of infection (p = 0.45) and deep vein thrombosis (DVT; p = 0.80) between the groups. Meta-analysis indicated that the application of thrombin-based hemostatic agent before wound closure decreased postoperative hemoglobin decline, drainage volume, total blood loss, and transfusion rate and did not increase the risk of infection, DVT, or other complications. Therefore, the reviewers believe that thrombin-based hemostatic agent is effective and safe in primary TKA.

  3. Effects of the glycoprotein IIb/IIIa receptor antagonist c7E3 Fab and anticoagulants on platelet aggregation and thrombin potential under high coagulant challenge in vitro.

    PubMed

    Koestenberger, M; Gallistl, S; Cvirn, G; Roschitz, B; Muntean, W

    2000-07-01

    The present study was performed to investigate the combined effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab (abciximab) and the anticoagulants unfractionated heparin (UH), low molecular weight heparin (LMWH), and recombinant hirudin (rH) on platelet aggregation and thrombin generation under high coagulant challenge by extrinsic activation of platelet-rich plasma. Platelet aggregation and thrombin generation were assessed simultaneously in the presence of different concentrations of abciximab and anticoagulants. Increasing concentrations of abciximab resulted in a dose-dependent anti-aggregating effect with a maximum at 20 microg/ml. Doses of 5, 10, and 20 microg/ml abciximab prolonged the lag phase until the onset of platelet aggregation, but this effect was independent of the dosage used. Abciximab had no influence on the thrombin potential under our high coagulant challenge. UH, LMWH, and rH showed a dose-dependent prolongation of the lag phase until the onset of platelet aggregation and decreased the thrombin potential. Addition of anticoagulants did not contribute to further inhibition of platelet aggregation in the presence of abciximab, but the combination of abciximab and anticoagulants exhibited an additive effect on prolongation of the lag phase until the onset of platelet aggregation. Addition of abciximab to anticoagulants did not result in further decrease of the thrombin potential. Our study demonstrates the respective specific effects of abciximab and anticoagulants on platelet aggregation and thrombin potential under high coagulant challenge, and also an additive effect of abciximab and the anticoagulants UH, LMWH, and rH on the lag phase until the onset of platelet aggregation.

  4. Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered fluorescence protein-functionalized monolithic capillary column.

    PubMed

    Lin, Lihua; Liu, Shengquan; Nie, Zhou; Chen, Yingzhuang; Lei, Chunyang; Wang, Zhen; Yin, Chao; Hu, Huiping; Huang, Yan; Yao, Shouzhuo

    2015-04-21

    Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

  5. GBF-dependent family genes morphologically suppress the partially active Dictyostelium STATa strain.

    PubMed

    Shimada, Nao; Kanno-Tanabe, Naoko; Minemura, Kakeru; Kawata, Takefumi

    2008-02-01

    Transcription factor Dd-STATa, a functional Dictyostelium homologue of metazoan signal transducers and activators of transcription proteins, is necessary for culmination during development. We have isolated more than 18 putative multicopy suppressors of Dd-STATa using genetic screening. One was hssA gene, whose expression is known to be G-box-binding-factor-dependent and which was specific to prestalk A (pstA) cells, where Dd-STATa is activated. Also, hssA mRNA was expressed in pstA cells in the Dd-STATa-null mutant. At least 40 hssA-related genes are present in the genome and constitute a multigene family. The tagged HssA protein was translated; hssA encodes an unusually high-glycine-serine-rich small protein (8.37 kDa), which has strong homology to previously reported cyclic-adenosine-monophosphate-inducible 2C and 7E proteins. Overexpression of hssA mRNA as well as frame-shifted versions of hssA RNA suppressed the phenotype of the partially active Dd-STATa strain, suggesting that translation is not necessary for suppression. Although overexpression of prespore-specific genes among the family did not suppress the parental phenotype, prestalk-specific family members did. Although overexpression of the hssA did not revert the expression of Dd-STATa target genes, and although its suppression mechanism remains unknown, morphological reversion implies functional relationships between Dd-STATa and hssA.

  6. Functional identification of neural stem cell-derived oligodendrocytes by means of calcium transients elicited by thrombin.

    PubMed

    Grade, Sofia; Agasse, Fabienne; Bernardino, Liliana; Silva, Carla G; Cortes, Luísa; Malva, João O

    2010-02-01

    Current immunosuppressive treatments for central nervous system demyelinating diseases fail to prevent long-term motor and cognitive decline in patients. Excitingly, glial cell transplantation arises as a promising complementary strategy to challenge oligodendrocytes loss occurring in myelination disorders. A potential source of new oligodendrocytes is the subventricular zone (SVZ) pool of multipotent neural stem cells. However, this approach has been handicapped by the lack of functional methods for identification and pharmacological analysis of differentiating oligodendrocytes, prior to transplantation. In this study, we questioned whether SVZ-derived oligodendrocytes could be functionally discriminated due to intracellular calcium level ([Ca(2+)](i)) variations following KCl, histamine, and thrombin stimulations. Previously, we have shown that SVZ-derived neurons and immature cells can be discriminated on the basis of their selective [Ca(2+)](i) rise upon KCl and histamine stimulation, respectively. Herein, we demonstrate that O4+ and proteolipid protein-positive (PLP+) oligodendrocytes do not respond to these stimuli, but display a robust [Ca(2+)](i) rise following thrombin stimulation, whereas other cell types are thrombin-insensitive. Thrombin-induced Ca(2+) increase in oligodendrocytes is mediated by protease-activated receptor-1 (PAR-1) activation and downstream signaling through G(q/11) and phospholipase C (PLC), resulting in Ca(2+) recruitment from intracellular compartments. This method allows the analysis of functional properties of oligodendrocytes in living SVZ cultures, which is of major interest for the development of effective grafting strategies in the demyelinated brain. Additionally, it opens new perspectives for the search of new pro-oligodendrogenic factors to be used prior grafting.

  7. Clotting factor VIII (FVIII) and thrombin generation in camel plasma: A comparative study with humans

    PubMed Central

    Abdel Gader, Abdel Galil M.; Al Momen, Abdul Karim M.; Alhaider, Abdulqader; Brooks, Marjory B.; Catalfamo, James L.; Al Haidary, Ahmed A.; Hussain, Mansour F.

    2013-01-01

    The objective of this study was to characterize the highly elevated levels of clotting factor VIII (FVIII) in camel plasma. Whole blood was collected from healthy camels and factor VIII clotting activity (FVIII:C) assays were conducted using both the clotting and the chromogenic techniques. The anticoagulant citrate phosphate dextrose adenine (CPDA) produced the highest harvest of FVIII:C, the level of plasma factor VIII, compared to heparin:saline and heparin:CPDA anticoagulants. Camel FVIII can be concentrated 2 to 3 times in cryoprecipitate. There was a significant loss of camel FVIII when comparing levels of FVIII in camel plasma after 1 h of incubation at 37°C (533%), 40°C (364%), and 50°C (223%). Thrombin generation of camel plasma is comparable to that of human plasma. It was concluded that camel plasma contains very elevated levels of FVIII:C, approaching 8 times the levels in human plasma, and that these elevated levels could not be attributed to excessive thrombin generation. Unlike human FVIII:C, camel FVIII:C is remarkably heat stable. Taken together, these unique features of camel FVIII could be part of the physiological adaptation of hemostasis of the Arabian camel in order to survive in the hot desert environment. PMID:24082408

  8. Analysis of factors for poor activated partial thromboplastin time control after dabigatran administration.

    PubMed

    Kose, E; Sato, R; An, T; Kikkawa, A; Matsumoto, Y; Kawakubo, T; Hayashi, H

    2015-11-01

    In this study, we compared patients whose activated partial thromboplastin time (APTT) was prolonged excessively with those whose APTT was controlled within the normal range after dabigatran administration. We analyzed the factors for the APTT prolongation. We divided the patients into two groups: those whose APTTs prolonged to more than 65 s and those whose APTTs were less than 65 s after dabigatran administration. There were 130 patients from March 2011 to July 2013, and we analyzed the background features and laboratory data of these patients. Results showed that there were no significant differences in the patients' background and laboratory data except for the high-density lipoprotein cholesterol (HDL-C) level. However, details of the relationship between the APTT prolongation and the HDL-C level are currently unknown. We hypothesize that the reason for the APTT prolongation is the variability in such parameters as the time of blood drawing, internal time of dabigatran, individual variability, and blood concentration. Therefore, we consider that these parameters need to be carefully evaluated even if APTT does not show prolongation.

  9. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization.

    PubMed

    Fritsma, George A; Dembitzer, Francine R; Randhawa, Ankush; Marques, Marisa B; Van Cott, Elizabeth M; Adcock-Funk, Dorothy; Peerschke, Ellinor I

    2012-06-01

    The activated partial thromboplastin time (aPTT) is widely used as a screening coagulation test and for monitoring unfractionated heparin therapy. Various commercial reagents are available, with different performance characteristics, particularly responsiveness to the lupus anticoagulant (LA). Because aPTT reagent selection significantly affects the interpretation of results, we reviewed College of American Pathologists proficiency testing data involving approximately 4,000 coagulation laboratories, and conducted a survey of coagulation laboratories (n = 93) using The Fritsma Factor hemostasis Web site to determine the basis for aPTT reagent selection. The data demonstrate that for routine aPTT testing, most laboratories use reagents with high/moderate responsiveness to LA. Significant misunderstanding was apparent regarding the use of appropriate aPTT reagent for routine testing and LA identification. We recommend aPTT reagents with low LA responsiveness to screen for coagulation factor deficiencies and heparin monitoring, and suggest continued education of laboratory professionals and reagent manufacturers about appropriate aPTT reagent use.

  10. Fluorescence-based blood coagulation assay device for measuring activated partial thromboplastin time.

    PubMed

    Dudek, Magdalena M; Kent, Nigel; Gustafsson, Kerstin M; Lindahl, Tomas L; Killard, Anthony J

    2011-01-01

    The measurement of blood clotting time is important in a range of clinical applications such as assessing coagulation disorders and controlling the effect of various anticoagulant drug therapies. Clotting time tests essentially measure the onset of clot formation which results from the formation of fibrin fibers in the blood sample. However, such assays are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. This work describes a clotting time measurement assay which uses a fluorescent probe to very precisely detect the onset of fibrin clot formation. It uses a microstructured surface which enhances the formation of multiple localized clot loci and which results in the abrupt redistribution of the fluorescent label at the onset of clot formation in both whole blood and plasma. This methodology was applied to the development of an activated partial thromboplastin time (aPTT) test in a lateral flow microfluidic platform and used to monitor the effect of heparin dosage where it showed linearity from 0 to 2 U/mL in spiked plasma samples (R(2)=0.996, n = 3), correlation against gold standard coagulometry of 0.9986, and correlation against standard hospital aPTT in 32 patient samples of 0.78.

  11. Exogenous Magnesium Chloride Reduces the Activated Partial Thromboplastin Times of Lupus Anticoagulant-Positive Patients

    PubMed Central

    Tokutake, Takayoshi; Baba, Hisami; Shimada, Yuji; Takeda, Wataru; Sato, Keijiro; Hiroshima, Yuki; Kirihara, Takehiko; Shimizu, Ikuo; Nakazawa, Hideyuki; Kobayashi, Hikaru; Ishida, Fumihiro

    2016-01-01

    The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of calcium chloride. It is used to screen for various coagulation disorders. Several previous reports have suggested that magnesium (Mg) might contribute to coagulation reactions by binding to specific coagulation proteins. We investigated the effects of Mg on the APTT. In healthy controls, the APTT was significantly prolonged in proportion to the increase in the concentration of magnesium chloride in the range from 2.1 to 16.7 mmol/L. Among eight samples from patients with various disorders that exhibited prolonged APTT, two samples demonstrated shorter APTT when Mg was added, both of which were from patients that were positive for lupus anticoagulant. When we examined 206 clinical APTT samples, we found that Mg shortened the APTT of two samples. These two samples were also from lupus anticoagulant-positive patients (p-value: <0.003). Our findings regarding the unique effects of exogenous Mg on the APTT of lupus anticoagulant-positive patients might shed light on the role of Mg in APTT assays and lead to the development of a novel screening method for lupus anticoagulant. PMID:27355205

  12. [Acquired partial lipodystrophy. Insulin resistance, hepatic lipase activity and small and dense LDL particles].

    PubMed

    Paglione, A M; Ferrari, N; Berg, G; Frechtel, G; Taverna, M; Fasulo, V; Lopez, G I; Gomez, R M; Bruno, O; Ruiz, M; Wikinski, R L

    2001-01-01

    Partial lipodystrophy (PLD) is an infrequent condition characterized by symmetric loss of subcutaneous adipose tissue in the upper or lower part of the body, although occasionally it affects only the extremities. In all cases it appears along with acantosis nigricans (AN), insulin resistance and impairment in the metabolism of lipids and carbohydrates. The case depicted pertains to a 49 year old female with no family history involving loss of adipose tissue in face and upper body. No fat in lower part of body was observed. The patient showed facial thinning at age 8, AN at 11 and gestational diabetes during her fourth pregnancy at 33. At present, the patient presents severe hyperglycemia and hyperinsulinemia with a marked insulin resistance. Type IV hyperlipoproteinemia (OMS), declined C-HDL and Apo A1 and low C-LDL but with a high proportion of small and dense LDL particles were present. Non esterified fatty acids were high. Lipoprotein lipase and hepatic lipase activities are in the lower limit and increased respectively. Fraction C3 of the complement was diminished. No mutations were observed either in codons 170, 809 and 972 of the IRS-1 receptor or in codon 276 of the adrenergic beta 2 gene.

  13. Effects of partial decerebration and hypophyseal allograft in the thymus of chicken embryos: thymostimulin localization and enzymatic activities.

    PubMed

    Aita, M; Romano, N

    2006-01-01

    Changes in chicken embryo thymus after partial decerebration (including the hypophysis) and hypophyseal allograft were investigated. Chicken embryos were partially decerebrated at 36-40 hr of incubation and on day 12 received a hypophyseal allograft from 18-day-old donor embryos. The embryonic thymuses were collected on day 18 and examined with histological methods, tested for the anti-thymostimulin-like immune-reaction, and for histoenzymatic activities and compared with normal and sham-operated embryos at the same age. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-thymostimulin, succinic dehydrogenase and ATPase enzymatic activities tested, yielded negative reactions. In partially decerebrated hypophyseal allografted embryos, the same thymic compartments improved and anti-thymostimulin-like immune-reaction and enzymatic activities partially recovered. These findings confirmed the key role of hypophysis in thymic ontogenic development and provided new information in metabolic enzymatic pathways and synthesis of a thymostimulin-like substance in the thymus.

  14. [Effects of partial root excision on the growth, photosynthesis, and antioxidant enzyme activities of maize under salt stress].

    PubMed

    Zhang, Hong; Cui, Li-Na; Meng, Jia-Jia; Zhang, Hai-Yan; Shi, De-Yang; Dong, Shu-Ting; Zhang, Ji-Wang; Liu, Peng

    2012-12-01

    A pot experiment was conducted to study the effects of partial root excision on the growth of two maize cultivars (Zhengdan 958 and Denghai 9) throughout their growth period and the photosynthesis and leaf antioxidant enzyme activities at grain-filling stage under salt stress. Four treatments were installed, i. e., control (no salt), low salt (0.2% NaCl), moderate salt (0.4% NaCl), and high salt (0.6% NaCl). Under low salt stress, the grain yield of Zhengdan 958 and Denghai 9 with partial root excision was increased by 13.1% and 31.4%, respectively, as compared with that of the cultivars with no root excision. At jointing stage, the growth of the cultivars with partial root excision was restrained, the root- and shoot dry masses under the same salt stresses being lesser than those of the cultivars with no root excision, but the growth under the conditions of no salt and low salt recovered quickly. At milk-ripe stage and under no salt and low salt conditions, the root- and shoot dry masses, leaf area, total root length, total root surface area, root activity, leaf chlorophyll content, and ear leaf net photosynthetic rate, stomatal conductance, transpiration rate, and CAT and POD activities of the cultivars with partial root excision were significantly larger than those of the cultivars with no root excision, while the shoot diameter and ear leaf MDA content were in adverse. Moderate and high salt stresses had greater effects on the cultivars with partial root excision. The root- and shoot dry masses, root morphology, and photosynthesis indices of the cultivars with partial root excision were smaller than those of the cultivars with no root excision, so did the grain yields. Throughout the growth period of the cultivars, the growth of the cultivars with partial root excision depended on the salt concentration, i. e., was promoted under no and low salt, and inhibited under moderate and high salt conditions.

  15. Identification of PPARgamma Partial Agonists of Natural Origin (II): In Silico Prediction in Natural Extracts with Known Antidiabetic Activity

    PubMed Central

    Guasch, Laura; Sala, Esther; Mulero, Miquel; Valls, Cristina; Salvadó, Maria Josepa; Pujadas, Gerard; Garcia-Vallvé, Santiago

    2013-01-01

    Background Natural extracts have played an important role in the prevention and treatment of diseases and are important sources for drug discovery. However, to be effectively used in these processes, natural extracts must be characterized through the identification of their active compounds and their modes of action. Methodology/Principal Findings From an initial set of 29,779 natural products that are annotated with their natural source and using a previously developed virtual screening procedure (carefully validated experimentally), we have predicted as potential peroxisome proliferators-activated receptor gamma (PPARγ) partial agonists 12 molecules from 11 extracts known to have antidiabetic activity. Six of these molecules are similar to molecules with described antidiabetic activity but whose mechanism of action is unknown. Therefore, it is plausible that these 12 molecules could be the bioactive molecules responsible, at least in part, for the antidiabetic activity of the extracts containing them. In addition, we have also identified as potential PPARγ partial agonists 10 molecules from 16 plants with undescribed antidiabetic activity but that are related (i.e., they are from the same genus) to plants with known antidiabetic properties. None of the 22 molecules that we predict as PPARγ partial agonists show chemical similarity with a group of 211 known PPARγ partial agonists obtained from the literature. Conclusions/Significance Our results provide a new hypothesis about the active molecules of natural extracts with antidiabetic properties and their mode of action. We also suggest plants with undescribed antidiabetic activity that may contain PPARγ partial agonists. These plants represent a new source of potential antidiabetic extracts. Consequently, our work opens the door to the discovery of new antidiabetic extracts and molecules that can be of use, for instance, in the design of new antidiabetic drugs or functional foods focused towards the

  16. Effect of dermatan sulphate on activated partial thromboplastin time determined with different reagents.

    PubMed

    Iorio, A; Nucciarelli, F; Renga, C; Nenci, G G; Gianese, F; Agnelli, G

    1997-01-01

    Five widely used activated partial thromboplastin time (APTT) reagents (Actin-FS, Actin-FSL, Hemolab Silimat, IL-Test APTT Ellagic Acid and Thrombofax Activated) were compared for their sensitivity and precision in measuring the effect of dermatan sulphate on blood coagulation. On each of 4 days, aliquots of the same normal human plasma pool were mixed with dermatan sulphate (MF701) at concentrations ranging from 10 to 100 micrograms/ml, and APTT was measured in duplicate with all reagents by a photo-optical coagulometer. The order of testing between and within reagents was changed every day. The relationship of APTT ratio to dermatan sulphate concentration was linear with all the reagents. There were statistically significant differences between reagents in their sensitivity to DS, as reflected by linear regression slopes. IL-Test was the most sensitive reagent. At dermatan sulphate concentrations of 20, 50, and 80 micrograms/ml APTT ratio ranged from 1.5 to 1.7, 1.9 to 2.3 and 2.3 to 2.9, respectively, according to the reagent. The lambda coefficient and coefficient of variation derived from regression analysis, both reflecting assay precision, ranged from 0.57 to 0.71 and from 4.6 to 5.1%, respectively, with all but the least precise reagent. The best sensitivity/precision balance was displayed by IL-Test. The APTT reagent should therefore be standardized, with special regard to sensitivity to DS, when testing the relationship of dermatan sulphate clinical effects to APTT response.

  17. The activated partial thromboplastin time of diluted plasma: variability due to the method of fibrin detection.

    PubMed Central

    Johnstone, I B

    1984-01-01

    The purpose of this study was to determine the effects of the dilution of plasma (1/3 in saline) on the kinetics of fibrin generation in the activated partial thromboplastin time (APTT) assay. The diluted APTT is considered to increase the sensitivity of the APTT test however, studies in our laboratory using an electro-optical fibrin detection system failed to show significant differences in APTT values obtained with diluted and undiluted canine plasma. Seventeen plasmas, including plasmas moderately and markedly deficient in intrinsic factor activity were assayed in the undiluted and diluted APTT assay using two methods for fibrin endpoint detection; a visual "tilt-tube" technique and an electro-optical detection system. In the former technique the endpoint was the formation of a visible fibrin web or clot; in the latter procedure the end point was the first detection of a change in optical density of the plasma. Optical density changes during fibrin formation were also recorded ( thrombokinetograms ). The results indicated that the electro-optical fibrin detection system failed to identify a prolongation of the APTT as a result of 1/3 plasma dilution; a prolongation that was consistently observed with the visual fibrin detection technique. Plasma dilution however, did significantly reduce the rate of fibrin production as indicated by the thrombokinetogram profile. It was concluded that the dilution of plasma with saline, as has been used to increase the sensitivity of the APTT assay procedure, has little effect on the time of onset of fibrin formation in a given plasma. The major effect appears to be on the way in which fibrin forms in that the polymerization/crosslinkage events associated with macroscopic fibrin production are delayed. PMID:6722646

  18. An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification.

    PubMed

    Bai, Lijuan; Yan, Bin; Chai, Yaqin; Yuan, Ruo; Yuan, Yali; Xie, Shunbi; Jiang, Liping; He, Ying

    2013-11-07

    In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.

  19. Rates of thrombin acylation and deacylation upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylating agents.

    PubMed

    Brown, A D; Powers, J C

    1995-08-01

    Acylated derivatives of thrombin have been made using low molecular weight acylating agents, carbamylating agents and carbonylating agents. The compounds used to acylate the active site serine include isatoic anhydrides, benzoxazinones, benzylisocyanate, N-(benzylcarbonyloxy)succinimide and p-(dimethylamino)benzoylimidazolide. The rates of acylation and deacylation were determined. The best overall inhibitors of thrombin are 2-ethoxy-4H-3,1-benzoxazin-4-one, isatoic anhydride and tert-butyl-2,4-dioxo-2H-3,1-benzoxazine-1(4H)-acetate, which have k2/Ki values of 52,700 M-1s-1, 48,900 M-1s-1 and 5400 M-1s-1, respectively. The carbamyl derivative of thrombin formed with benzylisocyanate had the slowest rate of deacylation (2.3 x 10(-7) s-1), while the ester derivative formed with 2-(N,N-dimethylamino)methylimino-4H-3,1-benzoxazin-4-one had the fastest rate of deacylation (1.9 x 10(-4) s-1).

  20. Thrombin Maybe Plays an Important Role in MK Differentiation into Platelets

    PubMed Central

    Yang, Xiao-Lei; Ge, Meng-Kai; Mao, De-Kui; Lv, Ying-Tao; Sun, Shu-Yan; Yu, Ai-Ping

    2016-01-01

    Objectives. After development and differentiation, megakaryocytes (MKs) can produce platelets. As is well known, thrombopoietin (TPO) can induce MKs to differentiate. The effect of thrombin on MKs differentiation is not clear. In this study, we used a human megakaryoblastic leukemia cell line (Meg-01) to assess the effect of thrombin on MKs differentiation. Methods. In order to interrogate the role of thrombin in Meg-01 cells differentiation, the changes of morphology, cellular function, and expression of diverse factors were analyzed. Results. The results show that thrombin suppresses Meg-01 cells proliferation and induces apoptosis and cell cycle arrest. Thrombin upregulates the expression of CD41b, which is one of the most important MK markers. Globin transcription factor 1 (GATA-1), an important transcriptional regulator, controls MK development and maturation. The expression of GATA-1 is also upregulated by thrombin in Meg-01 cells. The expression of B-cell lymphoma 2 (Bcl-2), an apoptosis-inhibitory protein, is downregulated by thrombin. Phosphorylated protein kinase B (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) were upregulated by thrombin in Meg-01 cells. All the results are consistent with Meg-01 cells treated with TPO. Discussion and Conclusion. In conclusion, all these data indicate that thrombin maybe plays an important role in MK differentiation into platelets. However, whether the platelet-like particles are certainly platelets remains unknown. PMID:27064425

  1. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  2. Antifactor Xa levels versus activated partial thromboplastin time for monitoring unfractionated heparin.

    PubMed

    Vandiver, Jeremy W; Vondracek, Thomas G

    2012-06-01

    Intravenous unfractionated heparin (UFH) remains an important therapeutic agent, particularly in the inpatient setting, for anticoagulation. Historically, the activated partial thromboplastin time (aPTT) has been the primary laboratory test used to monitor and adjust UFH. The aPTT test has evolved since the 1950s, and the historical goal range of 1.5-2.5 times the control aPTT, which first gained favor in the 1970s, has fallen out of favor due to a high degree of variability in aPTT readings from one laboratory to another, and even from one reagent to another. As a result, it is now recommended that the aPTT goal range be based on a corresponding heparin concentration of 0.2-0.4 unit/ml by protamine titration or 0.3-0.7 unit/ml by antifactor Xa assay. Given that several biologic factors can influence the aPTT independent of the effects of UFH, many institutions have transitioned to monitoring heparin with antifactor Xa levels, rather than the aPTT. Clinical data from the last 10-20 years have begun to show that a conversion from aPTT to antifactor Xa monitoring may offer a smoother dose-response curve, such that levels remain more stable, requiring fewer blood samples and dosage adjustments. Given the minimal increased acquisition cost of the antifactor Xa reagents, it can be argued that the antifactor Xa is a cost-effective method for monitoring UFH. In this review, we discuss the relative advantages and disadvantages of the aPTT, antifactor Xa, and protamine titration tests, and provide a clinical framework to guide practitioners who are seeking to optimize UFH monitoring within their own institutions.

  3. Topical hemostatic agents to reduce bleeding from cancellous bone. A comparison of microcrystalline collagen, thrombin, and thrombin-soaked gelatin foam.

    PubMed

    Cobden, R H; Thrasher, E L; Harris, W H

    1976-01-01

    In fifty-three dogs microcrystalline collagen, thrombin-soaked gelatin foam, and thrombin powder were evaluated as hemostatic agents when applied to bleeding cancellous surfaces after osteotomy of the greater trochanter using two quantitative models: one, a single osteotomy; the other, a double osteotomy. All three agents significantly reduced bleeding compared with the controls, the microcrystalline collagen being most effective. At three months there was no evidence that microcrystalline collagen and thrombin-gelatin interfered with bone healing after the greater trochanter was reattached with two wires.

  4. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

  5. A comparison of red blood cell transfusion utilization between anti-activated factor X and activated partial thromboplastin monitoring in patients receiving unfractionated heparin.

    PubMed

    Belk, K W; Laposata, M; Craver, C

    2016-11-01

    Essentials Anti-activated factor X (Anti-Xa) monitoring is more precise than activated partial thromboplastin (aPTT). 20 804 hospitalized cardiovascular patients monitored with Anti-Xa or aPTT were analyzed. Adjusted transfusion rates were significantly lower for patients monitored with Anti-Xa. Adoption of Anti-Xa protocols could reduce transfusions among cardiovascular patients in the US.

  6. Differential inhibitory action of apixaban on platelet and fibrin components of forming thrombi: Studies with circulating blood and in a platelet-based model of thrombin generation

    PubMed Central

    Arellano-Rodrigo, Eduardo; Reverter, Joan Carles; Lopez-Farre, Antonio; Diaz-Ricart, Maribel; Badimon, Juan Jose; Escolar, Gines

    2017-01-01

    Introduction Mechanisms of action of direct oral anticoagulants (DOAC) suggest a potential therapeutic use in the prevention of thrombotic complications in arterial territories. However, effects of DOACs on platelet activation and aggregation have not been explored in detail. We have investigated the effects of apixaban on platelet and fibrin components of thrombus formation under static and flow conditions. Methods We assessed the effects of apixaban (10, 40 and 160 ng/mL) on: 1) platelet deposition and fibrin formation onto a thrombogenic surface, with blood circulating at arterial shear-rates; 2) viscoelastic properties of forming clots, and 3) thrombin generation in a cell-model of coagulation primed by platelets. Results In studies with flowing blood, only the highest concentration of apixaban, equivalent to the therapeutic Cmax, was capable to significantly reduce thrombus formation, fibrin association and platelet-aggregate formation. Apixaban significantly prolonged thromboelastometry parameters, but did not affect clot firmness. Interestingly, results in a platelet-based model of thrombin generation under more static conditions, revealed a dose dependent persistent inhibitory action by apixaban, with concentrations 4 to 16 times below the therapeutic Cmax significantly prolonging kinetic parameters and reducing the total amount of thrombin generated. Conclusions Our studies demonstrate the critical impact of rheological conditions on the antithrombotic effects of apixaban. Studies under flow conditions combined with modified thrombin generation assays could help discriminating concentrations of apixaban that prevent excessive platelet accumulation, from those that deeply impair fibrin formation and may unnecessarily compromise hemostasis. PMID:28192448

  7. Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times.

    PubMed

    Kershaw, Geoffrey; Orellana, Daniel

    2013-04-01

    Mixing tests are a relatively simple procedure used in the hemostasis laboratory as a first-line investigation into the cause of an abnormal screening test, typically a prolonged activated partial thromboplastin time and/or a prolonged prothrombin time. The mixing test involves combining the test plasma with normal plasma, then repeating the screening test on the mixture to assess whether the clotting time becomes normal or remains prolonged. The primary purpose of a mixing test is to guide further investigations. When mixing test results "normalize," this suggests the test plasma is deficient in clotting factor(s) and thus specific factor assays can be performed to determine which are reduced. When the mixing test result does not "normalize," this suggests the presence of an inhibitor or other type of interference (e.g., the presence of an anticoagulant such as high-dose heparinoids), and so the laboratory needs to determine if this is a lupus anticoagulant or a specific coagulation factor inhibitor, or another type of inhibitor. Because these follow-up investigations are more costly and time-consuming than the basic screening tests, the appropriate performance and interpretation of mixing tests is advantageous for the laboratory. Moreover, the correct laboratory approach is also clinically relevant, as patient management is ultimately affected, and an incorrect interpretation may lead to inappropriate therapies being established. Components of a mixing test that can influence result interpretation include the sensitivity of the used screening reagents to various factor deficiencies and inhibitors, the source or composition of the normal plasma, and the setting of cutoffs for the formula used in expressing mixing test results. Numerous and differing criteria for mixing test interpretation have been suggested historically, which can lead to confusion as to which approach is the most appropriate. The use of differing criteria will also lead to differing

  8. Platelet factor 4 enhances generation of activated protein C in vitro and in vivo.

    PubMed

    Slungaard, Arne; Fernandez, Jose A; Griffin, John H; Key, Nigel S; Long, Janel R; Piegors, Donald J; Lentz, Steven R

    2003-07-01

    Platelet factor 4 (PF4), an abundant platelet alpha-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 microg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 microg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 +/- 5 ng/mL and 39 +/- 2 ng/mL, respectively (P <.001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 +/- 6 seconds in PF4-treated animals and by 9 +/- 1 seconds in control animals at 30 minutes (P <.001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of controls from 10 through 120 minutes (P < or =.04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation.

  9. Therapeutic Correction of Thrombin Generation in Dilution-Induced Coagulopathy: Computational Analysis Based on a Data Set of Healthy Subjects

    DTIC Science & Technology

    2012-01-01

    Thrombophilia Study. For every simulated thrombin curve, we calculated and analyzed five standard thrombin generation parameters. RESULTS: The three...model of in vitro thrombin accumulation to generate and analyze thrombin curves for the 472 control subjects in the Leiden Thrombophilia Study (LETS).15... Thrombophilia Study (LETS). Thromb Haemost. 1997;78: 631Y635. 16. Brummel-Ziedins KE, Orfeo T, Rosendaal FR, Undas A, Rivard GE, Butenas S, Mann KG

  10. Charged particle detectors with active detector surface for partial energy deposition of the charged particles and related methods

    DOEpatents

    Gerts, David W; Bean, Robert S; Metcalf, Richard R

    2013-02-19

    A radiation detector is disclosed. The radiation detector comprises an active detector surface configured to generate charge carriers in response to charged particles associated with incident radiation. The active detector surface is further configured with a sufficient thickness for a partial energy deposition of the charged particles to occur and permit the charged particles to pass through the active detector surface. The radiation detector further comprises a plurality of voltage leads coupled to the active detector surface. The plurality of voltage leads is configured to couple to a voltage source to generate a voltage drop across the active detector surface and to separate the charge carriers into a plurality of electrons and holes for detection. The active detector surface may comprise one or more graphene layers. Timing data between active detector surfaces may be used to determine energy of the incident radiation. Other apparatuses and methods are disclosed herein.

  11. Juvenile and adult metachromatic leukodystrophy: partial restoration of arylsulfatase A (cerebroside sulfatase) activity by inhibitors of thiol proteinases.

    PubMed Central

    von Figura, K; Steckel, F; Hasilik, A

    1983-01-01

    Arylsulfatase A polypeptides were examined in cultured fibroblasts from a patient with juvenile metachromatic leukodystrophy and three patients with the adult form of the disease, with the aid of metabolic labeling and immunoprecipitation. The mutant cells were severely deficient in the arylsulfatase polypeptides. The apparent rate of synthesis, however, as estimated from the secretion of polypeptides or activity by cells incubated in the presence of 10 mM NH4Cl was 20-50% of control. In the absence of NH4Cl, the mutant enzyme was rapidly degraded upon transport into lysosomes. In the presence of inhibitors of thiol proteinases arylsulfatase A polypeptides were partially protected from degradation, and the catalytic activity of arylsulfatase A was increased. In addition, the treatment partially corrected the capacity of the cells to degrade cerebroside sulfates. Inhibitors of thiol proteinases may be of therapeutic value in variants of metachromatic leukodystrophy, in which an unstable arylsulfatase A is synthesized. Images PMID:6136972

  12. Tissue factor-dependent pathway is not involved in exercise-induced formation of thrombin and fibrin.

    PubMed

    Weiss, Claus; Bierhaus, Angelika; Kinscherf, Ralf; Hack, Volker; Luther, Thomas; Nawroth, Peter Paul; Bärtsch, Peter

    2002-01-01

    In healthy individuals, prolonged intensive physical exercise leads to an activation of blood coagulation that results in the formation of thrombin and fibrin. This study investigated whether oxidative stress during intensive physical exercise induces tissue factor (TF) via activation of the redox-responsive transcription factor nuclear factor-kappaB (NF-kappaB). Twelve young men performed a standardized 1-h maximal run on a treadmill that gave rise to significant increases of markers of thrombin and fibrin formation. The ratio of intracellular reduced to oxidized glutathione as measured by HPLC decreased from 23.3 +/- 10.7 to 14.2 +/- 6.5 (P < 0.05), indicating the generation of free radicals during exercise. Electrophoretic mobility shift assays from nuclear extracts of peripheral blood mononuclear cells revealed that exercise testing increased NF-kappaB (p50/p65) binding activity to a NF-kappaB consensus sequence by 105 +/- 68% (P < 0.01) but did not affect NF-kappaB (p65/c-Rel) binding to a nonconsensus-kappaB-like site present in the TF promoter. Consistently, there was no exercise-induced increase in TF expression as demonstrated by TF-specific immunofluorescence staining and ELISA. Thus selective activation of NF-kappaB (p50/p65) during intensive physical exercise does not result in the expression of TF, suggesting that the TF-dependent pathway in peripheral blood mononuclear cells does not account for exercise-induced formation of thrombin and fibrin.

  13. Effect of the oral thrombin inhibitor dabigatran on allergic lung inflammation induced by repeated house dust mite administration in mice.

    PubMed

    de Boer, Johannes D; Berkhout, Lea C; de Stoppelaar, Sacha F; Yang, Jack; Ottenhoff, Roelof; Meijers, Joost C M; Roelofs, Joris J T H; van't Veer, Cornelis; van der Poll, Tom

    2015-10-15

    Asthma is a chronic disease of the airways; asthma patients are hampered by recurrent symptoms of dyspnoea and wheezing caused by bronchial obstruction. Most asthma patients suffer from chronic allergic lung inflammation triggered by allergens such as house dust mite (HDM). Coagulation activation in the pulmonary compartment is currently recognized as a feature of allergic lung inflammation, and data suggest that coagulation proteases further drive inflammatory mechanisms. Here, we tested whether treatment with the oral thrombin inhibitor dabigatran attenuates allergic lung inflammation in a recently developed HDM-based murine asthma model. Mice were fed dabigatran (10 mg/g) or placebo chow during a 3-wk HDM airway exposure model. Dabigatran treatment caused systemic thrombin inhibitory activity corresponding with dabigatran levels reported in human trials. Surprisingly, dabigatran did not lead to inhibition of HDM-evoked coagulation activation in the lung as measured by levels of thrombin-antithrombin complexes and D-dimer. Repeated HDM administration caused an influx of eosinophils and neutrophils into the lungs, mucus production in the airways, and a T helper 2 response, as reflected by a rise in bronchoalveolar IL-4 and IL-5 levels and a systemic rise in IgE and HDM-IgG1. Dabigatran modestly improved HDM-induced lung pathology (P < 0.05) and decreased IL-4 levels (P < 0.01), without influencing other HDM-induced responses. Considering the limited effects of dabigatran in spite of adequate plasma levels, these results argue against clinical evaluation of dabigatran in patients with asthma.

  14. In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste

    PubMed Central

    Suleria, Hafiz Ansar Rasul; Hines, Barney M.; Addepalli, Rama; Chen, Wei; Masci, Paul; Gobe, Glenda; Osborne, Simone A.

    2016-01-01

    Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin. PMID:28042854

  15. Folding versus charge: understanding selective target recognition by the thrombin aptamers.

    PubMed

    Marson, Giuseppe; Palumbo, Manlio; Sissi, Claudia

    2012-01-01

    The use of nucleic acids as drugs represents a consistently growing approach. Different therapeutical strategies take advantage of the biological and biophysical properties of DNA and RNA to properly modulate activity of selected targets. A peculiar characteristic of these molecules is their structural flexibility which allows them to assume distinct foldings depending upon their sequence and/or environment. During the last twenty years this has led to the theoretical and experimental development of oligonucleotide aptamers, short sequences which can recognize a target with specificity and affinity comparable to antibodies. A leading example is represented by the Thrombin aptamer (15fTBA), a 15-mer DNA selected by its high affinity for the exosite I (fibrinogen binding site) of the coagulation factor. The very stable protein-DNA complex formation is the result of complementarities between the two macromolecules promoted by the aptamer sequence and folding as well as of electrostatic interactions generated by the charge balance at the binding site/s. Here, we investigated the relative role of these contributions and their involvement in defining the biological properties of the resulting complex. Thus we compared the Thrombin binding and inhibition properties of TBA to those of unrelated single stranded oligonucleotides. Additionally, the differences between the two protein exosites were assessed by using 29hTBA, a longer (29-mer) aptamer known to bind exosite II (heparin binding site). A subtle balance of aptamer folding and sequence is shown to cooperate with charge density for effective and selective recognition of exosite I or exosite II by TBAs.

  16. Partial Fractionation of Venoms from Two Iranian Vipers, Echis carinatus and Cerastes persicus Fieldi and Evaluation of Their Antiplatelet Activity.

    PubMed

    Mehdizadeh Kashani, Toktam; Vatanpour, Hossein; Zolfagharian, Hossein; Hooshdar Tehrani, Hasan; Heydari, Mohammad Hossein; Kobarfard, Farzad

    2012-01-01

    Platelet aggregation inhibitory effect and anticoagulant properties of fractions separated from the venoms of Cerastes persicus fieldi and Echis carinatus were investigated. The partial fractionation was performed on a Sephadex G-100 column. Two fractions separated from Cerastes persicus fieldi showed anti platelet aggregation activity on ADP (200 μM)-induced platelet aggregation (ca 80% inhibition). Attempts to measure the antiplatelet aggregation activity of crude Echis carinatus venom and its fractions were not successful due to the protein coagulation of the plasma samples after the addition of venom. Anticoagulant activities of venoms were also evaluated. Total venom of Echis carinatus showed anti coagulant activity in PT test, while its fractions showed procoagulant activity.

  17. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  18. A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin.

    PubMed

    Jiao, Xiao Xia; Chen, Jing Rong; Zhang, Xi Yuan; Luo, Hong Qun; Li, Nian Bing

    2013-10-15

    A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1-18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.

  19. Ligand binding stepwise disrupts water network in thrombin: enthalpic and entropic changes reveal classical hydrophobic effect.

    PubMed

    Biela, Adam; Sielaff, Frank; Terwesten, Felix; Heine, Andreas; Steinmetzer, Torsten; Klebe, Gerhard

    2012-07-12

    Well-ordered water molecules are displaced from thrombin's hydrophobic S3/4-pocket by P3-varied ligands (Gly, d-Ala, d-Val, d-Leu to d-Cha with increased hydrophobicity and steric requirement). Two series with 2-(aminomethyl)-5-chlorobenzylamide and 4-amidinobenzylamide at P1 were examined by ITC and crystallography. Although experiencing different interactions in S1, they display almost equal potency. For both scaffolds the terminal benzylsulfonyl substituent differs in binding, whereas the increasingly bulky P3-groups address S3/4 pocket similarly. Small substituents leave the solvation pattern unperturbed as found in the uncomplexed enzyme while increasingly larger ones stepwise displace the waters. Medium-sized groups show patterns with partially occupied waters. The overall 40-fold affinity enhancement correlates with water displacement and growing number of van der Waals contacts and is mainly attributed to favorable entropy. Both Gly derivatives deviate from the series and adopt different binding modes. Nonetheless, their thermodynamic signatures are virtually identical with the homologous d-Ala derivatives. Accordingly, unchanged thermodynamic profiles are no reliable indicator for conserved binding modes.

  20. Role of feedback in AGN-host coevolution: A study from partially obscured active galactic nuclei

    NASA Astrophysics Data System (ADS)

    Wang, J.

    2015-05-01

    Partially obscured AGNs within a redshift range z = 0.011 ∼ 0.256 are used to re-study the role of feedback in the AGN-host coevolution issue in terms of their [OIII] λ 5007 emission line profile. The spectra of these objects enable us to determine the AGN's accretion properties directly from their broad H α emission. This is essential for getting rid of the "circular reasoning" in our previous study of narrow emission-line galaxies, in which the [OIII] emission line was used not only as a proxy of AGN's bolometric luminosity, but also as a diagnostic of outflow. In addition, the measurement of Dn (4000) index is improved by removing an underlying AGN's continuum according to the corresponding broad H α emission. With these improvements, we confirm and reinforce the correlation between L /LEdd and stellar population age. More important is that this correlation is found to be related to both [OIII] line blue asymmetry and bulk blueshift velocity, which suggests a linkage between SMBH growth and host star formation through the feedback process. The current sample of partially obscured AGNs shows that the composite galaxies have younger host stellar population, higher Eddington ratio, less significant [OIII] blue wing and smaller bulk [OIII] line shift than do the Seyfert galaxies.

  1. Sulfation, anticoagulant and antioxidant activities of polysaccharide from green algae Enteromorpha linza.

    PubMed

    Wang, Xiaomei; Zhang, Zhongshan; Yao, Zhiyun; Zhao, Mingxing; Qi, Huimin

    2013-07-01

    Sulfated polysaccharides exerted potent biological property which was relative to degree of sulfation, molecular weight, substitution position and chain conformation. In present study, the polysaccharide with low molecular weight (LEP) from Enteromorpha linza was sulfated with chlorosulfuric acid in formamide. The obtained polysaccharide sulfate was selected to evaluate their antioxidant activities and the anticoagulant activity in the coagulation assays, activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT). The data obtained in vitro models indicated that high DS and moderate Mw showed the best anticoagulant and antioxidant activities.

  2. 76 FR 67142 - Certain Activated Carbon From the People's Republic of China: Final Results and Partial...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-31

    ...-activation chemical treatment (chemical or water washing, chemical impregnation or other treatment), or... dehydrates molecules in the raw material, and results in the formation of water that is removed from the raw... analysis memoranda. \\13\\ CCT submitted Active Carbon India Private Limited's (``Active Carbon'')...

  3. A new dicoumarin and anticoagulant activity from Viola yedoensis Makino.

    PubMed

    Zhou, Hai Yan; Hong, Jun Li; Shu, Pan; Ni, Yue Juan; Qin, Min Jian

    2009-07-01

    A new dicoumarin, named as dimeresculetin (1), together with another dicoumarin, euphorbetin (2) and esculetin (3) were isolated from the ethyl acetate extract of the dried whole plants of Viola yedoensis Makino. The structure of 1 was elucidated as 7-hydroxy-6-[(6,7-dihydroxy-2-oxo-2H-1-benzopyran-5-yl)oxy]-2H-1-benzopyran-2-one on the basis of extensive NMR, as well as the other spectral analysis. Compounds 1-3 exhibited anticoagulant activities with respect to activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT).

  4. TORC1 activity is partially reduced under nitrogen starvation conditions in sake yeast Kyokai no. 7, Saccharomyces cerevisiae.

    PubMed

    Nakazawa, Nobushige; Sato, Aya; Hosaka, Masahiro

    2016-03-01

    Industrial yeasts are generally unable to sporulate but treatment with the immunosuppressive drug rapamycin restores this ability in a sake yeast strain Kyokai no. 7 (K7), Saccharomyces cerevisiae. This finding suggests that TORC1 is active under sporulation conditions. Here, using a reporter gene assay, Northern and Western blots, we tried to gain insight into how TORC1 function under nitrogen starvation conditions in K7 cells. Similarly to a laboratory strain, RPS26A transcription was repressed and Npr1 was dephosphorylated in K7 cells, indicative of the expected loss of TORC1 function under nitrogen starvation. The expression of nitrogen catabolite repression-sensitive genes, however, was not induced, the level of Cln3 remained constant, and autophagy was more slowly induced than in a laboratory strain, all suggestive of active TORC1. We conclude that TORC1 activity is partially reduced under nitrogen starvation conditions in K7 cells.

  5. Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect.

    PubMed

    Yoshinaga, Hidefumi; Masumoto, Shuji; Koyama, Koji; Kinomura, Naoya; Matsumoto, Yuji; Kato, Taro; Baba, Satoko; Matsumoto, Kenji; Horisawa, Tomoko; Oki, Hitomi; Yabuuchi, Kazuki; Kodo, Toru

    2017-01-01

    We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT1A weak partial agonistic activity, which could work as a 5-HT1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.

  6. Intracerebral propagation of interictal activity in partial epilepsy: implications for source localisation.

    PubMed Central

    Alarcon, G; Guy, C N; Binnie, C D; Walker, S R; Elwes, R D; Polkey, C E

    1994-01-01

    The hypothesis that focal scalp EEG and MEG interictal epileptiform activity can be modelled by single dipoles or by a limited number of dipoles was examined. The time course and spatial distribution of interictal activity recorded simultaneously by surface electrodes and by electrodes next to mesial temporal structures in 12 patients being assessed for epilepsy surgery have been studied to estimate the degree of confinement of neural activity present during interictal paroxysms, and the degree to which volume conduction and neural propagation take part in the diffusion of interictal activity. Also, intrapatient topographical correlations of ictal onset zone and deep interictal activity have been studied. Correlations between the amplitudes of deep and surface recordings, together with previous reports on the amplitude of scalp signals produced by artificially implanted dipoles suggest that the ratio of deep to surface activity recorded during interictal epileptiform activity on the scalp is around 1:2000. This implies that most such activity recorded on the scalp does not arise from volume conduction from deep structures but is generated in the underlying neocortex. Also, time delays of up to 220 ms recorded between interictal paroxysms at different recording sites show that interictal epileptiform activity can propagate neuronally within several milliseconds to relatively remote cortex. Large areas of archicortex and neocortex can then be simultaneously or sequentially active via three possible mechanisms: (1) by fast association fibres directly, (2) by fast association fibres that trigger local phenomena which in turn give rise to sharp/slow waves or spikes, and (3) propagation along the neocortex. The low ratio of deep-to-surface signal on the scalp and the simultaneous activation of large neocortical areas can yield spurious equivalent dipoles localised in deeper structures. Frequent interictal spike activities can also take place independently in areas other

  7. An Investigation of the Characteristics of the Enzyme Thrombin, Suitable for Classwork

    ERIC Educational Resources Information Center

    Blofield, B. Ann

    1972-01-01

    Shows how a simple investigation of the enzyme, thrombin, can provide a series of experiments giving information on enzyme characteristics. The results also provide a basis for discussion of the coagulation mechanism and related phenomena. (Author/AL)

  8. Direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Pagba, Cynthia V.; Lane, Stephen M.; Cho, Hansang; Wachsmann-Hogiu, Sebastian

    2010-07-01

    In this study, we exploit the sensitivity offered by surface-enhanced Raman scattering (SERS) for the direct detection of thrombin using the thrombin-binding aptamer (TBA) as molecular receptor. The technique utilizes immobilized silver nanoparticles that are functionalized with thiolated thrombin-specific binding aptamer, a 15-mer (5'-GGTTGGTGTGGTTGG-3') quadruplex forming oligonucleotide. In addition to the Raman vibrational bands corresponding to the aptamer and blocking agent, new peaks (mainly at 1140, 1540, and 1635 cm-1) that are characteristic of the protein are observed upon binding of thrombin. These spectral changes are not observed when the aptamer-nanoparticle assembly is exposed to a nonbinding protein such as bovine serum albumin (BSA). This methodology could be further used for the development of label-free biosensors for direct detection of proteins and other molecules of interest for which aptamers are available.

  9. Theoretical modeling and experimental validation of surface stress in thrombin aptasensor.

    PubMed

    Lim, Yang Choon; Kouzani, Abbas Z; Kaynak, Akif; Dai, Xiujuan J; Littlefair, Guy; Duan, Wei

    2014-12-01

    Adsorption of target molecules on the immobilized microcantilever surface produced beam displacement due to the differential surface stress generated between the immobilized and non-immobilized surface. Surface stress is caused by the intermolecular forces between the molecules. Van der Waals, electrostatic forces, hydrogen bonding, hydrophobic effect and steric hindrance are some of the intermolecular forces involved. A theoretical framework describing the adsorption-induced microcantilever displacement is derived in this paper. Experimental displacement of thrombin aptamer-thrombin interactions was carried out. The relation between the electrostatic interactions involved between adsorbates (thrombin) as well as adsorbates and substrates (thrombin aptamer) and the microcantilever beam displacement utilizing the proposed mathematical model was quantified and compared to the experimental value. This exercise is important to aid the designers in microcantilever sensing performance optimization.

  10. Pseudoaneurysm After Spontaneous Rupture of Renal Angiomyolipoma in Tuberous Sclerosis: Successful Treatment with Percutaneous Thrombin Injection

    SciTech Connect

    Corso, Rocco Carrafiello, Gianpaolo; Rampoldi, Antonio; Leni, Davide; Ticca, Cristiana; Vercelli, Ruggero; Vanzulli, Angelo

    2005-04-15

    We report a case of a large perinephric pseudoaneurysm due to spontaneous rupture of renal angiomyolipoma, occluded by percutaneous thrombin injection under ultrasound guidance in a young woman affected by tuberous sclerosis.

  11. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Influence of posterior dental arch length on brain activity during chewing in patients with mandibular distal extension removable partial dentures.

    PubMed

    Shoi, K; Fueki, K; Usui, N; Taira, M; Wakabayashi, N

    2014-07-01

    It is well known that shortened dental arch decreases masticatory function. However, its potential to change brain activity during mastication is unknown. The present study investigates the effect of a shortened posterior dental arch with mandibular removable partial dentures (RPDs) on brain activity during gum chewing. Eleven subjects with missing mandibular molars (mean age, 66.1 years) on both sides received experimental RPDs with interchangeable artificial molars in a crossover trial design. Brain activity during gum chewing with RPDs containing (full dental arch) and lacking artificial molars (shortened dental arch) was measured using functional magnetic resonance imaging. Additionally, masticatory function was evaluated for each dental arch type. Food comminuting and mixing ability and the perceived chewing ability were significantly lower in subjects with a shortened dental arch than those with a full dental arch (P < 0.05). Brain activation during gum chewing with the full dental arch occurred in the middle frontal gyrus, primary sensorimotor cortex extending to the pre-central gyrus, supplementary motor area, putamen, insula and cerebellum. However, middle frontal gyrus activation was not observed during gum chewing with the shortened dental arch. These results suggest that shortened dental arch affects human brain activity in the middle frontal gyrus during gum chewing, and the decreased middle frontal gyrus activation may be associated with decreased masticatory function.

  13. Computational Analysis of Intersubject Variability and Thrombin Generation in Dilutional Coagulopathy

    DTIC Science & Technology

    2012-11-01

    healthy subjects in the Leiden Thrombophilia Study. Individual thrombin curves were calculated for undiluted blood and for different dilution...performing computa- tional thrombin generation analyses on a data set obtained as a part of the Leiden Thrombophilia Study (LETS).24 Computer modeling...the results. Thromb Res 2007; 119:663-5. 24. van der Meer FJ, Koster T, Vandenbroucke JP, Briët E, Rosendaal FR. The Leiden thrombophilia study (LETS

  14. Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function.

    PubMed

    Lu, H; Menashi, S; Garcia, I; Cramer, E M; Li, H; Tenza, D; De Romeuf, C; Soria, J; Soria, C

    1993-09-01

    Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01-0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with < 0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min, indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31 +/- 0.90 for resting platelets, down-regulated to 0.84 +/- 0.13 (P < 0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63 +/- 2.21 (P > 0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.

  15. Efficacy of fibrinogen/thrombin-coated equine collagen patch in controlling lymphatic leaks.

    PubMed

    Vida, Vladimiro L; Padalino, Massimo A; Barzon, Elisa; Stellin, Giovanni

    2012-07-01

    We report the use of fibrinogen/thrombin-coated equine collagen patch (Tachosil(®) ) as a sealant agent in six patients who underwent heart surgery for congenital heart disease (CHD) and developed an intraoperative lymphatic leakage detected at the time of surgery. The use of fibrinogen/thrombin-coated equine collagen patch proved to be safe and effective in preventing the development of postoperative chylothorax.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

    SciTech Connect

    Wei, Wenqing; Zhao, Wei; Wang, Xiaoping; Teng, Maikun Niu, Liwen

    2007-08-01

    The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.

  17. Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis.

    PubMed

    Liu, Yanan; Liu, Nan; Ma, Xinhua; Li, Xiaoli; Ma, Jia; Li, Ya; Zhou, Zhijiang; Gao, Zhixian

    2015-04-21

    A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human α-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA-thrombin to form an aptamer-thrombin-aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1-thrombin) and 15.5 ± 0.637 nM (TBA2-thrombin), which demonstrated the high affinities of TBA-thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37-554.31 nM, and the coefficients of determination R(2) were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6-114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields.

  18. Human Protein C produces anticoagulation and increased fibrinolytic activity in the cat

    SciTech Connect

    Burdick, M.D.; Schaub, R.G.

    1986-03-05

    The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the Nembutal anesthetized cat was assessed. Human Protein C was activated by incubation with thrombin or by passage over a column of thrombin immobilized on CNBr Sepharose 4B. Cats were given bolus i.v. injections of either vehicle or PCa in a dose range of 3-16 ..mu..g/mL of calculated whole body volume. Citrated blood samples (9:1) were taken from a femoral vein prior to and at 5, 10, 20, 30, 60, 120, and 180 min. after PCa. Activated partial thromboplastin time (APTT), thrombin time (TT) euglobulin clot lysis (ECLT) and I-125 fibrin release (FR) was measured. Vehicle treated cats had no change in any parameter. PCa produced a dose and time dependent prolongation of APTT while TT was unchanged. Anticoagulation was evident immediately after PCa infusion and began to normalize within 20 min. Fibrinolytic activity measured by ECLT and FR was also stimulated by PCa but was not evident until 40-60 minutes after PCa injection. The results show that human PCa induces anticoagulation effects in the cat similar to other species. However, stimulation of fibrinolysis requires a longer period of time before expression. This delay of fibrinolytic stimulation should be considered when assessing the effects of human Protein C in other species.

  19. Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer-AuNPs-HRP conjugates.

    PubMed

    Zhao, Jie; Zhang, Youyu; Li, Haitao; Wen, Yanqing; Fan, Xiaoyu; Lin, Fanbo; Tan, Liang; Yao, Shouzhuo

    2011-01-15

    Successful development of an ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer-gold nanoparticles-horseradish peroxidase (aptamer-AuNPs-HRP) conjugates was reported. In this electrochemical protocol, aptamer1 (Apt1) was immobilized on core/shell Fe(3)O(4)/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs-Apt1/thrombin/Apt2-AuNPs-HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1-60 pM and lower detection limit down to 30 fM (S/N=3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.

  20. Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex

    PubMed Central

    Gosai, Agnivo; Ma, Xiao; Balasubramanian, Ganesh; Shrotriya, Pranav

    2016-01-01

    The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer. PMID:27874042

  1. A reusable aptasensor of thrombin based on DNA machine employing resonance light scattering technique.

    PubMed

    Hou, Yining; Liu, Jifeng; Hong, Min; Li, Xia; Ma, Yanhua; Yue, Qiaoli; Li, Chen-Zhong

    2017-06-15

    The design of molecular nanodevices attracted great interest in these years. Herein, a reusable, sensitive and specific aptasensor was constructed based on an extension-contraction movement of DNA interconversion for the application of human thrombin detection. The present biosensor was based on resonance light scattering (RLS) using magnetic nanoparticles (MNPs) as the RLS probe. MNPs coated with streptavidin can combine with biotin labeled thrombin aptamers. The combined nanoparticles composite is monodispersed in aqueous medium. When thrombin was added a sandwich structure can form on the surface of MNPs, which induced MNPs aggregation. RLS signal was therefore enhanced, and there is a linear relationship between RLS increment and thrombin concentration in the range of 60pM-6.0nM with a limit of detection at 3.5pM (3.29SB/m, according to the recent recommendation of IUPAC). The present aptasensor can be repeatedly used for at least 6 cycling times by heat to transfer G-quadruplex conformation to single strand of DNA sequence and release thrombin. MNPs can be captured by applying the external magnetic field. Furthermore, the proposed biosensor was successfully applied to detect thrombin in human plasma.

  2. Surface plasmon resonance analysis of immobilized fibrinogen and fibrin and their interaction with thrombin and fibrinogen

    NASA Astrophysics Data System (ADS)

    Dyr, Jan E.; Jirouskova, Marketa; Rysava, Jitka; Tichy, Ivo; Tobiska, Petr; Slavik, Radan; Homola, Jiri; Suttnar, Jiri

    1999-01-01

    The exploitation of surface plasmon resonance optical sensor for the study of the interaction of immobilized fibrinogen and fibrin monomer with soluble fibrinogen and thrombin is reported. Soluble fibrinogen was mostly reversible, the bound thrombin could be inhibited by milimolar concentration of phenylmethylsulphonyl fluoride (PMSF). At lease three sets of different thrombin binding sites were found. There was a residual fraction of thrombin bound to washed fibrin (ogin) (to about a five to ten percent of fibron monomer units) suggesting that a known naturally occurring fibrinogen variant differing in the gamma chain was the target. Surface bound fibrinogen was converted by thrombin to fibrin monomer that interacted with fibrinogen in solution. At low fibrin monomer surface density the second layer was formed that contained about the same amount of protein as the first layer, at higher fibrin monomer concentration less than one molecule of fibrinogen per molecule of fibrin monomer was captured. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen a fibrin network of predetermined composition, size, and arrangement could be formed.

  3. Thrombin use in surgery: an evidence-based review of its clinical use

    PubMed Central

    Ham, Sung W; Lew, Wesley K; Weaver, Fred A

    2010-01-01

    When surgical ligation of bleeding fails, or is not possible, surgeons rely on a number of hemostatic aids, including thrombin. This review discusses the history, pharmacology and clinical application of thrombin as a surgical hemostat. The initial thrombin was bovine in origin, but its use has been complicated by the formation of antibodies that cross-react with human coagulation factors. This has been associated with life-threatening bleeding and in some circumstances anaphylaxis and death. Human thrombin, isolated from pooled plasma of donors, was developed in an effort to minimize these risks, but its downsides are its limited availability and the potential for transmitting blood-borne pathogens. Recently a recombinant thrombin has been developed, and approved for use by the FDA. It has the advantage of being minimally antigenic and devoid of the risk of viral transmission. Thrombin is often used in conjunction with other hemostatic aids, including absorbable agents such as Gelfoam, and with fibrinogen in fibrin glues. The last part of this review will discuss these agents in detail, and review their clinical applications. PMID:22282693

  4. Design and X-ray crystal structures of human thrombin with synthetic cyanopeptide-analogues.

    PubMed

    Radau, G; Fokkens, J

    2007-02-01

    Based on the X-ray crystals of cocrystallized cyanopeptide-trypsin and cyanopeptide-thrombin-com-plexes, a rational drug design succeeded in the establishment of suitable lead structures for the development of new potential inhibitors of thrombin. This report deals with the design and X-ray crystallography data of new synthetic, low-molecular weight cyanopeptide-analogues, RA-1008 and RA-1014, complexed with human alpha-thrombin at 1.85 A resolution. The crystal structures of the complexes reveal, by analogy with modeling studies, that the salt bridge of Asp189 to this type of synthetic thrombin inhibitors leads to an almost identically binding into the S1 specificity pocket in comparison to the complex of the natural products, whereas in the overall binding modes the P2-P4 substructures differ from those of the leads. The strongest member of the second series of described thrombin inhibitors, RA-1014, shows in the crystal complex with thrombin a slightly higher affinity towards the enzyme than RA-1008 as confirmed by inhibition tests. This result and other key informations will be helpful to design a more potent series of inhibitors.

  5. Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex

    NASA Astrophysics Data System (ADS)

    Gosai, Agnivo; Ma, Xiao; Balasubramanian, Ganesh; Shrotriya, Pranav

    2016-11-01

    The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer.

  6. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

    PubMed Central

    Edwards, Judson Vincent; Prevost, Nicolette

    2011-01-01

    Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing). Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze). A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration. PMID:24956451

  7. Sulfated polysaccharides from Loligo vulgaris skin: potential biological activities and partial purification.

    PubMed

    Abdelmalek, Baha Eddine; Sila, Assaâd; Krichen, Fatma; Karoud, Wafa; Martinez-Alvarez, Oscar; Ellouz-Chaabouni, Semia; Ayadi, Mohamed Ali; Bougatef, Ali

    2015-01-01

    The characteristics, biological properties, and purification of sulfated polysaccharides extracted from squid (Loligo vulgaris) skin were investigated. Their chemical and physical characteristics were determined using X-ray diffraction and infrared spectroscopic analysis. Sulfated polysaccharides from squid skin (SPSS) contained 85.06% sugar, 2.54% protein, 1.87% ash, 8.07% sulfate, and 1.72% uronic acid. The antioxidant properties of SPSS were investigated based on DPPH radical-scavenging capacity (IC50 = 19.42 mg mL(-1)), hydrogen peroxide-scavenging activity (IC50 = 0.91 mg mL(-1)), and β-carotene bleaching inhibition (IC50 = 2.79 mg mL(-1)) assays. ACE-inhibitory activity of SPSS was also investigated (IC50 = 0.14 mg mL(-1)). Further antimicrobial activity assays indicated that SPSS exhibited marked inhibitory activity against the bacterial and fungal strains tested. Those polysaccharides did not display hemolytic activity towards bovine erythrocytes. Fractionation by DEAE-cellulose column chromatography showed three major absorbance peaks. Results of this study suggest that sulfated polysaccharides from squid skin are attractive sources of polysaccharides and promising candidates for future application as dietary ingredients.

  8. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    NASA Astrophysics Data System (ADS)

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-10-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

  9. Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma

    PubMed Central

    Verma, Mahendra Kumar; Xavier, Francies; Verma, Yogendra Kumar; Sobha, Kota

    2013-01-01

    Objective To isolate, partially purify and evaluate the cytotoxic and antitumor activity of a serine protease from the chosen Indian earthworm Pheretima posthuma. Methods Whole animal extract was prepared and purified its protein constituents by size and charge based chromatographic separation techniques using Sephadex G-50 and DEAE-Cellulose resin respectively. Average molecular weight of the protein isolate was determined and analyzed for its cytotoxic property against Vero cells in different dilutions (1: 20 and 1: 40) and anti-tumor activity by MTT assay (a colorimetric assay) using breast cancer cell line MCF-7, with tamoxifen as standard. Results One of the protein constituents after purification was characterized as serine protease by Caseinolytic plate diffusion assay. Average molecular weight of this purified isolate was determined, by SDS-PAGE analysis with standard protein ladder, as of 15 kDa. The performed tests suggested that the 15kDa fraction has potent cytotoxic activity and satisfactory antitumor activity as well in vitro. Conclusions Exact molecular mechanism of the cytotoxic and antitumor activities is yet to be explored and currently we are working on ultra-purification and biophysical characterization of this fraction. Further investigation into the mechanism(s) of cytotoxic and antitumor activities at molecular level would be useful in treatment of various classes of cancer and viral infections in future.

  10. Increase in ovulation rate after active immunization of sheep with inhibin partially purified from bovine follicular fluid.

    PubMed

    Henderson, K M; Franchimont, P; Lecomte-Yerna, M J; Hudson, N; Ball, K

    1984-09-01

    Four Romney ewes were actively immunized with a partially purified preparation of inhibin derived from bovine follicular fluid and their ovulation rates in four successive oestrous cycles were compared with those of four ewes receiving adjuvant alone. The ovulation rates of the ewes immunized with the inhibin preparation were significantly higher than those of the control ewes (2.06 +/- 0.16 (S.E.M.) vs 1.31 +/- 0.06 ovulations/ewe, n = 4). Plasma concentrations of FSH and LH, measured in blood samples taken three times a week for 11 weeks, during which time each ewe was immunized three times, were not significantly different between the two treatment groups. These results suggest that active immunization with inhibin-enriched follicular fluid may be a potential means of increasing fecundity in sheep.

  11. Partial deficit of pantothenate kinase 2 catalytic activity in a case of tremor-predominant neurodegeneration with brain iron accumulation.

    PubMed

    Liang, Tsao-Wei; Truax, Adam C; Trojanowski, John Q; Lee, Virginia M-Y; Stern, Matthew B; Kotzbauer, Paul T

    2006-05-01

    We describe an atypical case of pantothenate kinase-associated neurodegeneration (PKAN) in which slowly progressive arm tremor was the predominant symptom beginning at the age of 25, with late-onset dystonia and dysarthria developing at the age of 50. Compound heterozygous mutations resulting in missense amino acid substitutions G521R and I529V were identified in the pantothenate kinase (PANK2) gene. We demonstrate that while the G521R mutation results in an unstable and inactive protein, the previously unreported I529V substitution has no apparent effect on the stability or catalytic activity of PanK2. The phenotype that results from this combination of mutations suggests that atypical presentations of PKAN may arise from partial deficits in PanK2 catalytic activity.

  12. Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

    PubMed Central

    Li, Quan; Wang, Yanzhen; Cai, Guangsheng; Kong, Fange; Wang, Xiaohan; Liu, Yang; Yang, Chuanbin; Wang, Di; Teng, Lirong

    2015-01-01

    Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM) and liquid cultured mycelia (TM) in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP), antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5′-AMP-activated protein kinase (AMPK), and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α) and phosphofructokinase-1 (PFK-1) in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases. PMID:26697489

  13. Phospholipase C Activity in Human Polymorphonuclear Leukocytes: Partial Characterization and Effect of Indomethacin

    DTIC Science & Technology

    1988-12-01

    phospholipase C activity alone, and in the presence of 0.5 mM and I mM indomethacin, is plotted according to Lineweaver and Burke as described previously...The data were plotted according to the method of Lineweaver and Burke (26). The values represent the mean + S.E.M. of values derived from neutrophils of 4 subjects. 18

  14. Growth-inhibitory activity of lymphoid cell plasma membranes. II. Partial characterization of the inhibitor

    PubMed Central

    1984-01-01

    We have shown that plasma membranes from lymphoid cells have inhibitory activity for the growth of normal lymphocytes and lymphoid tumor cells (Stallcup, K. C., A. Dawson, and M. F. Mescher, J. Cell Biol. 99:1221- 1226). This growth-inhibitory activity has been found to co-purify with major histocompatibility complex class I antigens (H-2K and D) when these cell surface glycoproteins are isolated from detergent lysates of cells by affinity chromatography on monoclonal antibody columns. When incorporated into liposomes, the affinity-purified H-2 antigens inhibited the growth of both normal lymphocytes and tumor cells at concentrations of 1-3 micrograms/ml. Inhibition was readily reversed upon removal of the liposomes from the cell cultures, even after several days of exposure of cells to the inhibitor. Inhibitory activity was insensitive to protease digestion or heat treatment, indicating that it was not due to the H-2 glycoproteins. This was confirmed by the demonstration that inhibitory activity could be separated from the H-2 protein by gel filtration in the presence of deoxycholate and could be extracted from membranes or H-2 antigen preparations with organic solvents. The results demonstrate that the growth-inhibitory component(s) of the plasma membrane is a minor lipid or lipid-like molecule which retains activity in the absence of other membrane components. The findings reported here and in the preceding article suggest that this novel membrane component may have a role in control of lymphoid cell growth, possibly mediated by cell contacts. PMID:6332814

  15. Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

    PubMed

    Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Mendoza, Julio; Inga, Rosalina R; Gontijo, Silea; Richardson, Michael; Eble, Johannes A; Yarleque, Armando; Sanchez, Eladio F

    2013-07-01

    The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

  16. Isolated pregnancy-induced anti-thrombin deficiency in a woman with twin pregnancy.

    PubMed

    Kawabata, Kosuke; Morikawa, Mamoru; Yamada, Takahiro; Minakami, Hisanori

    2016-06-01

    A woman with twin pregnancy had a gradual decline in anti-thrombin (AT) activity from 72% at gestational week (GW) 29(-3/7) , to 53% at GW31(-2/7) , and to 41% at GW32(-2/7) , at which time hypertension (148/90 mmHg) and proteinuria (protein-to-creatinine ratio [P/Cr], 0.79 mg/mg) developed in the presence of normal platelet count (159 × 10(9) /L) and serum aspartate aminotransferase/lactate dehydrogenase (22/164 IU/L). AT product was given three times to maintain AT activity >50% and blood pressure was maintained below 155/95 mmHg with no treatment, but generalized edema with a weekly weight gain of 4.9 kg and increased proteinuria (to P/Cr, 7.6 mg/mg) required cesarean section at GW33(-3/7) . This case highlights the occurrence of pregnancy-induced AT deficiency alone in the absence of any other abnormality, including hypertension, proteinuria, or thrombocytopenia. Measurement of AT activity was considered helpful for determination of the appropriate time for delivery in this patient.

  17. The impact of thrombin generation and rotation thromboelastometry on assessment of severity of factor XI deficiency.

    PubMed

    Livnat, Tami; Shenkman, Boris; Martinowitz, Uri; Zivelin, Ariella; Dardik, Rima; Tamarin, Ilia; Mansharov, Rachel; Budnik, Ivan; Salomon, Ophira

    2015-08-01

    The phenotype of bleeding in patients with severe FXI deficiency is unpredictable and unlike other bleeding disorders, it is not directly correlated with levels of FXI. In this study we analyzed whether the global coagulation assays can serve as a clinical tool in predicting bleeding tendency in patients with severe FXI deficiency undergoing surgery, taking into account the large inter-individual variability of FXI levels and genotypes. Thrombin generation (TG) was measured in 39 platelet-poor plasma with or without tissue factor (TF) and in the presence or absence of corn trypsin inhibitor (CTI). Rotation thromboelastometry (ROTEM) was performed with fresh whole blood of 26 patients applying NATEM and INTEM tests. TG induced by recalcification can distinguish between bleeding and non-bleeding patients with severe FXI deficiency particularly among those with FXI activity of 2-20IU/dl. The addition of TF or TF and CTI to the TG assay masked the ability to differentiate between XI activity, genotype as well as bleeding and non-bleeding patients. ROTEM assays failed to distinguish bleeding from non-bleeding patients but could do so between different FXI activity levels and genotypes. In conclusion, in the current study we found a sensitive tool to distinguish between bleeding and non-bleeding patients. In order to recommend TG as a predictive tool for treatment tailoring, a larger patient group is required.

  18. Partial in vitro analysis of toxic and antigenic activities of eleven Peruvian pitviper snake venoms.

    PubMed

    Guerra-Duarte, C; Lopes-Peixoto, J; Fonseca-de-Souza, B R; Stransky, S; Oliveira, D; Schneider, F S; Lopes-de-Souza, L; Bonilla, C; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-12-15

    This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and L-amino acid oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, through ELISA and Western Blotting assays, were determined. Results show that the activities tested in this study were not similar amongst the venoms and each species present their own peculiarities, highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms.

  19. Glycosaminoglycans as naturally occurring combinatorial libraries: developing a mass spectrometry-based strategy for characterization of anti-thrombin interaction with low molecular weight heparin and heparin oligomers.

    PubMed

    Abzalimov, Rinat R; Dubin, Paul L; Kaltashov, Igor A

    2007-08-15

    Heparin is a densely charged polysaccharide, which is best known for its anticoagulant activity, although it also modulates a plethora of other biological processes. Unlike biopolymers whose synthesis is strictly controlled by a unique genetic template, heparin molecules exhibit a remarkable degree of structural heterogeneity, which poses a serious challenge for studies of heparin-protein interactions. This analytical challenge is often dealt with by reducing the enormous structural repertoire of heparin to a model small molecule. In this paper, we describe a different approach inspired by the experimental methodologies from the arsenal of combinatorial chemistry. Interaction of anti-thrombin III (AT) with heparinoids is studied using a mixture of oligoheparin molecules of fixed degree of polymerization, but varying chemical composition (heparin hexasaccharides obtained by size exclusion chromatography of an enzymatic digest of porcine intestinal heparin with bacterial heparinase), as well as a heparin-derived pharmaceutical preparation Tinzaparin (heparin oligosaccharides up to a 22-mer). AT binders are identified based on the results of ESI MS measurements of complexes formed by protein-oligoheparin association. Additionally, differential depletion of free heparin oligomers in solution in the presence of AT is used to verify the binding preferences. ESI MS characterization of oligoheparin-AT interaction under partially denaturing conditions allowed the conformer specificity of the protein-polyanion binding to be monitored. A model emerging from these studies invokes the notion of a well-defined binding site on AT, to which a flexible partner (heparin) adapts to maximize favorable intermolecular electrostatic interactions. This study demonstrates the enormous potential of ESI MS as an analytical tool to study the interactions of highly heterogeneous glycosaminoglycans with their cognate proteins outside of the commonly accepted reductionist paradigm, which reduces

  20. Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking.

    PubMed

    de Ullivarri, Miguel Fernández; Mendoza, Lucía M; Raya, Raúl R

    2014-11-01

    Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15-25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8-M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5-12 %) and SO2 (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.

  1. Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes.

    PubMed

    Springer, Michael; Wykoff, Dennis D; Miller, Nicole; O'Shea, Erin K

    2003-11-01

    A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

  2. Partially Phosphorylated Pho4 Activates Transcription of a Subset of Phosphate-Responsive Genes

    PubMed Central

    Miller, Nicole

    2003-01-01

    A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin–CDK complex Pho80–Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80–Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80–Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs. PMID:14624238

  3. Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

    PubMed Central

    Capelli, Davide; Cerchia, Carmen; Montanari, Roberta; Loiodice, Fulvio; Tortorella, Paolo; Laghezza, Antonio; Cervoni, Laura; Pochetti, Giorgio; Lavecchia, Antonio

    2016-01-01

    The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design. PMID:27708429

  4. Reconstitution of glucotoxic HIT-T15 cells with somatostatin transcription factor-1 partially restores insulin promoter activity.

    PubMed

    Harmon, J S; Tanaka, Y; Olson, L K; Robertson, R P

    1998-06-01

    activity after STF-1 transfection. We conclude that loss of RIPE-3b1 activity precedes loss of STF-1 activity in glucotoxic HIT-T15 cells and that defective promoter activity can be partially restored by STF-1 transfection and predict that eventual cloning of the RIPE-3b1 gene will allow cotransfection studies with both factors that will allow full reconstitution of insulin promoter activity.

  5. Thrombin-Anti-Thrombin Levels and Patency of Arterio-Venous Fistula in Patients Undergoing Haemodialysis Compared to Healthy Volunteers: A Prospective Analysis

    PubMed Central

    Milburn, James A.; Ford, Isobel; Mutch, Nicola J.; Fluck, Nicholas; Brittenden, Julie

    2013-01-01

    Background Patients on haemodialysis (HD) are at an increased risk of sustaining thrombotic events especially to their vascular access which is essential for maintenance of HD. Objectives To assess whether 1) markers of coagulation, fibrinolysis or endothelial activation are increased in patients on HD compared to controls and 2) if measurement of any of these factors could help to identify patients at increased risk of arteriovenous (AVF) access occlusion. Patients/Methods Venous blood samples were taken from 70 patients immediately before a session of HD and from 78 resting healthy volunteers. Thrombin-antithrombin (TAT), D-dimer, von Willebrand factor (vWF), plasminogen activator inhibitor-1 antigen (PAI-1) and soluble p-selectin were measured by ELISA. C-reactive protein (hsCRP) was measured by an immunonephelometric kinetic assay. Determination of the patency of the AVF was based upon international standards and was prospectively followed up for a minimum of four years or until the AVF was non-functioning. Results A total of 70 patients were studied with a median follow-up of 740 days (range 72-1788 days). TAT, D-dimer, vWF, p-selectin and hsCRP were elevated in patients on HD compared with controls. At one year follow-up, primary patency was 66% (46 patients). In multivariate analysis TAT was inversely associated with primary assisted patency (r= -0.250, p= 0.044) and secondary patency (r = -0.267, p= 0.031). Conclusions The novel finding of this study is that in patients on haemodialysis, TAT levels were increased and inversely correlated with primary assisted patency and secondary patency. Further evaluation is required into the possible role of TAT as a biomarker of AVF occlusion. PMID:23844096

  6. Robustness of persistent spiking to partial synchronization in a minimal model of synaptically driven self-sustained activity

    NASA Astrophysics Data System (ADS)

    Novikov, Nikita; Gutkin, Boris

    2016-11-01

    We study the behavior of a minimal model of synaptically sustained persistent activity that consists of two quadratic integrate-and-fire neurons mutually coupled via excitatory synapses. Importantly, each of the neurons is excitable, as opposed to an oscillator; hence when uncoupled it sits at a subthreshold rest state. When the constituent neurons are mutually coupled via sufficiently strong fast excitatory synapses, the system demonstrates bistability between a fixed point (quiescent background state) and a limit cycle (memory state with synaptically driven spiking activity). Previous work showed that this persistent activity can be stopped by an excitatory input that synchronizes the network. Here we analyzed how this persistent state reacts to partial synchronization. We considered three types of progressively more complex excitatory synaptic kernels: delta pulse, square, and exponential. The first two cases were treated analytically, and the latter case numerically. Using phase-plane methods, we characterized the shape of the region, such that all orbits starting within it correspond to infinite spike trains; this constitutes the persistent activity region. In the case of instant coupling, all such active orbits were neutrally stable; in the case of noninstant coupling, the activity region contained a unique stable limit cycle (so the activity region was the basin of attraction for the limit cycle). This limit cycle corresponded to purely antiphase spiking of two neurons. Increasing synchronization shifted the system toward the border of the activity region, eventually terminating spiking activity. We calculated three measures of robustness of the active state: width of the activity region in the phase plane, critical level of synchronization that can be tolerated by the persistent spiking activity, and speed of reconvergence to the limit cycle. Our analysis revealed that the self-sustained activity is more robust to synchronization when each individual neuron

  7. The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form

    PubMed Central

    Hauryliuk, Vasili; Mitkevich, Vladimir A.; Eliseeva, Natalia A.; Petrushanko, Irina Yu.; Ehrenberg, Måns; Makarov, Alexander A.

    2008-01-01

    Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the γ-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an “activity chimera,” with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its “ratcheted state,” with hybrid tRNA binding sites. PMID:18836081

  8. Partial purification and characterization of an antimicrobial activity from the wood extract of mangrove plant Ceriops decandra

    PubMed Central

    Simlai, Aritra; Mukherjee, Kalishankar; Mandal, Anurup; Bhattacharya, Kashinath; Samanta, Amalesh; Roy, Amit

    2016-01-01

    The development of resistance towards the antibiotics in use today has been a source of growing concern in the modern healthcare system around the world. To counter this major threat, there is an urgent need for discovery of new antimicrobials. Many plants, like mangroves, possess highly diversified list of natural phytochemicals which are known to have wide range of bioactivities. These phytochemicals can be good sources for the discovery of new drugs. In this study, we report the partial phytochemical characterization and antimicrobial activities of a semi-purified fraction isolated from the wood tissue of Ceriops decandra, a mangrove plant. This fraction named CD-3PM was chromatographically separated from C. decandra wood extract and was subjected to different spectral analyses to determine its partial chemical nature. The structural investigation indicates the presence of two diterpenoids, i) 3β, 13β-Dihydroxy-8-abietaen-7-one and ii) 3β-Hydroxy-8,13-abietadien-7-one in the CD-3PM fraction. The antimicrobial potential of this fraction was evaluated by microdilution-MTT assay against several organisms. Among the nine microorganisms found to be sensitive to the CD-3PM fraction, six organisms are reported to be pathogenic in nature. The CD-3PM fraction with broad spectrum antimicrobial efficacy revealed the presence of two diterpenoids and possesses potential applications in drug discovery process and food processing industries. PMID:27065777

  9. Partial purification and characterization of an antimicrobial activity from the wood extract of mangrove plant Ceriops decandra.

    PubMed

    Simlai, Aritra; Mukherjee, Kalishankar; Mandal, Anurup; Bhattacharya, Kashinath; Samanta, Amalesh; Roy, Amit

    2016-01-01

    The development of resistance towards the antibiotics in use today has been a source of growing concern in the modern healthcare system around the world. To counter this major threat, there is an urgent need for discovery of new antimicrobials. Many plants, like mangroves, possess highly diversified list of natural phytochemicals which are known to have wide range of bioactivities. These phytochemicals can be good sources for the discovery of new drugs. In this study, we report the partial phytochemical characterization and antimicrobial activities of a semi-purified fraction isolated from the wood tissue of Ceriops decandra, a mangrove plant. This fraction named CD-3PM was chromatographically separated from C. decandra wood extract and was subjected to different spectral analyses to determine its partial chemical nature. The structural investigation indicates the presence of two diterpenoids, i) 3β, 13β-Dihydroxy-8-abietaen-7-one and ii) 3β-Hydroxy-8,13-abietadien-7-one in the CD-3PM fraction. The antimicrobial potential of this fraction was evaluated by microdilution-MTT assay against several organisms. Among the nine microorganisms found to be sensitive to the CD-3PM fraction, six organisms are reported to be pathogenic in nature. The CD-3PM fraction with broad spectrum antimicrobial efficacy revealed the presence of two diterpenoids and possesses potential applications in drug discovery process and food processing industries.

  10. Characterization of DicB by partially masking its potent inhibitory activity of cell division

    PubMed Central

    Yang, Shaoyuan; Pei, Hairun; Zhang, Xiaoying; Wei, Qiang; Zhu, Jia; Zheng, Jimin; Jia, Zongchao

    2016-01-01

    DicB, a protein encoded by the Kim (Qin) prophage in Escherichia coli, inhibits cell division through interaction with MinC. Thus far, characterization of DicB has been severely hampered owing to its potent activity which ceases cell division and leads to cell death. In this work, through fusing maltose-binding protein to the N-terminus of DicB (MBP–DicB), we successfully expressed and purified recombinant DicB that enabled in vitro analysis for the first time. More importantly, taking advantage of the reduced inhibitory activity of MBP–DicB, we were able to study its effects on cell growth and morphology. Inhibition of cell growth by MBP–DicB was systematically evaluated using various DicB constructs, and their corresponding effects on cell morphology were also investigated. Our results revealed that the N-terminal segment of DicB plays an essential functional role, in contrast to its C-terminal tail. The N-terminus of DicB is of critical importance as even the first amino acid (following the initial Met) could not be removed, although it could be mutated. This study provides the first glimpse of the molecular determinants underlying DicB's function. PMID:27466443

  11. Semi-active control of a sandwich beam partially filled with magnetorheological elastomer

    NASA Astrophysics Data System (ADS)

    Dyniewicz, Bartłomiej; Bajkowski, Jacek M.; Bajer, Czesław I.

    2015-08-01

    The paper deals with the semi-active control of vibrations of structural elements. Elastomer composites with ferromagnetic particles that act as magnetorheological fluids are used. The damping coefficient and the shear modulus of the elastomer increases when it is exposed to an electro-magnetic field. The control of this process in time allows us to reduce vibrations more effectively than if the elastomer is permanently exposed to a magnetic field. First the analytical solution for the vibrations of a sandwich beam filled with an elastomer is given. Then the control problem is defined and applied to the analytical formula. The numerical solution of the minimization problem results in a periodic, perfectly rectangular control function if free vibrations are considered. Such a temporarily acting magnetic field is more efficient than a constantly acting one. The surplus reaches 20-50% or more, depending on the filling ratio of the elastomer. The resulting control was verified experimentally in the vibrations of a cantilever sandwich beam. The proposed semi-active control can be directly applied to engineering vibrating structural elements, for example helicopter rotors, aircraft wings, pads under machines, and vehicles.

  12. Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

    PubMed Central

    Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de los Santos, Berta; Arroyo, Francisco T.; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L.

    2016-01-01

    Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen. PMID:27471515

  13. Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

    PubMed

    Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de Los Santos, Berta; Arroyo, Francisco T; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L

    2016-01-01

    Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

  14. P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

    PubMed Central

    Tackett, Bryan C.; Sun, Hongdan; Mei, Yu; Maynard, Janielle P.; Cheruvu, Sayuri; Mani, Arunmani; Hernandez-Garcia, Andres; Vigneswaran, Nadarajah; Karpen, Saul J.

    2014-01-01

    Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2−/−) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24–72 h) in response to 70% PH were impaired in P2Y2−/− mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2−/− remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2−/− mice were treated with ATP or ATPγS for 5–120 min and 12–24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH. PMID:25301185

  15. Intracellular carbonic anhydrase activity sensitizes cancer cell pH signaling to dynamic changes in CO2 partial pressure.

    PubMed

    Hulikova, Alzbeta; Aveyard, Nicholas; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

    2014-09-12

    Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO2, HCO3(-) and H(+). Intracellular CA (CAi) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CAi activity, measured as cytoplasmic CO2 hydration rate (kf), ranged from high in colorectal HCT116 (∼2 s(-1)), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous kf = 0.18 s(-1)) in cervical HeLa and breast MDA-MB-468 cells. CAi activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CAi catalysis was not obligatory for supporting acid extrusion by H(+) efflux or HCO3(-) influx, nor for maintaining intracellular pH (pHi) uniformity. However, in the absence of CAi activity, acid loading from a highly alkaline pHi was rate-limited by HCO3(-) supply from spontaneous CO2 hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO2 partial pressure (pCO2) that disturb cytoplasmic CO2-HCO3(-)-H(+) equilibrium. In cancer cells with high CAi activity, extracellular pCO2 fluctuations evoked faster and larger pHi oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca(2+)] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pHi of cells with low CAi activity was less responsive to pCO2 fluctuations. Such low pass filtering would "buffer" cancer cell pHi from non-steady-state extracellular pCO2. Thus, CAi activity determines the coupling between pCO2 (a function of tumor perfusion) and pHi (a potent modulator of cancer cell physiology).

  16. Construction of photoelectrochemical thrombin aptasensor via assembling multilayer of graphene-CdS nanocomposites.

    PubMed

    Shangguan, Li; Zhu, Wei; Xue, Yanchun; Liu, Songqin

    2015-02-15

    A photoelectrochemical (PEC) aptasensor for highly sensitive and specific detection of thrombin was developed by using graphene–CdS nanocomposites multilayer as photoactive species and electroactive mediator hexaammineruthenium(III) chloride (Ru(NH(3))(6)(3+)) as signal enhancer. Graphene–CdS nanocomposites (G–CdS) were synthesized by one-pot reduction of oxide graphene and CdCl2 with thioacetamide. The photoactive multilayer was prepared by alternative assembly of the negatively charged 3-mercaptopropionic acid modified graphene–CdS nanocomposites (MPA-G–CdS) and the positively charged polyethylenimine (PEI) on ITO electrode. This layer-by-layer assembly method enhanced the stability and homogeneity of the photocurrent readout of G–CdS. Thrombin aptamer was covalently bound to the multilayer by using glutaraldehyde as cross-linking. Electroactive mediator (Ru(NH(3))(6)(3+)) could interact with the DNA phosphate backbone and thus facilitated the electron transfer between G–CdS multilayer and electrode and enhanced the photocurrent. Hybridizing of a long complementary DNA with thrombin aptamer could increase the adsorption amount of (Ru(NH(3))(6)(3+)), which in turn boosted the signal readout. In the presence of target thrombin, the affinity interaction between thrombin and its aptamer resulted in the long complementary DNA releasing from the G–CdS multilayer and decreasing of photocurrent signal. On the basis of G–CdS multilayer as the photoactive species, (Ru (NH(3))(6)(3+)) as an electroactive mediator, and aptamer as a recognition module, a high sensitive PEC aptasensor for thrombin detection was proposed. The thrombin aptasensor displayed a linear range from 2.0 pM to 600.0 pM and a detection limit of 1.0 pM. The present strategy provided a promising ideology for the future development of PEC biosensor.

  17. Unusual Initial Manifestation of Acquired Hemophilia A: A Normal Activated Partial Thromboplastin Time, Intramuscular Hematoma and Cerebral Hemorrhage.

    PubMed

    Tsuyama, Nobuaki; Ichiba, Toshihisa; Naito, Hiroshi

    We herein present a case of acquired hemophilia A with a normal activated partial thromboplastin (aPTT), intramuscular hematoma and cerebral hemorrhage occurring in a 73-year-old man. The patient visited our emergency department with gait disturbance, pain and swelling in his right leg. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed intramuscular hematoma and intracranial hemorrhage. The results of initial coagulation studies were normal, but repeated coagulation studies revealed an isolated prolongation of the aPTT. Additional laboratory tests confirmed the diagnosis of acquired hemophilia A. If the initial aPTT is normal, we should therefore repeat the aPTT and also perform other coagulation studies including a mixing study, factor VIII level and inhibitor, to investigate the underlying diseases in elderly patients with spontaneous hemorrhaging of unknown etiology.

  18. Effect of isotretinoin on prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT).

    PubMed

    Kaptanoglu, Asli Feride; Uncu, Murat; Ozyurt, Selcuk; Hincal, Evren

    2013-08-01

    Patients with severe acne may need elective/urgent surgical interventions during treatment with isotretinoin and it is critical for the surgeon to consider the possible effects of this medication on coagulation systems. The aim of this study is to determine the changes in prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) during isotretinoin treatment. PT, aPTT, and INR values of 51 severe acne patients were evaluated during routine pre-treatment biochemical analysis. Only patients with normal values were included in the study. The results of before and after 1 month treatment were compared statistically. There were no statistically significant change in mean alanine aminotranferease (ALT), aspartate aminotransferase (AST), PT, and INR values after treatment. A significant increase in aPTT was detected. The INR values, which are more trusted and safe, showed no difference. Isotretinoin seems to have no effect on these coagulation parameters.

  19. Thrombin Injection for Treatment of Brachial Artery Pseudoaneurysm at the Site of a Hemodialysis Fistula: Report of Two Patients

    SciTech Connect

    Clark, Timothy W.I.; Abraham, Robert J.

    2000-09-15

    We report two patients with arteriovenous hemodialysis fistulas that were complicated by brachial artery pseudoaneurysms. Each pseudoanerysm was percutaneously thrombosed with an injection of thrombin, using techniques to prevent escape of thrombin into the native brachial artery. In one patient, an angioplasty balloon was inflated across the neck of the aneurysm during thrombin injection. In the second patient, thrombin was injected during ultrasound-guided compression of the neck of the pseudoaneurysm. Complete thrombosis of each pseudoaneurysm was achieved within 30 sec. No ischemic or embolic events occurred. This technique may be useful in treating pseudoaneurysms of smaller peripheral arteries.

  20. An electrochemical label-free and sensitive thrombin aptasensor based on graphene oxide modified pencil graphite electrode.

    PubMed

    Ahour, F; Ahsani, M K

    2016-12-15

    In this work, we tactfully constructed a novel label-free electrochemical aptasensor for rapid and facile detection of thrombin using graphene oxide (GO) and thrombin binding aptamer (TBA). The strategy relies on the preferential adsorption of single-stranded DNA (ssDNA) to GO over aptamer-target complexes. The TBA-thrombin complex formation was monitored by differential pulse voltammetry (DPV) using the guanine oxidation signal. In the absence of thrombin, the aptamers adsorbed onto the surface of GO leading to a strong background guanine oxidation signal. Conversely, in the presence of thrombin, the conformational transformation of TBA after incubating with the thrombin solution and formation of the aptamer-thrombin complexes which had weak binding ability to GO, leads to the desorption of TBA-thrombin complex from electrode surface and significant oxidation signal decrease. The selectivity of the biosensor was studied using other biological substances. The biosensor's signal was proportional to the thrombin concentration from 0.1 to 10nM with a detection limit of 0.07nM. Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes.

  1. Selective anxiolytics: are the actions related to partial "agonist" activity or a preferential affinity for benzodiazepine receptor subtypes?

    PubMed

    Gee, K W; Yamamura, H I

    1983-01-01

    Both pharmacological and biochemical evidence support the existence of BZ receptor subtypes. Determination of the molecular basis of BZ receptor heterogeneity requires additional research. The physiological significance of BZ receptor subtypes is not currently understood. One hypothesis presented to explain the unique pharmacological effects of CL 218872 suggests that CL 218872 has preferential affinity for a BZ receptor subtype (i.e., type I sites) that mediates the anxiolytic effects of the clinically active BZs. An alternative hypothesis has been proposed to account for these observations and is based upon the possibility that CL 218872 may act as a partial agonist at the BZ receptor. The partial agonist theory is supported by behavioral evidence and the relatively small differences in affinity of the BZ receptor subtypes discriminated by CL 218872 at physiological temperatures. In addition, in vivo binding studies suggest that occupancy of type II BZ receptor subtypes (i.e., those with low affinity for CL 218872) is necessary for CL 218872 to produce minimal anticonflict activity (4). Unlike certain other neurotransmitter systems, it is difficult to correlate the heterogeneous binding properties of BZ receptor ligands with their agonist/antagonist potential at BZ receptor. For example, CL 218872 discriminates BZ receptor subtypes and acts as an agonist at the BZ receptor. Beta-carbolines such as PCC also discriminate receptor subtypes, yet they act as antagonists at the BZ receptor. Compounding the complexity, neither the nature nor the existence of an endogenous ligand is known. So, the designation of agonist or antagonist effects is made on a purely functional basis.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

    PubMed Central

    Cotârleţ, Mihaela; Negoiţă, Teodor Gh.; Bahrim, Gabriela E.; Stougaard, Peter

    2011-01-01

    The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20°C, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20°C. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures. PMID:24031702

  3. Estimating Measurement Error of the Patient Activation Measure for Respondents with Partially Missing Data.

    PubMed

    Linden, Ariel

    2015-01-01

    The patient activation measure (PAM) is an increasingly popular instrument used as the basis for interventions to improve patient engagement and as an outcome measure to assess intervention effect. However, a PAM score may be calculated when there are missing responses, which could lead to substantial measurement error. In this paper, measurement error is systematically estimated across the full possible range of missing items (one to twelve), using simulation in which populated items were randomly replaced with missing data for each of 1,138 complete surveys obtained in a randomized controlled trial. The PAM score was then calculated, followed by comparisons of overall simulated average mean, minimum, and maximum PAM scores to the true PAM score in order to assess the absolute percentage error (APE) for each comparison. With only one missing item, the average APE was 2.5% comparing the true PAM score to the simulated minimum score and 4.3% compared to the simulated maximum score. APEs increased with additional missing items, such that surveys with 12 missing items had average APEs of 29.7% (minimum) and 44.4% (maximum). Several suggestions and alternative approaches are offered that could be pursued to improve measurement accuracy when responses are missing.

  4. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors

    PubMed Central

    Mrozkova, Petra; Spicarova, Diana; Palecek, Jiri

    2016-01-01

    Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception. PMID:27755539

  5. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity

    SciTech Connect

    Lee, Y.-H.; Cheng, C.-M.; Chang, Y.-F.; Wang, T.-Y.; Yuo, C.-Y.; E-mail: m815006@kmu.edu.tw

    2007-03-09

    Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. In addition, Apoptin also exhibits tumor-specific nuclear localization and tumor-specific phosphorylation on threonine 108 (T108). Here, we studied the effects of T108 phosphorylation on the tumor-specific nuclear localization and apoptotic activity of Apoptin. We first showed that a hemagglutinin (HA)-tagged Apoptin, but not the green fluorescent protein-fused Apoptin used in many previous studies, exhibited the same intracellular distribution pattern as native Apoptin. We then made and analyzed an HA-Apoptin mutant with its T108 phosphorylation site abolished. We found that Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization and abolishing the T108 phosphorylation of Apoptin does affect its apoptotic activity in tumor cells but only partially. Our results support the previous finding that Apoptin contains two distinct apoptosis domains located separately at the N- and C-terminal regions and suggest that the T108 phosphorylation may only be required for the apoptotic activity mediated through the C-terminal apoptosis domain.

  6. Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: purification and partial characterization.

    PubMed

    Choi, Jun-Hui; Kim, Dae-Won; Park, Se-Eun; Choi, Bong-Suk; Sapkota, Kumar; Kim, Seung; Kim, Sung-Jun

    2014-10-01

    A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn(2+). The Km and Vmax values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis.

  7. Low intraindividual variability of activated partial thromboplastin time revealed in a population of 10,487 control individuals.

    PubMed

    Ma, Youngeun; Huh, Hee Jae; Kim, Sun-Hee; Kim, Hee-Jin

    2013-10-01

    The activated partial thromboplastin time (aPTT) is a routine coagulation test that reflects the activities of multiple coagulation proteins. Given the known genetic elements underlying the different coagulation factor activities, a low intraindividual variability is expected in aPTT values, but has not been demonstrated in a large population. In this regard, we evaluated the intraindividual variability of aPTT by analyzing serial aPTTs from a large population. The study population consisted of control individuals who had three or more consecutive aPTT values at at least 6-month intervals at a single institution. The coefficient of variation of serial aPTT values was determined in each control individual, and the mean value of the coefficient of variations in the control population was calculated. The aPTT values from a total of 10,487 individuals [mean age 57 years (range 21-93 years); male-to-female ratio 1 : 0.9] were included. The mean value of the coefficient of variation of aPTTs in those individuals was 3.75%, which indicates a very low intraindividual variability. This is the first study to demonstrate a low intraindividual variability of aPTT in a large population. The result supports the previous notion that aPTT is a genetically determined parameter and has potential clinical implications.

  8. Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway.

    PubMed

    Huang, Y Q; Li, J J; Karpatkin, S

    2000-03-03

    Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.

  9. A multifunctional label-free electrochemical impedance biosensor for Hg(2+), adenosine triphosphate and thrombin.

    PubMed

    Chen, Lifen; Chen, Zhong-Ning

    2015-01-01

    A multifunctional label-free biosensor for the detection of Hg(2+), adenosine triphosphate and thrombin has been developed based on the changing of the electrochemical impedance spectroscopy (EIS) from the modified electrodes when nucleic acid subunits interacting with different targets. The modified electrode consists of three interaction sections, including DNA with T-T mismatch recognizing Hg(2+) to form T-Hg(2+)-T complex, split DNA chip against ATP, and DNA domin against thrombin to form G-quadruplex. Upon DNA interaction with thrombin or ATP, an increased charge transfer resistance (Rct) had been detected. However, a decreased Rct against Hg(2+) was obtained. The Rct difference (ΔRct) has relationship with the concentration of the different targets, Hg(2+), ATP and thrombin can be selectively detected with the detection limit of 0.03, 0.25, and 0.20 nmol L(-1), respectively. To separately detect the three analytes existing in the same sample, ATP aptamer, G-rich DNA strands and EDTA were applied to mask ATP, Hg(2+) or thrombin separately.

  10. A model of thrombin inactivation in heparinized and nonheparinized tubes with consequences for thrombus formation.

    PubMed

    Basmadjian, D; Sefton, M V

    1986-01-01

    The role of flow and mass transport in determining procoagulant concentration at the wall of synthetic and natural cylindrical blood vessels is analyzed theoretically. The model assumes steady laminar flow and considers, in addition to the fluid dynamic parameters, three rate-determining steps: production of procoagulant (thrombin) and its inactivation at the wall, as well as inactivation in the fluid bulk. The ratio of thrombin wall concentration to production rate Cw/N emerges as a critical parameter in characterizing the behavior of the tube wall. With a wall-inactivation rate typical of heparinized materials, Cw/N = 11.1 s/cm, independent of flow (shear rate) and axial position. This is significantly less than the range of Cw/N (50-500 s/cm) for which the thrombin concentration is high enough to result in significant fibrin formation and thrombosis. Hence little fibrin formation and a high degree of thromboresistance is expected for heparinized materials. Nonheparinized materials have Cw/N values above this range, which are only weakly dependent on shear rate and diameter, suggesting that flow-induced dispersion of thrombin (or other procoagulants) has limited impact on the thrombin wall concentration. These latter results appear to refute the conventional wisdom that attributes the relative patency of large-diameter vessels and differences between venous and arterial thrombi to such flow effects. It is likely that additional factors such as flow pulsatility and wall geometry must be considered to account for these observations.

  11. Three different signal amplification strategies for the impedimetric sandwich detection of thrombin.

    PubMed

    Ocaña, Cristina; del Valle, Manel

    2016-03-17

    In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM).

  12. Development of an efficient G-quadruplex stabilized thrombin binding aptamer containing 3-carbon spacer molecule.

    PubMed

    Naduvile Veedu, Rakesh; Aaldering, Lukas; Poongavanam, Vasanthanathan; Langkjaer, Niels; Murugan, Arul; Jørgensen, Per Trolle; Wengel, Jesper

    2017-02-02

    Thrombin binding aptamer (TBA) that shows anticoagulant properties is one of the most studied G-quadruplex forming aptamers. In this study, we investigated the impact of different chemical modifications such as Spacer-C3, unlocked nucleic acid (UNA) and 3'-amine-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7 and T12) of the TBA G-quadruplex structure rendering a series of TBA variants and studied their stability by thermal denaturation studies, folding by circular dichroism spectroscopy and thrombin clotting time. The results showed that Spacer-C3 introduction at T7 loop position (TBA-SP7) significantly improved the stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights to the experimental observation that further supported the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

  13. Telmisartan protects against diabetic vascular complications in a mouse model of obesity and type 2 diabetes, partially through peroxisome proliferator activated receptor-{gamma}-dependent activity

    SciTech Connect

    Toyama, Kensuke; Nakamura, Taishi; Kataoka, Keiichiro; Yasuda, Osamu; Fukuda, Masaya; Tokutomi, Yoshiko; Dong, Yi-Fei; Ogawa, Hisao; Kim-Mitsuyama, Shokei

    2011-07-08

    Highlights: {yields} Telmisartan, an angiotensin receptor blocker, acts as a partial PPAR{gamma} agonist. {yields} The protective effects of telmisartan against diabetic vascular injury were associated with attenuation of vascular NF{kappa}B activation and TNF {alpha}. {yields} PPAR{gamma} activity of telmisartan was involved in the normalization of vascular PPAR{gamma} downregulation in diabetic mice. {yields} We provided the first evidence indicating that PPAR{gamma} activity of telmisartan contributed to the protective effects of telmisartan against diabetic vascular complication. -- Abstract: Experimental and clinical data support the notion that peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) activation is associated with anti-atherosclerosis as well as anti-diabetic effect. Telmisartan, an angiotensin receptor blocker (ARB), acts as a partial PPAR{gamma} agonist. We hypothesized that telmisartan protects against diabetic vascular complications, through PPAR{gamma} activation. We compared the effects of telmisartan, telmisartan combined with GW9662 (a PPAR{gamma} antagonist), and losartan with no PPAR{gamma} activity on vascular injury in obese type 2 diabetic db/db mice. Compared to losartan, telmisartan significantly ameliorated vascular endothelial dysfunction, downregulation of phospho-eNOS, and coronary arterial remodeling in db/db mice. More vascular protective effects of telmisartan than losartan were associated with greater anti-inflammatory effects of telmisartan, as shown by attenuation of vascular nuclear factor kappa B (NF{kappa}B) activation and tumor necrosis factor {alpha}. Coadministration of GW9662 with telmisartan abolished the above mentioned greater protective effects of telmisartan against vascular injury than losartan in db/db mice. Thus, PPAR{gamma} activity appears to be involved in the vascular protective effects of telmisartan in db/db mice. Moreover, telmisartan, but not losartan, prevented the downregulation of

  14. Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

    SciTech Connect

    Baldassare, J.J.; Henderson, P.A.; Fisher, G.J. )

    1989-01-10

    The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulated IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.

  15. Variability of the thrombin- and ADP-induced Ca2+ response among human platelets measured using fluo-3 and fluorescent videomicroscopy.

    PubMed

    Tao, J; Rose, B; Haynes, D H

    1996-05-28

    The intracellular free Ca2+ concentration ([Ca2+]cyt) of individual human platelets localized between siliconized glass cover slips was determined at rest and after stimulation with thrombin and ADP using the Ca2+ indicator fluo-3 (0.97 +/- 0.30 mmol/l cell volume) with fluorescence video microscopy. Resting [Ca2+]cyt in the presence of 2 mM external Ca2+ showed only small inter-platelet variability ([Ca2+]cyt = 86 +/- 30 (S.D.) nM). Resting [Ca2+]cyt of individual fluo-3-loaded platelets measured as a function of time had a S.D. of 10 nM or 12% (S.D./mean). Individual platelets showed no affinity for the siliconized support and their [Ca2+]cyt showed no tendency to oscillate in either the resting or in the activated state. When 0.2 U/ml thrombin or 20 microM ADP were added, all platelets showed a characteristic Ca2+ transient whereby [Ca2+]cyt increased to peak values within 8-12 sec and then declined. The Ca2+ transients measured with fluo-3 were in approximate synchrony but peak [Ca2+]cyt values showed large inter-platelet variability. The ensemble average peak [Ca2+]cyt for thrombin and ADP were 672 +/- 619 (S.D.) nM and 640 +/- 642 (S.D.) nM, respectively. Thus inter-platelet variations (S.D./mean) were 92% or 100% as large as the average measured values. Mathematically-constructed averages of the single platelet experiments agreed reasonably well with platelet-averaged values obtained in parallel experiments with stirred platelet suspensions in a plastic cuvette, measured with a conventional spectrofluorometer. Peak [Ca2+]cyt values reflecting dense tubular Ca2+ release alone (external Ca2+ removed) also showed large interplatelet variation (171 +/- 105 (S.D.) nM with thrombin and 183 +/- 134 (S.D.) nM with ADP). Dense tubular Ca2+ release induced by cyclopiazonic acid (a dense tubular Ca2+-ATPase inhibitor) gave peak [Ca2+]cyt of 289 +/- 170 nM. Thus the size of the dense tubular Ca2+ pool has an inter-platelet variation of 59% (S.D./mean). Variability of

  16. Partial Sleep Restriction Activates Immune Response-Related Gene Expression Pathways: Experimental and Epidemiological Studies in Humans

    PubMed Central

    Rantanen, Ville; Kronholm, Erkki; Surakka, Ida; van Leeuwen, Wessel M. A.; Lehto, Maili; Matikainen, Sampsa; Ripatti, Samuli; Härmä, Mikko; Sallinen, Mikael; Salomaa, Veikko; Jauhiainen, Matti; Alenius, Harri; Paunio, Tiina; Porkka-Heiskanen, Tarja

    2013-01-01

    Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases. PMID:24194869

  17. Early Changes in the Antithrombin and Thrombin-Antithrombin Complex in Patients With Paroxysmal Atrial Fibrillation

    PubMed Central

    Negreva, Mariya; Georgiev, Svetoslav; Prodanova, Krasimira; Nikolova, Julia

    2016-01-01

    Background Data on coagulation changes in paroxysmal atrial fibrillation (PAF) are scarce. The aim of this study was to examine plasma antithrombin (AT) levels and activity as well as thrombin-antithrombin (TAT) complex levels in the early hours of the clinical manifestation of PAF. Methods Fifty-one patients (26 men and 25 women; mean age 59.84 ± 1.60 years) were consecutively selected with PAF duration < 24 hours, and 52 controls (26 men and 26 women; mean age 59.50 ± 1.46 years) matched the patients in terms of gender, age and comorbidities. Plasma levels and activity of AT and levels of the covalent TAT complex were studied once in each study participant. Results AT plasma levels in PAF patients were statistically significantly lower compared to controls (164.69 ± 10.51 vs. 276.21 ± 8.29 μg/mL, P < 0.001). Plasma activity of the anticoagulant was also significantly lower in PAF (71.33±4.87 vs. 110.72±3.09%, P < 0.001). TAT complex concentration in plasma was higher in the patient group (5.32 ± 0.23 vs. 3.20 ± 0.14 μg/L, P < 0.001). Conclusion We can say that PAF is associated with significantly reduced AT levels and activity and increased levels of TAT complex during the first 24 hours after its manifestation. These changes indicate a reduced activity of AT anticoagulant system, which is a probable prerequisite for the established enhanced coagulation (high TAT complex levels). PMID:28197274

  18. Effects of Chilling and Partial Freezing on Rigor Mortis Changes of Bighead Carp (Aristichthys nobilis) Fillets: Cathepsin Activity, Protein Degradation and Microstructure of Myofibrils.

    PubMed

    Lu, Han; Liu, Xiaochang; Zhang, Yuemei; Wang, Hang; Luo, Yongkang

    2015-12-01

    To investigate the effects of chilling and partial freezing on rigor mortis changes in bighead carp (Aristichthys nobilis), pH, cathepsin B, cathepsin B+L activities, SDS-PAGE of sarcoplasmic and myofibrillar proteins, texture, and changes in microstructure of fillets at 4 °C and -3 °C were determined at 0, 2, 4, 8, 12, 24, 48, and 72 h after slaughter. The results indicated that pH of fillets (6.50 to 6.80) was appropriate for cathepsin function during the rigor mortis. For fillets that were chilled and partially frozen, the cathepsin activity in lysosome increased consistently during the first 12 h, followed by a decrease from the 12 to 24 h, which paralleled an increase in activity in heavy mitochondria, myofibrils and sarcoplasm. There was no significant difference in cathepsin activity in lysosomes between fillets at 4 °C and -3 °C (P > 0.05). Partially frozen fillets had greater cathepsin activity in heavy mitochondria than chilled samples from the 48 to 72 h. In addition, partially frozen fillets showed higher cathepsin activity in sarcoplasm and lower cathepsin activity in myofibrils compared with chilled fillets. Correspondingly, we observed degradation of α-actinin (105 kDa) by cathepsin L in chilled fillets and degradation of creatine kinase (41 kDa) by cathepsin B in partially frozen fillets during the rigor mortis. The decline of hardness for both fillets might be attributed to the accumulation of cathepsin in myofibrils from the 8 to 24 h. The lower cathepsin activity in myofibrils for fillets that were partially frozen might induce a more intact cytoskeletal structure than fillets that were chilled.

  19. Profile of thrombin generation in children with acute lymphoblastic leukemia treated by Berlin-Frankfurt-Münster (BFM) protocols.

    PubMed

    Lejhancova-Tousovska, Katerina; Zapletal, Ondrej; Vytiskova, Sona; Strbackova, Petra; Sterba, Jaroslav

    2012-03-01

    Treatment with L-asparaginase is associated with coagulation disturbances with deep venous thrombosis being the most common clinical consequence. Use of the calibrated automated thrombogram allows precise estimation of thrombin generated in vitro. We show the first data on thrombin generation, measured by calibrated automated thrombography (CAT), in children with acute lymphoblastic leukemia treated with L-asparaginase. Thrombin generation was measured by means of CAT in 23 children treated for acute lymphoblastic leukemia. Samples were obtained at predefined time points during the induction and reinduction phase of acute lymphoblastic leukemia-intercontinental Berlin-Frankfurt-Münster (BFM) 2000 or Associazione Italiana Ematologica Oncologia Pedaitrica Interim BFM 2000 protocols. Antihrombin and fibrinogen were measured on the same sample. Twenty-eight sets of thrombin generation measurements were collected from 23 patients. We observed no significant effect of antithrombin deficiency and/or hypofibrinogenemia on thrombin generation. Endogenous thrombin generation and peak thrombin were significantly higher during induction than in the reinduction phase (P < 0.001). Four patients with severe infection experienced an increase in thrombin generation, reaching maximum in a median of 7.5 days after the onset of infection. Two of those patients developed deep venous thrombosis at the time of peaked endogenous thrombin generation. Thrombin generation in children with acute lymphoblastic leukemia treated according to BFM protocols is significantly higher during the induction phase compared with reinduction and is not substantially affected by hypofibrinogenemia and/or antithrombin deficiency. Severe infection during the induction phase enhances thrombin generation with subsequent risk of thrombosis.

  20. Alpha-1 proteinase inhibitor M358R reduces thrombin generation when displayed on the surface of cells expressing tissue factor.

    PubMed

    Gierczak, Richard F; Pepler, Laura; Bhagirath, Vinai; Liaw, Patricia C; Sheffield, William P

    2014-11-01

    The M358R variant of alpha-1-proteinase inhibitor (API) is a potent soluble inhibitor of thrombin. Previously we engineered AR-API M358R, a membrane-bound form of this protein and showed that it inhibited exogenous thrombin when expressed on transfected cells lacking tissue factor (TF). To determine the suitability of AR-API M358R for gene transfer to vascular cells to limit thrombogenicity, we tested the ability of AR-API M358R to inhibit endogenous thrombin generated in plasma via co-expression co-expressing it on the surface of cells expressing TF. Transfected AR-API M358R formed inhibitory complexes with thrombin following exposure of recalcified, defibrinated plasma to TF on T24/83 cells, but discontinuously monitored thrombin generation was unaffected. Similarly, AR-API M358R expression did not reduce continuously monitored thrombin generation by T24/83 cell suspensions exposed to recalcified normal plasma in a Thrombogram-Thrombinoscope-type thrombin generation assay (TGA); in contrast, 1 μM hirudin variant 3 or soluble API M358R abolished thrombin generation. Gene transfer of TF to HEK 293 conferred the ability to support TF-dependent thrombin generation on HEK 293 cells. Co-transfection of HEK 293 cells with a 9:1 excess of DNA encoding AR-API M358R to that encoding TF reduced peak thrombin generation approximately 3-fold compared to controls. These in vitro results suggest that surface display of API M358R inhibits thrombin generation when the tethered serpin is expressed in excess of TF, and suggest its potential to limit thrombosis in appropriate vascular beds in animal models.