Effects of exercise and conditioning on clotting and fibrinolytic activity in men

NASA Technical Reports Server (NTRS)

Ferguson, Earl W.; Bernier, Lani L.; Banta, Guy R.; Yu-Yahiro, Janet; Schoomaker, Eric B.

1987-01-01

Blood clotting and fibrinolytic activity in three groups of nonsmoking, nonobese, healthy men ranging from 19 to 59 years are studied. The groups consisted of (1) marathoners (men running more than 50 miles/week); (2) joggers (men running 5-15 miles/week; and (3) sedentary subjects (men who did not exercise routinely). It is observed that the rate of blood clotting is accelerated by exercise; marathoners had greater increases in fibrinolytic activity than the other two groups; and fibrin degradation products increased with exercise. The data reveal that the changes in clotting assays with exercise do not correlate with changes in whole blood lactate, blood pyruvate, or rectal temperatures. It is noted that the level of acceleration for fibrinolytic activity is directly related to the maximum aerobic capacity and work load of the individual, and that conditioning enhances the fibrinolytic response to exercise.

Whole blood clots are more resistant to lysis than plasma clots--greater efficacy of rivaroxaban.

PubMed

Varin, Rémi; Mirshahi, Shahsultan; Mirshahi, Pezhman; Klein, Christophe; Jamshedov, Jovid; Chidiac, Jean; Perzborn, Elisabeth; Mirshahi, Massoud; Soria, Claudine; Soria, Jeannette

2013-03-01

Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated. Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy. WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots. The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of enoxaparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent.

PubMed

Schaden, Eva; Schober, Andreas; Hacker, Stefan; Spiss, Christian; Chiari, Astrid; Kozek-Langenecker, Sibylle

2010-04-01

Drug monitoring of low molecular weight heparin is generally not recommended, but could be reasonable in critically ill patients, whose risk for bleeding or thrombosis shows a high interpatient variability. Anti-Xa assays are not available around the clock even in central hospitals, whereas rotational thrombelastometry (ROTEM) becomes increasingly used at the bedside. Prothrombinase-induced clotting time (PiCT) reagent allows determination of factor Xa-inhibition in plasma. The aim of our study was to evaluate enoxaparin determination in whole blood with the ROTEM using specific test modifications, including PiCT. After ethics committee's approval, citrated whole blood obtained from overall 16 healthy volunteers was incubated with enoxaparin at 16 different anti-Xa concentrations. Main endpoint was the clotting time (CT) in ROTEM representing initial activation of clot formation. CT was determined in the new PiCT-ROTEM test, in a low-tissue factor-activated modification (LowTF-ROTEM) as well as in the commercially available heparin-sensitive ROTEM assays (HEPTEM and INTEM). In the absence of enoxaparin, CT values were 168.6 +/- 6.1 s (PiCT-ROTEM), 247.3 +/- 18.6 s (LowTF-ROTEM), and -6.2 +/- 7.9 s (INTEM-HEPTEM). A linear dependency (P < 0.01) between anti-Xa concentration and CT was found for PiCT-ROTEM, LowTF-ROTEM, and for INTEM-HEPTEM with correlation coefficients of 0.93 for PiCT-ROTEM, 0.94 for LowTF-ROTEM, and 0.81 for INTEM-HEPTEM. This in-vitro experiment demonstrates a strong correlation between enoxaparin anti-Xa concentrations and specific ROTEM tests. These promising assays should be further evaluated for monitoring anticoagulation in high-risk patients in clinical studies.

Blood Clots

MedlinePlus

... News Physician Resources Professions Site Index A-Z Blood Clots Blood clots are semi-solid masses of blood ... evaluated? How are blood clots treated? What are blood clots? Blood clots are semi-solid masses of blood. ...

Honey Bee Venom (Apis mellifera) Contains Anticoagulation Factors and Increases the Blood-clotting Time

PubMed Central

Zolfagharian, Hossein; Mohajeri, Mohammad; Babaie, Mahdi

2015-01-01

Objectives: Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Methods: Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Results: Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs) = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. Conclusion: BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2) and melittin, and that can increase the blood clotting times in vitro. PMID:26998384

Honey Bee Venom (Apis mellifera) Contains Anticoagulation Factors and Increases the Blood-clotting Time.

PubMed

Zolfagharian, Hossein; Mohajeri, Mohammad; Babaie, Mahdi

2015-12-01

Bee venom (BV) is a complex mixture of proteins and contains proteins such as phospholipase and melittin, which have an effect on blood clotting and blood clots. The mechanism of action of honey bee venom (HBV, Apis mellifera) on human plasma proteins and its anti-thrombotic effect were studied. The purpose of this study was to investigate the anti-coagulation effect of BV and its effects on blood coagulation and purification. Crude venom obtained from Apis mellifera was selected. The anti-coagulation factor of the crude venom from this species was purified by using gel filtration chromatography (sephadex G-50), and the molecular weights of the anti-coagulants in this venom estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blood samples were obtained from 10 rabbits, and the prothrombin time (PT) and the partial thromboplastin time (PTT) tests were conducted. The approximate lethal dose (LD) values of BV were determined. Crude BV increased the blood clotting time. For BV concentrations from 1 to 4 mg/mL, clotting was not observed even at more than 300 seconds, standard deviations (SDs) = ± 0.71; however, clotting was observed in the control group 13.8 s, SDs = ± 0.52. Thus, BV can be considered as containing anti-coagulation factors. Crude BV is composed 4 protein bands with molecular weights of 3, 15, 20 and 41 kilodalton (kDa), respectively. The LD50 of the crude BV was found to be 177.8 μg/mouse. BV contains anti-coagulation factors. The fraction extracted from the Iranian bees contains proteins that are similar to anti-coagulation proteins, such as phospholipase A2 (PLA2) and melittin, and that can increase the blood clotting times in vitro.

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

PubMed Central

Hess, Katharina; Ajjan, Ramzi; Phoenix, Fladia; Dobó, József; Gál, Péter; Schroeder, Verena

2012-01-01

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation. PMID:22536427

Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism.

PubMed

Martinez, Marissa R; Cuker, Adam; Mills, Angela M; Crichlow, Amanda; Lightfoot, Richard T; Chernysh, Irina N; Nagaswami, Chandrasekaran; Weisel, John W; Ischiropoulos, Harry

2014-03-01

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.

Enhanced lysis and accelerated establishment of viscoelastic properties of fibrin clots are associated with pulmonary embolism

PubMed Central

Martinez, Marissa R.; Cuker, Adam; Mills, Angela M.; Crichlow, Amanda; Lightfoot, Richard T.; Chernysh, Irina N.; Nagaswami, Chandrasekaran; Weisel, John W.

2014-01-01

The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remains uncertain. Therefore, we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII cross-linking, and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation, or maximum absorbance of turbidity compared with DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin cross-linking by activated factor XIII were similar between clots from DVT and PE subjects. Electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared with those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization. PMID:24414255

Acute toxicity of diphacinone in Northern bobwhite: Effects on survival and blood clotting

USGS Publications Warehouse

Rattner, Barnett A.; Horak, Katherine E.; Warner, Sarah E.; Johnston, John J.

2010-01-01

The anticoagulant rodenticide diphacinone was slightly toxic (acute oral LD50 2014 mg/kg) to Northern bobwhite (Colinus virginianus) in a 14-day acute toxicity trial. Precise and sensitive assays of blood clotting (prothrombin time, Russell?s Viper venom time, and thrombin clotting time) were adapted for use in quail, and this combination of assays is recommended to measure the effects of anticoagulant rodenticides. A single oral sublethal dose of diphacinone (434 mg/kg body weight) prolonged clotting time at 48 h post-dose compared to controls. At 783 mg/kg (approximate LD02), clotting time was prolonged at both 24 and 48 h post-dose. Prolongation of in vitro clotting time reflects impaired coagulation complex activity, and was detected before overt signs of toxicity were apparent at the greatest dosages (2868 and 3666 mg/kg) in the acute toxicity trial. These clotting time assays and toxicity data will assist in the development of a pharmacodynamic model to predict toxicity, and also facilitate rodenticide hazard and risk assessments in avian species.

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction.

PubMed

Mor-Cohen, Ronit; Rosenberg, Nurit; Averbukh, Yulia; Seligsohn, Uri; Lahav, Judith

2014-05-01

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation. In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange. Copyright © 2014 Elsevier Ltd. All rights reserved.

Plasma clot formation and clot lysis to compare effects of different anticoagulation treatments on hemostasis in patients with atrial fibrillation.

PubMed

Königsbrügge, Oliver; Weigel, Günter; Quehenberger, Peter; Pabinger, Ingrid; Ay, Cihan

2018-02-07

The effect of direct oral anticoagulants (DOACs) on turbidimetric measurements of plasma clot formation and susceptibility to fibrinolysis may facilitate a comparison between different classes of anticoagulants in plasma samples. We obtained 424 citrate plasma samples from 226 atrial fibrillation patients on anticoagulation and 24 samples without anticoagulation serving as controls. As comparators, we measured the international normalized ratio (INR) for phenprocoumon samples (N = 166), anti-Xa for low molecular weight heparin (LMWH) samples (N = 42), and DOAC levels with mass spectrometry (dabigatran N = 40, rivaroxaban N = 110, apixaban N = 42). Plasma clot formation and lysis were recorded continuously on a photometer after addition of an activation mix (tissue factor 2 pmol/l and tissue plasminogen activator 333 ng/ml). We used linear regression and ANCOVA for correlation analysis. Clot formation lag phase was prolonged in the presence of anticoagulants in a concentration-dependent manner for DOACs (dabigatran Spearman r = 0.74; rivaroxaban r = 0.78; apixaban r = 0.72, all p < 0.0001), INR dependent for phenprocoumon (r = 0.59, p < 0.0001), anti-Xa level dependent in LMWH samples (r = 0.90, p < 0.0001). Maximum rate of clot formation and peak clot turbidity were reduced in the presence of anticoagulants, but correlated only moderately with the comparator measures of anticoagulation. The clot lysis time was inversely correlated with DOAC concentrations in the presence of recombinant thrombomodulin. A direct ex vivo comparison between the effects of different classes of anticoagulants is possible with turbidimetric measurement of plasma clot formation and lysis. Anticoagulation inhibited clot formation in a plasma concentration manner for DOACs, INR dependent for phenprocoumon, and anti-Xa dependent for LMWH. Susceptibility to fibrinolysis increased with increasing DOAC concentrations.

Blood clot detection using magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Khurshid, Hafsa; Friedman, Bruce; Berwin, Brent; Shi, Yipeng; Ness, Dylan B.; Weaver, John B.

2017-05-01

Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles' relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

Comparative lytic efficacy of rt-PA and ultrasound in porcine versus human clots.

PubMed

Huang, Shenwen; Shekhar, Himanshu; Holland, Christy K

2017-01-01

Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant. An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US. The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately.

[Guidelines for certification of Activated clotting time (ACT) according to the EN ISO 22870 standards].

PubMed

Lasne, Dominique; Bauters, Anne; Le Querrec, Agnès; Bourdin, Carole; Voisin, Sophie

2015-01-01

Point of care testing (POCT) must comply with regulatory requirements according to standard EN ISO 22870, which identify biologists as responsible for POCT. Activated clotting time (ACT) is mandatory to monitor on whole blood, anticoagulation achieved by unfractionated heparin during cardiopulmonary bypass (CPB) or cardiac catheterization. This test has no equivalent in the laboratory. With the aim to help the multidisciplinary groups for POCT supervision when they have to analyse the wish of medical departments to use ACT and to help the biologists to be in accordance with the standard, we present the guidelines of the GEHT (Groupe d'étude d'hémostase et thrombose) subcommittee "CEC et Biologie délocalisée" for the certification of ACT. These guidelines are based on the SFBC guidelines for the certification of POCT and on the analysis of the literature to ascertain the justification of clinical need and assess the analytical performance of main analyzers used in France, as well as on a survey conducted with French and Belgian biologists.

Blood Clots

MedlinePlus

... if you get hurt, your body forms a blood clot to stop the bleeding. After the bleeding stops ... some people get too many clots or their blood clots abnormally. Many conditions can cause the blood to ...

Contraction of Blood Clots Is Impaired in Acute Ischemic Stroke.

PubMed

Tutwiler, Valerie; Peshkova, Alina D; Andrianova, Izabella A; Khasanova, Dina R; Weisel, John W; Litvinov, Rustem I

2017-02-01

Obstructive thrombi or thrombotic emboli are the pathogenic basis of ischemic stroke. In vitro blood clots and in vivo thrombi can undergo platelet-driven contraction (retraction), resulting in volume shrinkage. Clot contraction can potentially reduce vessel occlusion and improve blood flow past emboli or thrombi. The aim of this work was to examine a potential pathogenic role of clot contraction in ischemic stroke. We used a novel automated method that enabled us to quantify time of initiation and extent and rate of clot contraction in vitro. The main finding is that clot contraction from the blood of stroke patients was reduced compared with healthy subjects. Reduced clot contraction correlated with a lower platelet count and their dysfunction, higher levels of fibrinogen and hematocrit, leukocytosis, and other changes in blood composition that may affect platelet function and properties of blood clots. Platelets from stroke patents were spontaneously activated and displayed reduced responsiveness to additional stimulation. Clinical correlations with respect to severity and stroke pathogenesis suggest that the impaired clot contraction has the potential to be a pathogenic factor in ischemic stroke. The changeable ability of clots and thrombi to shrink in volume may be a novel unappreciated mechanism that aggravates or alleviates the course and outcomes of ischemic stroke. The clinical importance of clot or thrombus transformations in vivo and the diagnostic and prognostic value of this blood test for clot contraction need further exploration. © 2016 American Heart Association, Inc.

Contraction of Blood Clots is Impaired in Acute Ischemic Stroke

PubMed Central

Tutwiler, Valerie; Peshkova, Alina D.; Andrianova, Izabella A.; Khasanova, Dina R.; Weisel, John W.; Litvinov, Rustem I.

2016-01-01

Objective Obstructive thrombi or thrombotic emboli are the pathogenic basis of ischemic stroke. In vitro blood clots and in vivo thrombi can undergo platelet-driven contraction (retraction), resulting in volume shrinkage. Clot contraction can potentially reduce vessel occlusion and improve blood flow past emboli or thrombi. The aim of this work was to examine a potential pathogenic role of clot contraction in ischemic stroke. Approach and Results We employed a novel automated method that enabled us to quantify time of initiation, extent and rate of clot contraction in vitro. The main finding is clot contraction from the blood of stroke patients was reduced compared to healthy subjects. Reduced clot contraction correlated with a lower platelet count and their dysfunction, higher levels of fibrinogen and hematocrit, leukocytosis and other changes in blood composition that may affect platelet function and properties of blood clots. Platelets from stroke patents were spontaneously activated and displayed reduced responsiveness to additional stimulation. Clinical correlations with respect to severity and stroke etiology suggest that the impaired clot contraction has the potential to be a pathogenic factor in ischemic stroke. Conclusions The changeable ability of clots and thrombi to shrink in volume may be a novel unappreciated mechanism that aggravates or alleviates the course and outcomes of ischemic stroke. The clinical importance of clot or thrombus transformations in vivo and the diagnostic and prognostic value of this blood test for clot contraction needs further exploration. PMID:27908894

Comparison of Hemostasis Times With a Kaolin-Based Hemostatic Pad (QuikClot Radial) vs Mechanical Compression (TR Band) Following Transradial Access: A Pilot Prospective Study.

PubMed

Roberts, Jonathan S; Niu, Jianli; Pastor-Cervantes, Juan A

2017-10-01

Hemostasis following transradial access (TRA) is usually achieved by mechanical compression. We investigated use of the QuikClot Radial hemostasis pad (Z-Medica) compared with the TR Band (Terumo Medical) to shorten hemostasis after TRA. Thirty patients undergoing TRA coronary angiography and/or percutaneous coronary intervention were randomized into three cohorts post TRA: 10 patients received mechanical compression with the TR Band, 10 patients received 30 min of compression with the QuikClot Radial pad, and 10 patients received 60 min of compression with the QuikClot Radial pad. Times to hemostasis and access-site complications were recorded. Radial artery patency was evaluated 1 hour after hemostasis by the reverse Barbeau's test. There were no differences in patient characteristics, mean dose of heparin (7117 ± 1054 IU), or mean activated clotting time value (210 ± 50 sec) at the end of procedure among the three groups. Successful hemostasis was achieved in 100% of patients with both the 30-min and 60-min compression groups using the QuikClot pad. Hemostasis failure occurred in 50% of patients when the TR Band was initially weaned at the protocol-driven time (40 min after sheath removal). Mean compression time for hemostasis with the TR Band was 149.4 min compared with 30.7 min and 60.9 min for the 30-min and 60-min QuikClot groups, respectively. No radial artery occlusion occurred in any subject at the end of the study. Use of the QuikClot Radial pad following TRA in this pilot trial significantly shortened hemostasis times when compared with the TR Band, with no increased complications noted.

Prediction of recurrent venous thromboembolism by clot lysis time: a prospective cohort study.

PubMed

Traby, Ludwig; Kollars, Marietta; Eischer, Lisbeth; Eichinger, Sabine; Kyrle, Paul A

2012-01-01

Venous thromboembolism (VTE) is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT]) and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity. 135 (19%) patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR) of recurrence was 1.13 (95% CI 1.02-1.25; p = 0.02) and was 1.08 (95% CI 0.98-1.20; p = 0.13) after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91-1.42, p = 0.22) for each increase in CLT of 10 minutes. CLT values in the 4(th) quartile of the female patient population, as compared to values in the 1(st) quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07-10.05; p = 0.04). No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only.

Prediction of Recurrent Venous Thromboembolism by Clot Lysis Time: A Prospective Cohort Study

PubMed Central

Traby, Ludwig; Kollars, Marietta; Eischer, Lisbeth; Eichinger, Sabine; Kyrle, Paul A.

2012-01-01

Venous thromboembolism (VTE) is a chronic disease, which tends to recur. Whether an abnormal fibrinolytic system is associated with an increased risk of VTE is unclear. We assessed the relationship between fibrinolytic capacity (reflected by clot lysis time [CLT]) and risk of recurrent VTE. We followed 704 patients (378 women; mean age 48 yrs) with a first unprovoked VTE for an average of 46 months after anticoagulation withdrawal. Patients with natural coagulation inhibitor deficiency, lupus anticoagulant, cancer, homozygosity for factor V Leiden or prothrombin mutation, or requirement for indefinite anticoagulation were excluded. Study endpoint was symptomatic recurrent VTE. For measurement of CLT, a tissue factor-induced clot was lysed by adding tissue-type plasminogen activator. Time between clot formation and lysis was determined by measuring the turbidity.135 (19%) patients had recurrent VTE. For each increase in CLT of 10 minutes, the crude relative risk (RR) of recurrence was 1.13 (95% CI 1.02–1.25; p = 0.02) and was 1.08 (95% CI 0.98–1.20; p = 0.13) after adjustment for age and sex. For women only, the adjusted RR was 1.14 (95% CI, 0.91–1.42, p = 0.22) for each increase in CLT of 10 minutes. CLT values in the 4th quartile of the female patient population, as compared to values in the 1st quartile, conferred a risk of recurrence of 3.28 (95% CI, 1.07–10.05; p = 0.04). No association between CLT and recurrence risk was found in men. Hypofibrinolysis as assessed by CLT confers a moderate increase in the risk of recurrent VTE. A weak association between CLT and risk of recurrence was found in women only. PMID:23240024

Comparative lytic efficacy of rt-PA and ultrasound in porcine versus human clots

PubMed Central

Shekhar, Himanshu; Holland, Christy K.

2017-01-01

Introduction Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant. Materials and methods An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US. Results and conclusions The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately. PMID:28545055

Sonothrombolysis is effective with recombinant tissue-type plasminogen activator, but not with Abciximab. Results from an in vitro study with whole blood clots and platelet-rich clots.

PubMed

Eggers, Jürgen; Ossadnik, Stefan; Hütten, Heiko; Seidel, Günter

2009-12-01

Transcranial "diagnostic" ultrasound (US) has been shown to accelerate thrombolysis related to recombinant tissue-type plasminogen activator (rt-PA). In this in vitro study, we evaluated the potential of US to increase clot dissolution mediated by Abciximab (Abc) compared to rt-PA. The effect of 1.8-MHz pulsed wave (PW) Doppler US on dissolution of whole venous blood clots (WBC) and platelet-rich clots (PRC) treated with Abc and rt-PA was investigated in an in vitro model. Clot dissolution was measured by weight loss. Abc-related WBC dissolution was enhanced by additional US, but the effect was not any more detectable when the US was attenuated by a human temporal bone (US-tb). In PRC there was no additional effect of US on the Abc-related clot lysis. Rt-PA-related clot dissolution was increased by US in WBC and PRC as well, however, US-tb was only effective in WBC. The effect of insonation on WBC dissolution treated with the combination of Abc plus rt-PA was lower compared with those treated with rt-PA. In this in vitro experiment, the additional effect of "diagnostic" US in combination with Abc was only present in WBC and less strong than with rt-PA. The results do not support the use of Abc for sonothrombolysis targeting both, fibrin-rich and platelet-rich clots. In contrast, US when combined with rt-PA increases dissolution in both, WBC and PRC as well.

A novel clot lysis assay for recombinant plasminogen activator.

PubMed

Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

2015-03-01

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

Assessment of Heparin Anticoagulation Measured Using i-STAT and Hemochron Activated Clotting Time.

PubMed

Maslow, Andrew; Chambers, Alison; Cheves, Tracey; Sweeney, Joseph

2018-01-31

Adequate anticoagulation, measured using activated clotting time (ACT), is important during vascular and cardiac surgeries. Unfractionated heparin is the most common anticoagulant used. The purpose of this analysis was to compare the i-STAT ACT (iACT) to the Hemochron ACT (hACT), both of which were then compared to anti-factor Xa (anti-Xa) assay, a representation of heparin level and activity. Prospective study. Tertiary care cardiovascular center. Eleven consecutive elective adult cardiac surgical patients. Prior to cardiopulmonary bypass, ACTs were measured using i-STAT and Hemochron technologies and compared to each other and to anti-Xa assay prior to and during a cumulative administration of heparin. Data were compared using bias analyses. Heparin (300 U/kg) was administered in quarterly doses. Coagulation labs were collected prior to and 3 minutes after each quarterly dose of heparin. The baseline ACTs for i-STAT and Hemochron were 147 and 142 seconds, respectively. A significant association was found between iACT and hACT (p = 0.002). The iACT measurements underestimated hACT at ACT levels >180 seconds or anti-Xa levels >0.75 U/mL. No significant difference was found between ACT data at anti-Xa levels <0.5 U/mL. There was a good association between the iACT and hACT; however, the 2 tests are not equivalent. Overall, the iACT underestimated the hACT. Agreement between the ACT technologies was good at lower ACTs and anti-Xa levels, but declined with an anti-Xa >0.75 U/mL. Copyright © 2018 Elsevier Inc. All rights reserved.

Pilot study of Alteplase (tissue plasminogen activator) for treatment of urinary clot retention in an in vitro model.

PubMed

Ritch, Chad R; Ordonez, Maria A; Okhunov, Zhamshid; Araujo, Juan; Walsh, Rhonda; Baudin, Vania; Lee, Daniel; Badani, Ketan K; Gupta, Mantu; Landman, Jaime

2009-08-01

The management of urinary clot retention and hematuria involves manual irrigation with sterile water or normal saline via a Foley catheter followed by continuous bladder irrigation. Irrigation may become difficult because of the formation of dense blood clots. Tissue plasminogen activator (t-PA/Alteplase) may be a useful pharmacological agent to improve the efficacy of manual irrigation of large, dense clots. The goal of the current study was to compare t-PA to sterile water for clot irrigation in an in vitro model. In vitro models of clot retention were created using 500-cc urinary leg bags each filled with 80 cc of unpreserved whole blood from a healthy volunteer. Each model was incubated at 25 degrees C for 24 hours to allow clot formation. Four models each with 25 mL solution of t-PA at concentrations of 2, 1, 0.5, and 0.25 mg/mL were evaluated and compared to a control (25 mL sterile water). Models were instilled with solution (t-PA or control) and incubated for 30 minutes at 37 degrees C, and then irrigated with sterile water via 18F Foley by a blinded investigator. Three separate experiments were conducted, and statistical analysis was performed comparing various irrigation parameters. Clot evacuation with 25 mL of t-PA at a concentration of 2 mg/mL (50 mg) was significantly easier (p = 0.05) and faster (p < 0.05) than the sterile water control. The mean time for clot evacuation in this model was 2.7 minutes for t-PA solution 2 mg/mL versus 7.3 minutes for the control (p < 0.05). Compared to the control, irrigation with t-PA solution 2 mg/mL also required less irrigant (180 mL vs. 500 mL) (p < 0.05) for complete evacuation. There was a similar trend in efficacy for the lower doses of t-PA, but this was not statistically significant. In this in vitro study, a single 25 mL instillation of t-PA solution 2 mg/mL is significantly better than sterile water alone for clot evacuation. In vivo animal studies are pending.

ClotChip: A Microfluidic Dielectric Sensor for Point-of-Care Assessment of Hemostasis.

PubMed

Maji, Debnath; Suster, Michael A; Kucukal, Erdem; Sekhon, Ujjal D S; Gupta, Anirban Sen; Gurkan, Umut A; Stavrou, Evi X; Mohseni, Pedram

2017-12-01

This paper describes the design, fabrication, and testing of a microfluidic sensor for dielectric spectroscopy of human whole blood during coagulation. The sensor, termed ClotChip, employs a three-dimensional, parallel-plate, capacitive sensing structure with a floating electrode integrated into a microfluidic channel. Interfaced with an impedance analyzer, the ClotChip measures the complex relative dielectric permittivity, ϵ r , of human whole blood in the frequency range of 40 Hz to 100 MHz. The temporal variation in the real part of the blood dielectric permittivity at 1 MHz features a time to reach a permittivity peak, , as well as a maximum change in permittivity after the peak, , as two distinct parameters of ClotChip readout. The ClotChip performance was benchmarked against rotational thromboelastometry (ROTEM) to evaluate the clinical utility of its readout parameters in capturing the clotting dynamics arising from coagulation factors and platelet activity. exhibited a very strong positive correlation ( r = 0.99, p < 0.0001) with the ROTEM clotting time parameter, whereas exhibited a strong positive correlation (r = 0.85,  p < 0.001) with the ROTEM maximum clot firmness parameter. This paper demonstrates the ClotChip potential as a point-of-care platform to assess the complete hemostatic process using <10 μL of human whole blood.

Real-time monitoring of human blood clotting using a lateral excited film bulk acoustic resonator

NASA Astrophysics Data System (ADS)

Chen, Da; Wang, Jingjng; Wang, Peng; Guo, Qiuquan; Zhang, Zhen; Ma, Jilong

2017-04-01

Frequent assay of hemostatic status is an essential issue for the millions of patients using anticoagulant drugs. In this paper, we presented a micro-fabricated film bulk acoustic sensor for the real-time monitoring of blood clotting and the measurement of hemostatic parameters. The device was made of an Au/ZnO/Si3N4 film stack and excited by a lateral electric field. It operated under a shear mode resonance with the frequency of 1.42 GHz and had a quality factor of 342 in human blood. During the clotting process of blood, the resonant frequency decreased along with the change of blood viscosity and showed an apparent step-ladder curve, revealing the sequential clotting stages. An important hemostatic parameter, prothrombin time, was quantitatively determined from the frequency response for different dilutions of the blood samples. The effect of a typical anticoagulant drug (heparin) on the prothrombin time was exemplarily shown. The proposed sensor displayed a good consistency and clinical comparability with the standard coagulometric methods. Thanks to the availability of direct digital signals, excellent potentials of miniaturization and integration, the proposed sensor has promising application for point-of-care coagulation technologies.

A targeted ferritin-microplasmin based thrombolytic nanocage selectively dissolves blood clots.

PubMed

Seo, Junyoung; Al-Hilal, Taslim A; Jee, Jun-Goo; Kim, Yong-Lim; Kim, Ha-Jeong; Lee, Byung-Heon; Kim, Soyoun; Kim, In-San

2018-04-01

The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (μPn); CLT recognizes fibrin-fibronectin complexes in clots, μPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-μPn) into nanocage structure protects the activated-μPn from its circulating inhibitors. Importantly, activated CLT-sFt-μPn thrombolytic nanocage showed a prolonged circulatory life over activated-μPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-μPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

Application of Adaptive DP-optimality to Design a Pilot Study for a Clotting Time Test for Enoxaparin.

PubMed

Gulati, Abhishek; Faed, James M; Isbister, Geoffrey K; Duffull, Stephen B

2015-10-01

Dosing of enoxaparin, like other anticoagulants, may result in bleeding following excessive doses and clot formation if the dose is too low. We recently showed that a factor Xa based clotting time test could potentially assess the effect of enoxaparin on the clotting system. However, the test did not perform well in subsequent individuals and effectiveness of an exogenous phospholipid, Actin FS, in reducing the variability in the clotting time was assessed. The aim of this work was to conduct an adaptive pilot study to determine the range of concentrations of Xa and Actin FS to take forward into a proof-of-concept study. A nonlinear parametric function was developed to describe the response surface over the factors of interest. An adaptive method was used to estimate the parameters using a D-optimal design criterion. In order to provide a reasonable probability of observing a success of the clotting time test, a P-optimal design criterion was incorporated using a loss function to describe the hybrid DP-optimality. The use of adaptive DP-optimality method resulted in an efficient estimation of model parameters using data from only 6 healthy volunteers. The use of response surface modelling identified a range of sets of Xa and Actin FS concentrations, any of which could be used for the proof-of-concept study. This study shows that parsimonious adaptive DP-optimal designs may provide both precise parameter estimates for response surface modelling as well as clinical confidence in the potential benefits of the study.

Ultrastructural characteristics of fibrin clots from canine and feline platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin.

PubMed

Silva, Raúl F; Carmona, Jorge U; Rezende, Cleuza M F

2013-04-15

The aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates (PC) that had been activated with calcium gluconate (CG) or CG plus batroxobin (CGB). Platelets from fibrin clots were classified according their morphological changes. The area of the intercellular space (μm2), the area of the fibrin fibers (μm2), and the width of the fibrin fibers (μm) were determined for the dog clots. The platelet area (μm2), the area of fibrin fibers (μm2), the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. Cat platelets displayed full activation. Dog platelets displayed lysis with loss of normal architecture. In both species, a statistically significant difference was found (P < 0.01) between the fibrin fiber measurements in the PC clots activated with CG and CGB. The findings suggest that activation with CG caused platelet alpha granules to release their contents. In cats, fibrin production was greater when the PC was activated with CG. In dogs, activation with CG produced thick fibrin fibers.

Effect of clot aging and cholesterol content on ultrasound-assisted thrombolysis.

PubMed

Zhou, Yufeng; Murugappan, Suresh Kanna; Sharma, Vijay Kumar

2014-10-01

Exposure to 2-MHz transcranial diagnostic ultrasound enhances the thrombolytic activity of intravenously administered tissue plasminogen activator (IV-tPA) in acute ischemic stroke (sonothrombolysis). However, rates of arterial recanalization vary widely, depending upon the clot burden, its location, and stroke subtype. We evaluated the influence of age and cholesterol level of the blood clots on sonothrombolysis in an in vitro model. To "age" the clots, serum was replaced by fresh blood periodically. We increased the cholesterol content of the clots by adding cholesterin to the blood. The clots were lysed by tPA and/or transcranial Doppler ultrasound sonication for 1 h. The extent of thrombolysis induced by various treatment protocols (controls, sonication, tPA, and sonothrombolysis) was evaluated with relative changes in the clot weights and in the clot structure by scanning electron microscopy (SEM) at end of the experiment. Sonothrombolysis induced significantly higher weight reduction in fresh clots (37.3 % in 2-h old clots versus 24.8 % in 10-h ones, p < 0.005) as well as the clots with higher cholesterol levels (41.7 versus 30.6 % in normal cholesterol clots, p < 0.005). SEM demonstrated patterns of clot dissolution among various treatment modalities. Sonothrombolysis induced better clot lysis in fresh thrombi with high cholesterol levels.

Pulsed focused ultrasound-induced displacements in confined in vitro blood clots.

PubMed

Wright, Cameron C; Hynynen, Kullervo; Goertz, David E

2012-03-01

Ultrasound has been shown to potentiate the effects of tissue plasminogen activator to improve clot lysis in a range of in vitro and in vivo studies as well as in clinical trials. One possible mechanism of action is acoustic radiation force-induced clot displacements. In this study, we investigate the temporal and spatial dynamics of clot displacements and strain initiated by focused ultrasound pulses. Displacements were produced by a 1.51 MHz f-number 1 transducer over a range of acoustic powers (1-85 W) in clots constrained within an agar vessel phantom channel. Displacements were tracked during and after a 5.45 ms therapy pulse using a 20 MHz high-frequency ultrasound imaging probe. Peak thrombus displacements were found to be linear as a function of acoustic power up to 60 W before leveling off near 128 μm for the highest transmit powers. The time to peak displacement and recovery time of blood clots was largely independent of acoustic powers with measured values near 2 ms. A linear relationship between peak axial strain and transmit power was observed, reaching a peak value of 11% at 35 W. The peak strain occurred ~0.75 mm from the focal zone for all powers investigated in both lateral and axial directions. These results indicate that substantial displacements can be induced by focused ultrasound in confined blood clots, and that the spatial and temporal displacement patterns are complex and highly dependent on exposure conditions, which has implications for future work investigating their link to clot lysis and for developing approaches to exploit these effects.

Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

PubMed Central

Elnager, Abuzar; Hassan, Rosline; Idris, Zamzuri; Mustafa, Zulkifli; Wan-Arfah, Nadiah; Sulaiman, S. A.; Gan, Siew Hua; Abdullah, Wan Zaidah

2015-01-01

Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted. PMID:25664321

Fibrinolytic activity and dose-dependent effect of incubating human blood clots in caffeic acid phenethyl ester: in vitro assays.

PubMed

Elnager, Abuzar; Hassan, Rosline; Idris, Zamzuri; Mustafa, Zulkifli; Wan-Arfah, Nadiah; Sulaiman, S A; Gan, Siew Hua; Abdullah, Wan Zaidah

2015-01-01

Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted.

Clot lysis time in platelet-rich plasma: method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid.

PubMed

Panes, Olga; Padilla, Oslando; Matus, Valeria; Sáez, Claudia G; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego

2012-01-01

Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is

Late clotted haemothorax after blunt chest trauma.

PubMed Central

Sinha, P; Sarkar, P

1998-01-01

A clotted haemothorax can develop any time after blunt chest trauma. Two cases are described in which late clotted haemothoraces developed which were treated by limited thoracotomy and evacuation of clots. Late clotted haemothorax may occur even in the absence of any abnormal initial clinical findings. Early detection and treatment is important to avoid the complications of fibrothorax and empyema with permanent pulmonary dysfunction. After blunt chest trauma patients should be advised to return to the accident and emergency department for assessment on development of any new chest symptom. Under these circumstances a chest x ray is mandatory to exclude a haemothorax. Images Figure 1 PMID:9639184

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

PubMed

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective

Blood clotting

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Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage

PubMed Central

Masomi-Bornwasser, Julia; Müller-Werkmeister, Hendrik; Kantelhardt, Sven Rainer; König, Jochem; Kempski, Oliver; Giese, Alf

2017-01-01

Hematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral hemorrhage (ICH). Optimal dose and schedule are still unclear. The aim of this study was to create a reliable in vitro blood clot model for investigation of optimal drug dose and timing. An in vitro clot model was established, using 25 mL and 50 mL of human blood. Catheters were placed into the clots and three groups, using intraclot application of rtPA, placebo, and catheter alone, were analyzed. Dose-response relationship, repetition, and duration of rtPA treatment and its effectiveness in aged clots were investigated. A significant relative end weight difference was found in rtPA treated clots compared to catheter alone (p = 0.002) and placebo treated clots (p < 0.001). Dose-response analysis revealed 95% effective dose around 1 mg rtPA in 25 and 50 mL clots. Approximately 80% of relative clot lysis could be achieved after 15 min incubation. Lysis of aged clots was less effective. A new clot model for in vitro investigation was established. Our data suggest that current protocols for rtPA based ICH therapy may be optimized by using less rtPA at shorter incubation times. PMID:28459065

Optimization of Catheter Based rtPA Thrombolysis in a Novel In Vitro Clot Model for Intracerebral Hemorrhage.

PubMed

Keric, Naureen; Masomi-Bornwasser, Julia; Müller-Werkmeister, Hendrik; Kantelhardt, Sven Rainer; König, Jochem; Kempski, Oliver; Giese, Alf

2017-01-01

Hematoma lysis with recombinant tissue plasminogen activator (rtPA) has emerged as an alternative therapy for spontaneous intracerebral hemorrhage (ICH). Optimal dose and schedule are still unclear. The aim of this study was to create a reliable in vitro blood clot model for investigation of optimal drug dose and timing. An in vitro clot model was established, using 25 mL and 50 mL of human blood. Catheters were placed into the clots and three groups, using intraclot application of rtPA, placebo, and catheter alone, were analyzed. Dose-response relationship, repetition, and duration of rtPA treatment and its effectiveness in aged clots were investigated. A significant relative end weight difference was found in rtPA treated clots compared to catheter alone ( p = 0.002) and placebo treated clots ( p < 0.001). Dose-response analysis revealed 95% effective dose around 1 mg rtPA in 25 and 50 mL clots. Approximately 80% of relative clot lysis could be achieved after 15 min incubation. Lysis of aged clots was less effective. A new clot model for in vitro investigation was established. Our data suggest that current protocols for rtPA based ICH therapy may be optimized by using less rtPA at shorter incubation times.

Evaluation of Collaterals and Clot Burden Using Time-Resolved C-Arm Conebeam CT Angiography in the Angiography Suite: A Feasibility Study.

PubMed

Yang, P; Niu, K; Wu, Y; Struffert, T; Doerfler, A; Holter, P; Aagaard-Kienitz, B; Strother, C; Chen, G-H

2017-04-01

The assessment of collaterals and clot burden in patients with acute ischemic stroke provides important information about treatment options and clinical outcome. Time-resolved C-arm conebeam CT angiography has the potential to provide accurate and reliable evaluations of collaterals and clot burden in the angiographic suite. Experience with this technique is extremely limited, and feasibility studies are needed to validate this technique. Our purpose was to present such a feasibility study. Ten C-arm conebeam CT perfusion datasets from 10 subjects with acute ischemic stroke acquired before endovascular treatment were retrospectively processed to generate time-resolved conebeam CTA. From time-resolved conebeam CTA, 2 experienced readers evaluated the clot burden and collateral flow in consensus by using previously reported scoring systems and assessed the clinical value of this novel imaging technique independently. Interobserver agreement was analyzed by using the intraclass correlation analysis method. Clot burden and collateral flow can be assessed by using the commonly accepted scoring systems for all eligible cases. Additional clinical information (eg, the quantitative dynamic information of collateral flow) can be obtained from this new imaging technique. Two readers agreed that time-revolved C-arm conebeam CTA is the preferred method for evaluating the clot burden and collateral flow compared with other conventional imaging methods. Comprehensive evaluations of clot burden and collateral flow are feasible by using time-resolved C-arm conebeam CTA data acquired in the angiography suite. This technique further enriches the imaging tools in the angiography suite to enable a "one-stop- shop" imaging workflow for patients with acute ischemic stroke. © 2017 by American Journal of Neuroradiology.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking.

PubMed

Byrnes, James R; Duval, Cédric; Wang, Yiming; Hansen, Caroline E; Ahn, Byungwook; Mooberry, Micah J; Clark, Martha A; Johnsen, Jill M; Lord, Susan T; Lam, Wilbur A; Meijers, Joost C M; Ni, Heyu; Ariëns, Robert A S; Wolberg, Alisa S

2015-10-15

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. © 2015 by The American Society of Hematology.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking

PubMed Central

Byrnes, James R.; Duval, Cédric; Wang, Yiming; Hansen, Caroline E.; Ahn, Byungwook; Mooberry, Micah J.; Clark, Martha A.; Johnsen, Jill M.; Lord, Susan T.; Lam, Wilbur A.; Meijers, Joost C. M.; Ni, Heyu; Ariëns, Robert A. S.

2015-01-01

Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. PMID:26324704

The euglobulin clot lysis time to assess the impact of nanoparticles on fibrinolysis

NASA Astrophysics Data System (ADS)

Minet, Valentine; Alpan, Lutfiye; Mullier, François; Toussaint, Olivier; Lucas, Stéphane; Dogné, Jean-Michel; Laloy, Julie

2015-07-01

Nanoparticles (NPs) are developed for many applications in various fields, including nanomedicine. The NPs used in nanomedicine may disturb homeostasis in blood. Secondary hemostasis (blood coagulation) and fibrinolysis are complex physiological processes regulated by activators and inhibitors. An imbalance of this system can either lead to the development of hemorrhages or thrombosis. No data are currently available on the impact of NPs on fibrinolysis. The objectives of this study are (1) to select a screening test to study ex vivo the impact of NPs on fibrinolysis and (2) to test NPs with different physicochemical properties. Euglobulin clot lysis time test was selected to screen the impact of some NPs on fibrinolysis using normal pooled plasma. A dose-dependent decrease in the lysis time was observed with silicon dioxide and silver NPs without disturbing the fibrin network. Carbon black, silicon carbide, and copper oxide did not affect the lysis time at the tested concentrations.

Biological variation in tPA-induced plasma clot lysis time.

PubMed

Talens, Simone; Malfliet, Joyce J M C; Rudež, Goran; Spronk, Henri M H; Janssen, Nicole A H; Meijer, Piet; Kluft, Cornelis; de Maat, Moniek P M; Rijken, Dingeman C

2012-10-01

Hypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis. Sufficient information about assay variation and biological variation in CLT is not yet available. Thus, this study aimed to determine the analytical, within-subject and between-subject variation in CLT. We collected blood samples from 40 healthy individuals throughout a period of one year (average 11.8 visits) and determined the CLT of each plasma sample in duplicate. The mean (± SD) CLT was 83.8 (± 11.1) minutes. The coefficients of variation for total variation, analytical variation, within-subject variation and between-subject variation were 13.4%, 2.6%, 8.2% and 10.2%, respectively. One measurement can estimate the CLT that does not deviate more than 20% from its true value. The contribution of analytical variation to the within-subject variation was 5.0%, the index of individuality was 0.84 and the reference change value was 23.8%. The CLT was longer in the morning compared to the afternoon and was slightly longer in older individuals (> 40 years) compared to younger (≤40 years) individuals. There was no seasonal variation in CLT and no association with air pollution. CLT correlated weakly with fibrinogen, C-reactive protein, prothrombin time and thrombin generation. This study provides insight into the biological variation of CLT, which can be used in future studies testing CLT as a potential risk factor for thrombosis.

Numerical Study of Blood Clots Influence on the Flow Pattern and Platelet Activation on a Stented Bifurcation Model.

PubMed

García Carrascal, P; García García, J; Sierra Pallares, J; Castro Ruiz, F; Manuel Martín, F J

2017-05-01

Stent implantation is a common procedure followed in arteries affected by atherosclerosis. This procedure can lead to other stenting-related problems. One of these is the deposition and accumulation of blood clots over stent struts. This process can have further consequences, in so far as it can introduce modifications to the flow pattern. This problem is especially critical in stented bifurcations, where resulting stent geometry is more complex. In this regard, a numerical study is presented of the effect on the flow pattern and platelet activation of blood clot depositions on the stent struts of a stented coronary bifurcation. The numerical model is first validated with experimental measurements performed for this purpose. Experiments considered a flow with suspended artificial thrombi, which naturally deposited on stent struts. The location and shape observed were used to create numerical thrombi. Following this, numerical simulations were performed to analyze the influence of the presence of thrombi depositions on parameters such as Time Averaged Wall Shear Stress, Oscillatory Shear Index or Relative Residence Time. Finally, a study was also carried out of the effect of different geometrical configurations, from a straight tube to a stented bifurcation model with thrombus depositions, on platelet activation.

Model predictions of deformation, embolization and permeability of partially obstructive blood clots under variable shear flow.

PubMed

Xu, Shixin; Xu, Zhiliang; Kim, Oleg V; Litvinov, Rustem I; Weisel, John W; Alber, Mark

2017-11-01

Thromboembolism, one of the leading causes of morbidity and mortality worldwide, is characterized by formation of obstructive intravascular clots (thrombi) and their mechanical breakage (embolization). A novel two-dimensional multi-phase computational model is introduced that describes active interactions between the main components of the clot, including platelets and fibrin, to study the impact of various physiologically relevant blood shear flow conditions on deformation and embolization of a partially obstructive clot with variable permeability. Simulations provide new insights into mechanisms underlying clot stability and embolization that cannot be studied experimentally at this time. In particular, model simulations, calibrated using experimental intravital imaging of an established arteriolar clot, show that flow-induced changes in size, shape and internal structure of the clot are largely determined by two shear-dependent mechanisms: reversible attachment of platelets to the exterior of the clot and removal of large clot pieces. Model simulations predict that blood clots with higher permeability are more prone to embolization with enhanced disintegration under increasing shear rate. In contrast, less permeable clots are more resistant to rupture due to shear rate-dependent clot stiffening originating from enhanced platelet adhesion and aggregation. These results can be used in future to predict risk of thromboembolism based on the data about composition, permeability and deformability of a clot under specific local haemodynamic conditions. © 2017 The Author(s).

mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets

PubMed Central

Weyrich, Andrew S.; Denis, Melvin M.; Schwertz, Hansjorg; Tolley, Neal D.; Foulks, Jason; Spencer, Eliott; Kraiss, Larry W.; Albertine, Kurt H.; McIntyre, Thomas M.

2007-01-01

New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34+ stem cell–derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamycin. In contrast, overexpression of Bcl-3 in a surrogate cell line enhanced clot retraction. These studies identify new features of post-transcriptional gene regulation and signal-dependant protein synthesis in activated platelets that may contribute to thrombus and wound remodeling and suggest that posttranscriptional pathways are targets for molecular intervention in thrombotic disorders. PMID:17110454

Comparison of fibrin clots derived from peripheral blood and bone marrow.

PubMed

Shoji, Takeshi; Nakasa, Tomoyuki; Yoshizuka, Masaaki; Yamasaki, Takuma; Yasunaga, Yuji; Adachi, Nobuo; Ochi, Mitsuo

2017-03-01

Autologous fibrin clots derived from peripheral blood (pb-fibrin clot) and bone marrow (bm-fibrin clot) are thought to be effective for tissue regeneration. However, there is no report detailing the amount of growth factors in pb-/bm-fibrin clot. In this study we evaluated the amount of growth factors in human pb-/bm-fibrin clot, and prove the validity of fibrin clot for clinical use. Human pb-/bm-fibrin clots were obtained during surgery. In the first experiment, enzyme-linked immunosorbent assay (ELISA) was performed for detecting the amount of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-β), platelet derived-growth factors-AB (PDGF-AB), and stromal cell-derived factor-1 (SDF-1). In the second experiment, the efficacy of fibrin clot on the osteogenic differentiation and fibroblast proliferation was evaluated. Pb-/bm-fibrin clots were incubated in human osteoblast derived from mesenchymal stromal cells (MSCs) or human skin fibroblast. Alizarin red staining and real-time PCR (COL1A1, RUNX2) were performed for the detection of osteogenic potential. Cell-growth assay (WST-8) and real-time PCR (COL1A1) were also performed for the detection of the potential of fibroblast proliferation. ELISA analysis revealed that the amount of VEGF, HGF, bFGF, IGF-1, and SDF-1 of bm-fibrin clot group is higher than that of pb-fibrin clot group with statistical differences. Besides, we confirmed that bm-fibrin clot has much potential for the osteogenic differentiation and fibroblast proliferation. The positive outcomes confirm the efficacy of pb-/bm-fibrin clot, and bm-fibrin clot was proved to have much potential for tissue regeneration compared with pb-fibrin clot. The current study showed the potential of a strategy for regenerative medicine using bm-fibrin clot.

Formation of blood clot on biomaterial implants influences bone healing.

PubMed

Shiu, Hoi Ting; Goss, Ben; Lutton, Cameron; Crawford, Ross; Xiao, Yin

2014-12-01

The first step in bone healing is forming a blood clot at injured bones. During bone implantation, biomaterials unavoidably come into direct contact with blood, leading to a blood clot formation on its surface prior to bone regeneration. Despite both situations being similar in forming a blood clot at the defect site, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Dental implantology has long demonstrated that the fibrin structure and cellular content of a peri-implant clot can greatly affect osteoconduction and de novo bone formation on implant surfaces. This article reviews the formation of a blood clot during bone healing in relation to the use of platelet-rich plasma (PRP) gels. It is implicated that PRP gels are dramatically altered from a normal clot in healing, resulting in conflicting effect on bone regeneration. These results indicate that the effect of clots on bone regeneration depends on how the clots are formed. Factors that influence blood clot structure and properties in relation to bone healing are also highlighted. Such knowledge is essential for developing strategies to optimally control blood clot formation, which ultimately alter the healing microenvironment of bone. Of particular interest are modification of surface chemistry of biomaterials, which displays functional groups at varied composition for the purpose of tailoring blood coagulation activation, resultant clot fibrin architecture, rigidity, susceptibility to lysis, and growth factor release. This opens new scope of in situ blood clot modification as a promising approach in accelerating and controlling bone regeneration.

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid.

PubMed

Kindell, Daniel Glenn; Keck, Rick Wayne; Jankun, Jerzy

2015-06-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC 50 (half maximal inhibitory concentration) of 8.8×10 -8 mol/l, while 6-aminocaproic acid showed an IC 50 of 1.6×10 -4 mol/l. However, at doses of >9.0×10 -7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy.

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid

PubMed Central

KINDELL, DANIEL GLENN; KECK, RICK WAYNE; JANKUN, JERZY

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC50 (half maximal inhibitory concentration) of 8.8×10−8 mol/l, while 6-aminocaproic acid showed an IC50 of 1.6×10−4 mol/l. However, at doses of >9.0×10−7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy. PMID:26136983

Impaired contraction of blood clots as a novel prothrombotic mechanism in systemic lupus erythematosus.

PubMed

Le Minh, Giang; Peshkova, Alina D; Andrianova, Izabella A; Sibgatullin, Timur B; Maksudova, Adelia N; Weisel, John W; Litvinov, Rustem I

2018-01-31

The aim of this work was to examine a possible role of clot contraction/retraction in thrombotic complications of systemic lupus erythematosus (SLE). Using a novel automated method, we investigated kinetics of clot contraction in the blood of 51 SLE patients and 60 healthy donors. The functionality of platelets in the SLE patients was assessed using flow cytometry by expression of P-selectin and fibrinogen-binding capacity. The rate and degree of clot contraction were significantly reduced in SLE patients compared with healthy subjects, especially in the patients with higher blood levels of anti-dsDNA antibodies. The reduced platelet contractility correlated with partial refractoriness of platelets isolated from the blood of SLE patients to stimulation induced by the thrombin receptor activating peptide. To test if the anti-dsDNA autoantibodies cause continuous platelet activation, followed by exhaustion and dysfunction of the cells, we added purified exogenous anti-dsDNA autoantibodies from SLE patients to normal blood before clotting. In support of this hypothesis, the antibodies first enhanced clot contraction and then suppressed it in a time-dependent manner. Importantly, a direct correlation of clot contraction parameters with the disease severity suggests that the reduced compactness of intravascular clots and thrombi could be a pathogenic factor in SLE that may exaggerate the impaired blood flow at the site of thrombosis. In conclusion, autoantibodies in SLE can affect platelet contractility, resulting in reduced ability of clots and thrombi to shrink in volume, which increases vessel obstruction and may aggravate the course and outcomes of thrombotic complications in SLE. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

PubMed

Deprés-Tremblay, Gabrielle; Chevrier, Anik; Tran-Khanh, Nicolas; Nelea, Monica; Buschmann, Michael D

2017-11-10

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study.

PubMed

Bowyer, A E; Hillarp, A; Ezban, M; Persson, P; Kitchen, S

2016-07-01

Essentials Validated assays are required to precisely measure factor IX (FIX) activity in FIX products. N9-GP and two other FIX products were assessed in various coagulation assay systems at two sites. Large variations in FIX activity measurements were observed for N9-GP using some assays. One-stage and chromogenic assays accurately measuring FIX activity for N9-GP were identified. Background Measurement of factor IX activity (FIX:C) with activated partial thromboplastin time-based one-stage clotting assays is associated with a large degree of interlaboratory variation in samples containing glycoPEGylated recombinant FIX (rFIX), i.e. nonacog beta pegol (N9-GP). Validation and qualification of specific assays and conditions are necessary for the accurate assessment of FIX:C in samples containing N9-GP. Objectives To assess the accuracy of various one-stage clotting and chromogenic assays for measuring FIX:C in samples containing N9-GP as compared with samples containing rFIX or plasma-derived FIX (pdFIX) across two laboratory sites. Methods FIX:C, in severe hemophilia B plasma spiked with a range of concentrations (from very low, i.e. 0.03 IU mL(-1) , to high, i.e. 0.90 IU mL(-1) ) of N9-GP, rFIX (BeneFIX), and pdFIX (Mononine), was determined at two laboratory sites with 10 commercially available one-stage clotting assays and two chromogenic FIX:C assays. Assays were performed with a plasma calibrator and different analyzers. Results A high degree of variation in FIX:C measurement was observed for one-stage clotting assays for N9-GP as compared with rFIX or pdFIX. Acceptable N9-GP recovery was observed in the low-concentration to high-concentration samples tested with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays. Similar patterns of FIX:C measurement were observed at both laboratory sites, with minor differences probably being attributable to the use of different analyzers. Conclusions These results suggest that, of the

Studies on cytotoxic and clot lysis activity of probiotically fermented cocktail juice prepared using Camellia sinensis and Punica grantum

NASA Astrophysics Data System (ADS)

Biswas, Ananya; Deori, Meenakshi; Nivetha, A.; Mohansrinivasan, V.

2017-11-01

In the current research the effect of probiotic microorganisms viz; Lactococcus lactis and Lactobacillus plantarum on fermentation of Camellia sinensis and Punica grantum was studied. In vitro test were done to analyze the anticancer, antioxidant and atherosclerosis (clot lysis) properties of fermented juice. The juice was fermented for 48 and 96h, during which concentration of phenolic content, total acid content and free radical scavenging activity of the sample was analyzed by DPPH assay (α, α-diphenyl-β-picrylhydrazyl). Dropping of pH was observed after 48 h of fermentation. The clot lysis activity was found to be 80 % in 100μl concentration of fermented cocktail juice. The 96 h fermented sample has shown around 70% inhibition against colon cancer cell lines. Analytical study of HPLC proves the organic acid production such as ascorbic acid in superior amount for 96h of fermented sample, Based on the retention time, the corresponding peaks were detected at 4.919 and 4.831 min.

Diagnosing human blood clotting deficiency.

PubMed

Ong, Chong Cheen; Gopinath, Subash C B; Rebecca, Leong Wei Xian; Perumal, Veeradasan; Lakshmipriya, Thangavel; Saheed, Mohamed Shuaib Mohamed

2018-05-15

There are different clotting factors present in blood, carries the clotting cascade and excessive bleeding may cause a deficiency in the clotting Diagnosis of this deficiency in clotting drastically reduces the potential fatality. For enabling a sensor to detect the clotting factors, suitable probes such as antibody and aptamer have been used to capture these targets on the sensing surface. Two major clotting factors were widely studied for the diagnosis of clotting deficiency, which includes factor IX and thrombin. In addition, factor IX is considered as the substitute for heparin and the prothrombotic associated with the increased thrombin generation are taking into account their prevalence. The biosensors, surface plasmon resonance, evanescent-field-coupled waveguide-mode sensor, metal-enhanced PicoGreen fluorescence and electrochemical aptasensor were well-documented and improvements have been made for high-performance sensing. We overviewed detecting factor IX and thrombin using these biosensors, for the potential application in medical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Limitations of using synthetic blood clots for measuring in vitro clot capture efficiency of inferior vena cava filters

PubMed Central

Robinson, Ronald A; Herbertson, Luke H; Das, Srilekha Sarkar; Malinauskas, Richard A; Pritchard, William F; Grossman, Laurence W

2013-01-01

The purpose of this study was first to evaluate the clot capture efficiency and capture location of six currently-marketed vena cava filters in a physiological venous flow loop, using synthetic polyacrylamide hydrogel clots, which were intended to simulate actual blood clots. After observing a measured anomaly for one of the test filters, we redirected the focus of the study to identify the cause of poor clot capture performance for large synthetic hydrogel clots. We hypothesized that the uncharacteristic low clot capture efficiency observed when testing the outlying filter can be attributed to the inadvertent use of dense, stiff synthetic hydrogel clots, and not as a result of the filter design or filter orientation. To study this issue, sheep blood clots and polyacrylamide (PA) synthetic clots were injected into a mock venous flow loop containing a clinical inferior vena cava (IVC) filter, and their captures were observed. Testing was performed with clots of various diameters (3.2, 4.8, and 6.4 mm), length-to-diameter ratios (1:1, 3:1, 10:1), and stiffness. By adjusting the chemical formulation, PA clots were fabricated to be soft, moderately stiff, or stiff with elastic moduli of 805 ± 2, 1696 ± 10 and 3295 ± 37 Pa, respectively. In comparison, the elastic moduli for freshly prepared sheep blood clots were 1690 ± 360 Pa. The outlying filter had a design that was characterized by peripheral gaps (up to 14 mm) between its wire struts. While a low clot capture rate was observed using large, stiff synthetic clots, the filter effectively captured similarly sized sheep blood clots and soft PA clots. Because the stiffer synthetic clots remained straight when approaching the filter in the IVC model flow loop, they were more likely to pass between the peripheral filter struts, while the softer, physiological clots tended to fold and were captured by the filter. These experiments demonstrated that if synthetic clots are used as a surrogate for animal or human blood

Limitations of using synthetic blood clots for measuring in vitro clot capture efficiency of inferior vena cava filters.

PubMed

Robinson, Ronald A; Herbertson, Luke H; Sarkar Das, Srilekha; Malinauskas, Richard A; Pritchard, William F; Grossman, Laurence W

2013-01-01

The purpose of this study was first to evaluate the clot capture efficiency and capture location of six currently-marketed vena cava filters in a physiological venous flow loop, using synthetic polyacrylamide hydrogel clots, which were intended to simulate actual blood clots. After observing a measured anomaly for one of the test filters, we redirected the focus of the study to identify the cause of poor clot capture performance for large synthetic hydrogel clots. We hypothesized that the uncharacteristic low clot capture efficiency observed when testing the outlying filter can be attributed to the inadvertent use of dense, stiff synthetic hydrogel clots, and not as a result of the filter design or filter orientation. To study this issue, sheep blood clots and polyacrylamide (PA) synthetic clots were injected into a mock venous flow loop containing a clinical inferior vena cava (IVC) filter, and their captures were observed. Testing was performed with clots of various diameters (3.2, 4.8, and 6.4 mm), length-to-diameter ratios (1:1, 3:1, 10:1), and stiffness. By adjusting the chemical formulation, PA clots were fabricated to be soft, moderately stiff, or stiff with elastic moduli of 805 ± 2, 1696 ± 10 and 3295 ± 37 Pa, respectively. In comparison, the elastic moduli for freshly prepared sheep blood clots were 1690 ± 360 Pa. The outlying filter had a design that was characterized by peripheral gaps (up to 14 mm) between its wire struts. While a low clot capture rate was observed using large, stiff synthetic clots, the filter effectively captured similarly sized sheep blood clots and soft PA clots. Because the stiffer synthetic clots remained straight when approaching the filter in the IVC model flow loop, they were more likely to pass between the peripheral filter struts, while the softer, physiological clots tended to fold and were captured by the filter. These experiments demonstrated that if synthetic clots are used as a surrogate for animal or human blood

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

PubMed

Rijken, D C; Abdul, S; Malfliet, J J M C; Leebeek, F W G; Uitte de Willige, S

2016-07-01

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the

Comparison of the milk-clotting properties of three plant extracts.

PubMed

Mazorra-Manzano, Miguel A; Perea-Gutiérrez, Teresa C; Lugo-Sánchez, María E; Ramirez-Suarez, Juan C; Torres-Llanez, María J; González-Córdova, Aarón F; Vallejo-Cordoba, Belinda

2013-12-01

Several proteases from plant sources have been proposed as milk coagulants, however, limited research has been done on their milk-clotting properties. The effect of temperature on the milk-clotting activity of kiwi fruit, melon and ginger extracts was evaluated, as well as the effects of the different extracts on curd properties. Melon extracts showed high milk-clotting activity over a broad temperature range (45-75 °C) while kiwi fruit and ginger extracts showed high activity over a narrower temperature range, with a maximum at 40 and 63 °C, respectively. Curds produced using kiwi extracts had textural properties comparable with those obtained using commercial rennet, while melon extracts produced a fragile gel and low curd yield. The milk-clotting behavior of the three plant extracts was related to the protease specificity present in these extracts. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Histotripsy Thrombolysis on Retracted Clots

PubMed Central

Zhang, Xi; Owens, Gabe E.; Cain, Charles A.; Gurm, Hitinder S.; Macoskey, Jonathan; Xu, Zhen

2016-01-01

Retracted blood clots have been previously recognized to be more resistant to drug-based thrombolysis methods, even with ultrasound and microbubble enhancements. Microtripsy, a new histotripsy approach, has been investigated as a non-invasive, drug-free, and image-guided method that uses ultrasound to break up clots with improved treatment accuracy and a lower risk of vessel damage when compared to the traditional histotripsy thrombolysis approach. Unlike drug-mediated thrombolysis, which is dependent on the permeation of the thrombolytic agents into the clot, microtripsy controls acoustic cavitation to fractionate clots. We hypothesize that microtripsy thrombolysis is effective on retracted clots and that the treatment efficacy can be enhanced using strategies incorporating electronic focal steering. To test our hypothesis, retracted clots were prepared in vitro and the mechanical properties were quantitatively characterized. Microtripsy thrombolysis was applied on the retracted clots in an in vitro flow model using three different strategies: single-focus, electronically-steered multi-focus, and a dual-pass multi-focus strategy. Results show that microtripsy was used to successfully generate a flow channel through the retracted clot and the flow was restored. The multi-focus and the dual-pass treatments incorporating the electronic focal steering significantly increased the recanalized flow channel size compared to the single-focus treatments. The dual-pass treatments achieved a restored flow rate up to 324 mL/min without cavitation contacting the vessel wall. The clot debris particles generated from microtripsy thrombolysis remained within the safe range. The results in this study show the potential of microtripsy thrombolysis for retracted clot recanalization with the enhancement of electronic focal steering. PMID:27166017

Quality of Clotting Factor Activity in Fresh Frozen Plasma at Thaw with a Microwave System and after Storage at 4 degrees C for 48 Hours.

PubMed

Kuta, Piotr; Hauck-Dlimi, Barbara; Strobel, Julian; Zimmermann, Robert; Eckstein, Reinhold

2016-01-01

Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma

Histotripsy Thrombolysis on Retracted Clots.

PubMed

Zhang, Xi; Owens, Gabe E; Cain, Charles A; Gurm, Hitinder S; Macoskey, Jonathan; Xu, Zhen

2016-08-01

Retracted blood clots have been previously recognized to be more resistant to drug-based thrombolysis methods, even with ultrasound and microbubble enhancements. Microtripsy, a new histotripsy approach, has been investigated as a non-invasive, drug-free and image-guided method that uses ultrasound to break up clots with improved treatment accuracy and a lower risk of vessel damage compared with the traditional histotripsy thrombolysis approach. Unlike drug-mediated thrombolysis, which is dependent on the permeation of the thrombolytic agents into the clot, microtripsy controls acoustic cavitation to fractionate clots. We hypothesize that microtripsy thrombolysis is effective on retracted clots and that the treatment efficacy can be enhanced using strategies incorporating electronic focal steering. To test our hypothesis, retracted clots were prepared in vitro and the mechanical properties were quantitatively characterized. Microtripsy thrombolysis was applied on the retracted clots in an in vitro flow model using three different strategies: single-focus, electronically-steered multi-focus and dual-pass multi-focus. Results show that microtripsy was used to successfully generate a flow channel through the retracted clot and the flow was restored. The multi-focus and the dual-pass treatments incorporating the electronic focal steering significantly increased the recanalized flow channel size compared to the single-focus treatments. The dual-pass treatments achieved a restored flow rate up to 324 mL/min without cavitation contacting the vessel wall. The clot debris particles generated from microtripsy thrombolysis remained within the safe range. The results of this study show the potential of microtripsy thrombolysis for retracted clot recanalization with the enhancement of electronic focal steering. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

[Contraction (retraction) of blood clots in patients with ischemic stroke].

PubMed

Peshkova, A D; Saikhunov, M V; Demin, T V; Lozhkin, A P; Panasyuk, M V; Litvinov, R I; Khasanova, D R

2016-01-01

To study a possible pathogenetic role of the blood clot contraction and its disturbances in the acute stage of ischemic stroke (IS). Using a new instrumental technique to study the dynamics of clot contraction in vitro, the authors have determined quantitative parameters of clot contraction (the extent and rate of contraction, duration of the lag-period) in the blood of 85 patients with acute IS. The contractile activity of blood clots was substantially reduced compared to the blood of healthy subjects. Correlations between hemostatic and contractile parameters suggest that the reduced clot contraction in stroke is due to the lower platelet count and impaired platelet functionality, higher levels of fibrinogen and antithrombin III as well as higher hematocrit and hemoglobin contents, leukocytosis, and changes in the biochemical blood composition. The results show that the reduced ability of clots may be a novel pathogenic mechanism that aggravates the course and outcomes of IS.

Gel point and fractal microstructure of incipient blood clots are significant new markers of hemostasis for healthy and anticoagulated blood.

PubMed

Evans, Phillip A; Hawkins, Karl; Morris, Roger H K; Thirumalai, Naresh; Munro, Roger; Wakeman, Lisa; Lawrence, Matthew J; Williams, P Rhodri

2010-10-28

Here we report the first application of a fractal analysis of the viscoelastic properties of incipient blood clots. We sought to ascertain whether the incipient clot's fractal dimension, D(f,) could be used as a functional biomarker of hemostasis. The incipient clot is formed at the gel point (GP) of coagulating blood, the GP demarcating a functional change from viscoelastic liquid to a viscoelastic solid. Incipient clots formed in whole healthy blood show a clearly defined value of D(f) within a narrow range that represents an index of clotting in health, where D(f) = 1.74 (± 0.07). A significant relationship is found between the incipient clot formation time, T(GP), and the activated partial thromboplastin time, whereas the association of D(f) with the microstructural characteristics of the incipient clot is supported by its significant correlation with fibrinogen. Our study reveals that unfractionated heparin not only prolongs the onset of clot formation but has a significant effect on its fractal microstructure. A progressive increase in unfractionated heparin concentration results in a linear decrease in D(f) and a corresponding prolongation in T(GP). The results represent a new, quantitative measure of clot quality derived from measurements on whole blood samples.

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates

PubMed Central

Wannez, Adeline; Bailly, Nicolas; Alpan, Lutfiye; Gheldof, Damien; Douxfils, Jonathan; Deneys, Véronique; Bihin, Benoît; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian; Mullier, François

2018-01-01

Background Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. Materials and methods Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. Results The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=−0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed. Discussion The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate. PMID:28287378

On-Chip Titration of an Anticoagulant Argatroban and Determination of the Clotting Time within Whole Blood or Plasma Using a Plug-Based Microfluidic System

PubMed Central

Song, Helen; Li, Hung-Wing; Munson, Matthew S.; Van Ha, Thuong G.; Ismagilov, Rustem F.

2006-01-01

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0–1.5 μg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 °C) and physiological temperature (37 °C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor’s blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 °C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other. PMID:16841902

Erythrocyte migration and gap formation in rabbit blood clots in vitro.

PubMed

Ueki, T; Yazama, F; Horiuchi, T; Yamada, M

2008-04-01

Thrombolytic agents must be carried by the blood circulation to thrombi to exert their functions. Structural gaps exist between blood vessels and thrombi or in the area surrounding thrombi. Therefore, information about fundamental gap formation at thrombotic areas is critically important for thrombolytic therapy. We previously reported that t-PA accelerates the activities of bovine erythrocytes and hemoglobin (Hb) towards bovine plasminogen activation. Here, we examined gap generation by observing morphological changes during thrombolytic processes in rabbit blood clots deformation of erythrocytes from blood clots and Hb transfer from erythrocytes to serum in vitro. Rabbit venous blood samples (1 ml) were stored under sterile conditions in glass tubes at 37 degrees C for 2, 24, 48 h, 1, and 2 weeks. We examined clot diameter, erythrocyte diameter and number as well as Hb volume in the serum, as well as histological changes in the clots. The diameter of blood clots did not change until 2 weeks after sampling. Erythrocyte diameter decreased within 48 h and at 2 weeks after sampling at the clot surface (p < 0.001) and interior (p < 0.001). The number of erythrocytes in the serum started to increase starting from 24 h after sampling (p < 0.01). Serum Hb volume also gradually increased from 24 h until 2 weeks after sampling (p < 0.01). The erythrocyte envelope became disrupted and cytoplasm started to flow through pores into the serum at 24 h. The results indicated that blood clots are reduced due to clot retraction, erythrocyte dissociation and cytoplasm leakage without a distinct fibrinolytic reaction. These results indicated that gaps start to form between 2 and 24 h after blood clotting.

[Decreased retraction of blood clots in patients with venous thromboembolic complications].

PubMed

Bredikhin, R A; Peshkova, A D; Maliasev, D V; Batrakova, M V; Le Min, J; Panasiuk, M V; Fatkhullina, L S; Ignat'ev, I M; Khaĭrullin, R N; Litvinov, R I

Haemostatic disorders play an important role in the pathogenesis of acute venous thrombosis. One of the least studied reactions of blood coagulation and thrombogenesis is spontaneous contraction of blood clots, which takes place at the expense of the contractility apparatus of activated blood platelets adhered to fibrin fibres. The work was aimed at studying the parameters of contraction of blood clots, formed in vitro, in blood of 41 patients with acute venous thromboses as compared with the same parameters in apparently healthy donors. We used a new instrumental method making it possible to determine the time from initiation to the beginning of contraction, as well as the degree and velocity of clot contraction. It was revealed that in patients with venous thrombosis the ability of clots to shrink was significantly reduced as compared with the control. We detected a statistically significant retardation of and decrease in of blood clot concentration in patients with venous thrombosis complicated by pulmonary artery thromboembolism as compared with contraction in patients with isolated deep vein thrombosis, witch may be important for early diagnosis and determination of the risk of thromboembolism. Besides, we revealed a statistically significant retardation of contraction in patients with proximal thrombosis as compared with contraction in patients with distal thrombosis, with similar values of the degree of contraction. Contraction was statistically significantly reduced in acute thrombosis (less than 21 days), whereas in subacute thrombosis (more than 21 days) the parameters of contraction were closer to normal values. The obtained findings suggest that reduction of blood clot contraction may be a new, hitherto unstudied pathogenetic mechanism deteriorating the course and outcome of venous thrombosis. The clinical significance of contraction and its impairments, as well as the diagnostic and prognostic value of the laboratory test for blood clot contraction

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo.

PubMed

Hérault, J-P; Cappelle, M; Bernat, A; Millet, L; Bono, F; Schaeffer, P; Herbert, J-M

2003-09-01

Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot-bound FXa and clot-bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non-specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC50) of Xa and IIa activity were, respectively: heparin 120 +/- 7 and 3 +/- 1 ng mL-1; SanOrg123781A 77 +/- 5 and 4 +/- 1 ng mL-1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC50 values for inhibition of FXa and FIIa activity were, respectively: heparin 100 +/- 5 and 800 +/- 40 ng mL-1; SanOrg123781A 10 +/- 5 and 30 +/- 3 ng mL-1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot-bound FXa and to some extent to clot-bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot-bound FXa with IC50 values of 2 +/- 0.5 micro g mL-1 and 0.6 +/- 0.2 micro g mL-1, respectively. Both compounds also inhibited clot-bound thrombin activity, the IC50 values of heparin and SanOrg123781A being 1 +/- 0.01 micro g mL-1 and 0.1 +/- 0.1 micro g mL-1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot-bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot-bound FXa with IC50 values of 4 +/- 0.6 and 1 +/- 0.1 micro g mL-1, respectively. As with clot-bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion

National Blood Clot Alliance

MedlinePlus

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Identification and characterization of α1 -antitrypsin in fibrin clots.

PubMed

Talens, S; Malfliet, J J M C; van Hal, P Th W; Leebeek, F W G; Rijken, D C

2013-07-01

Preliminary studies indicated that α1 -antitrypsin (A1AT) is the most abundant protein that is non-covalently bound to fibrin clots prepared from plasma. The aim of this study was to identify and characterize fibrin(ogen)-bound A1AT. Plasma clots were prepared and extensively washed with saline. Clot-bound A1AT could only be extracted using denaturing agents such as urea, thiourea or SDS, pointing to an apparently strong association. Purified fibrinogen, but still containing A1AT as a contaminant, was gel filtered, which showed that the A1AT was bound to fibrinogen. A specific ELISA detected the presence of A1AT-fibrinogen complexes in both purified fibrinogen and pooled normal plasma. Finally, fibrin(ogen)-Sepharose chromatography indicated that A1AT purified from plasma contained a small fraction of fibrin(ogen)-binding A1AT. To study the inhibitory activity of fibrin(ogen)-bound A1AT, both fibrinogen containing A1AT and washed plasma clots were incubated with increasing amounts of elastase. SDS-PAGE and Western blotting showed under both conditions the generation of the A1AT-elastase complex as well as cleaved A1AT. The inhibitory activity of fibrin(ogen)-bound A1AT was also demonstrated by measuring elastase-induced lysis of fibrin clots. Fibrin clots contain strongly bound A1AT, which is functionally active as a serine protease inhibitor (serpin). This A1AT might play a role in the local regulation of proteases involved in coagulation or fibrinolysis and represent a novel link between the inflammatory and hemostatic systems. © 2013 International Society on Thrombosis and Haemostasis.

Fibrin, γ’-fibrinogen, and trans-clot pressure gradient control hemostatic clot growth during human blood flow over a collagen/tissue factor wound

PubMed Central

Muthard, Ryan W.; Welsh, John D.; Brass, Lawrence F.; Diamond, Scott L.

2015-01-01

SUMMARY Objective Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin mediated clot growth. Approach and Results Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and ΔP, we found thrombin to be highly localized in the P-selectin+ core of hemostatic clots. Increasing ΔP from 9 to 29 mm-Hg (wall shear rate = 400 s−1) reduced P-selectin+ core size and total clot size due to enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mM GPRP dysregulated hemostasis by enhancing both P-selectin+ core size and clot size at 400 s−1 (20 mm-Hg). For whole blood flow (no GPRP), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s−1, 20 mm-Hg). Consistent with the antithrombin-1 activity of fibrin implicated with GPRP, anti-γ’-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s−1) but not arterial wall shear rates (2000 s−1). Pathological shear (15,000 s−1) and GPRP synergized to exacerbate clot growth. Conclusions Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the trans-clot ΔP. Also, γ’-fibrinogen had a role in venous but not arterial conditions. PMID:25614284

Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice

PubMed Central

Martin, Sara M.; Holle, Lori A.; Cooley, Brian C.; Flick, Matthew J.

2018-01-01

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIIIplasma) as a heterotetramer of A2 and B2 subunits and platelets (FXIIIplt) as an A2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIIIplasma, but not FXIIIplt, produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIIIplasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis. PMID:29344582

Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice.

PubMed

Kattula, Sravya; Byrnes, James R; Martin, Sara M; Holle, Lori A; Cooley, Brian C; Flick, Matthew J; Wolberg, Alisa S

2018-01-09

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIII plasma ) as a heterotetramer of A 2 and B 2 subunits and platelets (FXIII plt ) as an A 2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIII plasma , but not FXIII plt , produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIII plasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

PubMed

Mahboubi, Arash; Sadjady, Seyyed Kazem; Mirzaei Saleh Abadi, Mohammad; Azadi, Saeed; Solaimanian, Roya

2012-01-01

DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

Structure, Mechanics, and Instability of Fibrin Clot Infected with Staphylococcus epidermidis.

PubMed

Ma, Tianhui Maria; VanEpps, J Scott; Solomon, Michael J

2017-11-07

Health care-associated infection, over half of which can be attributed to indwelling medical devices, is a strong risk factor for thromboembolism. Although most experimental models of medical device infection draw upon isolated bacterial biofilms, in fact there is no infection without host protein contribution. Here we study, to our knowledge, a new model for medical device infection-that of an infected fibrin clot-and show that the common blood-borne pathogen Staphylococcus epidermidis influences this in vitro model of a blood clot mechanically and structurally on both microscopic and macroscopic scales. Bacteria present during clot formation produce a visibly disorganized microstructure that increases clot stiffness and triggers mechanical instability over time. Our results provide insight into the observed correlation between medical device infection and thromboembolism; the increase in model clot heterogeneity shows that S. epidermidis can rupture a fibrin clot. The resultant embolization of the infected clot can contribute to the systemic dissemination of the pathogen. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

[Isolation and fermentation condition of milk-clotting enzyme producing strain from glutinous rice wine].

PubMed

Cheng, Qiaoling; Bai, Xiaojia; Wang, Yanping

2008-06-01

Glutinous rice wine is a traditional food in south of China and it can coagulate milk. It has been proved that its function of coagulating milk is because of the presence of milk-clotting enzyme produced by microorganisms in glutinous rice wine. The aim of this work is to isolate milk-clotting enzyme producing strain from glutinous rice wine and study the fermentation condition. We screened out four bacteria and fungus by gradient dilution. It was proved that mold played the most important role in the production of milk-clotting enzyme. This is further confirmed by casein plate method. The optimization of fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The enzyme activity was increased 144% under the conditions established.

Intrapleural fibrinolysis in clotted haemothorax.

PubMed

Agarwal, R; Aggarwal, A N; Gupta, D

2006-11-01

The immediate treatment of haemothorax is thoracostomy tube insertion. One complication of haemothorax is retained blood and if improperly managed, this can lead to complications such as empyema and fibrothorax. The ideal management of clotted haemothorax is a matter of controversy. Video-assisted thoracoscopic surgery (VATS) is believed to be the best available modality for the management of clotted haemothorax. However, VATS is not routinely available in many centres. One easily available and effective alternative to VATS is the use of intrapleural fibrinolysis. We report the successful management of a post-traumatic clotted haemaothorax in a 34-year-old man, using intrapleural instillation of streptokinase, and review the literature on the management of clotted haemothorax.

Fluid Mechanics of Blood Clot Formation.

PubMed

Fogelson, Aaron L; Neeves, Keith B

2015-01-01

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

Fluid Mechanics of Blood Clot Formation

NASA Astrophysics Data System (ADS)

Fogelson, Aaron L.; Neeves, Keith B.

2015-01-01

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

Modelling the effect of laminar axially directed blood flow on the dissolution of non-occlusive blood clots.

PubMed

Sersa, I; Vidmar, J; Grobelnik, B; Mikac, U; Tratar, G; Blinc, A

2007-06-07

Axially directed blood plasma flow can significantly accelerate thrombolysis of non-occlusive blood clots. Viscous forces caused by shearing of blood play an essential role in this process, in addition to biochemical fibrinolytic reactions. An analytical mathematical model based on the hypothesis that clot dissolution dynamics is proportional to the power of the flowing blood plasma dissipated along the clot is presented. The model assumes cylindrical non-occlusive blood clots with the flow channel in the centre, in which the flow is assumed to be laminar and flow rate constant at all times during dissolution. Effects of sudden constriction on the flow and its impact on the dissolution rate are also considered. The model was verified experimentally by dynamic magnetic resonance (MR) microscopy of artificial blood clots dissolving in an in vitro circulation system, containing plasma with a magnetic resonance imaging contrast agent and recombinant tissue-type plasminogen activator (rt-PA). Sequences of dynamically acquired 3D low resolution MR images of entire clots and 2D high resolution MR images of clots in the axial cross-section were used to evaluate the dissolution model by fitting it to the experimental data. The experimental data fitted well to the model and confirmed our hypothesis.

Clot stability as a determinant of effective factor VIII replacement in hemophilia A.

PubMed

Leong, L; Chernysh, I N; Xu, Y; Sim, D; Nagaswami, C; de Lange, Z; Kosolapova, S; Cuker, A; Kauser, K; Weisel, J W

2017-10-01

Factor VIII (FVIII) replacement is standard of care for patients with hemophilia A (HemA); however, patient response does not always correlate with FVIII levels. We hypothesize this may be in part due to the physical properties of clots and contributions of fibrin, platelets, and erythrocytes, which may be important for hemostasis. To understand how FVIII contributes to effective hemostasis in terms of clot structure and mechanical properties. In vitro HemA clots in human plasma or whole blood were analyzed using turbidity waveform analysis, confocal microscopy, and rheometry with or without added FVIII. In vivo clots from saphenous vein puncture in wild-type and HemA mice with varying FVIII levels were examined using scanning electron microscopy. FVIII profoundly affected HemA clot structure and physical properties; added FVIII converted the open and porous fibrin meshwork and low stiffness of HemA clots to a highly branched and dense meshwork with higher stiffness. Platelets and erythrocytes incorporated into clots modulated clot properties. The clots formed in the mouse saphenous vein model contained variable amounts of compressed erythrocytes (polyhedrocytes), fibrin, and platelets depending on the levels of FVIII, correlating with bleeding times. FVIII effects on clot characteristics were dose-dependent and reached a maximum at ~25% FVIII, such that HemA clots formed with this level of FVIII resembled clots from unaffected controls. Effective clot formation can be achieved in HemA by replacement therapy, which alters the architecture of the fibrin network and associated cells, thus increasing clot stiffness and decreasing clot permeability.

Case series: Bladder clot evacuation using a prostate morcellation device.

PubMed

Doersch, Karen M; Navetta, Andrew F; Bird, Erin T; El Tayeb, Marawan M

2017-07-01

We sought to provide a technical update on the use of a prostate morcellator device (PMD) to manage organized blood clots of the bladder following laser prostatectomy. Herein, we describe our experience in using the Wolf Piranha morcellator in managing organized bladder blood clots supplemented with a retrospective chart review of the patients in whom this procedure was performed. Six patients, all male with a mean age of 75 ± 8.9 years, had organized bladder clots following either holmium laser enucleation or photoselective vaporization of the prostate managed with a PMD. Clots were recognized based on hematuria or urinary retention a median of 3.5 days following the aforementioned procedures. Initial management was attempted with more conservative measures, including a three-way Foley catheter, followed by cystoscopy with an Ellik evacuator, or a glass Tommey syringe. Morcellation times were a mean of 10.2 ± 6.15 minutes (range 2-18). This technique was able to manage clots that were an average of 173.3 ± 115.9 cc in size. The procedure was well-tolerated. No patients experienced intraoperative or morcellator-related complications. Benign prostatic hypertrophy frequently requires surgical endoscopic management and can be complicated by hematuria and bladder blood clot formation. When these clots become organized, this can lead to urinary retention and the required management, evacuation, may be difficult. The use of a Wolf Piranha PMD is a safe, well-tolerated, and effective in evacuating organized blood clots of the bladder.

Sensitivity of the activated partial thromboplastin time, the dilute Russell's viper venom time, and the kaolin clotting time for the detection of the lupus anticoagulant: a direct comparison using plasma dilutions.

PubMed

Martin, B A; Branch, D W; Rodgers, G M

1996-01-01

Increasing dilutions of lupus anticoagulant (LA) plasmas from twelve patients were used to directly compare the sensitivity of four tests for LA. The tests evaluated were the modified Bell and Alton activated partial thromboplastin time (APTT), an APTT using a commercially prepared partial thromboplastin (Platelin LS APTT), a modified dilute Russell's viper venom time (DRVVT), and a modified kaolin clotting time (KCT). LAs were detected in all twelve plasmas by each of three tests and eleven of twelve plasmas in a fourth test when undiluted patient plasma was used. Repeating the tests after diluting the LA plasmas with normal platelet-free plasma (PFP) showed that the KCT was the most sensitive test for LA, detecting eleven of twelve LAs at a dilution of 10% patient plasma and ten of twelve LAs at a dilution of 5% patient plasma. The modified Bell and Alton APTT and the modified DRVVT had similar sensitivities at a patient plasma concentration of 10%, detecting seven of twelve and eight of twelve LAs, respectively. The Platelin LS APTT detected only four of twelve LAs at a patient plasma concentration of 10%. Our results indicate that the modified KCT is a sensitive method for the detection of LAs. The modified Bell and Alton APTT and the DRVVT were less sensitive.

Computational analysis of blood clot dissolution using a vibrating catheter tip.

PubMed

Lee, Jeong Hyun; Oh, Jin Sun; Yoon, Bye Ri; Choi, Seung Hong; Rhee, Kyehan; Jho, Jae Young; Han, Moon Hee

2012-04-01

We developed a novel concept of endovascular thrombolysis that employs a vibrating electroactive polymer actuator. In order to predict the efficacy of thrombolysis using the developed vibrating actuator, enzyme (plasminogen activator) perfusion into a clot was analyzed by solving flow fields and species transport equations considering the fluid structure interaction. In vitro thrombolysis experiments were also performed. Computational results showed that plasminogen activator perfusion into a clot was enhanced by actuator vibration at frequencies of 1 and 5 Hz. Plasminogen activator perfusion was affected by the actuator oscillation frequencies and amplitudes that were determined by electromechanical characteristics of a polymer actuator. Computed plasminogen activator perfused volumes were compared with experimentally measured dissolved clot volumes. The computed plasminogen activator perfusion volumes with threshold concentrations of 16% of the initial plasminogen activator concentration agreed well with the in vitro experimental data. This study showed the effectiveness of actuator oscillation on thrombolysis and the validity of the computational plasminogen activator perfusion model for predicting thrombolysis in complex flow fields induced by an oscillating actuator.

Tissue factor-expressing monocytes inhibit fibrinolysis through a TAFI-mediated mechanism, and make clots resistant to heparins

PubMed Central

Semeraro, Fabrizio; Ammollo, Concetta T.; Semeraro, Nicola; Colucci, Mario

2009-01-01

Background Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro. Design and Methods Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator. Results LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/μL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (~50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity. Conclusions Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA

PubMed Central

Abraham, Libin; Kapopara, Piyushkumar R.; Lai, Benjamin F. L.; Shenoi, Rajesh A.; Rosell, Federico I.; Conway, Edward M.; Pryzdial, Edward L. G.; Haynes, Charles A.

2017-01-01

Anticoagulant therapy–associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis. PMID:28034889

Mechanics and contraction dynamics of single platelets and implications for clot stiffening

NASA Astrophysics Data System (ADS)

Lam, Wilbur A.; Chaudhuri, Ovijit; Crow, Ailey; Webster, Kevin D.; Li, Tai-De; Kita, Ashley; Huang, James; Fletcher, Daniel A.

2011-01-01

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

Zeolite-based hemostat QuikClot releases calcium into blood and promotes blood coagulation in vitro

PubMed Central

Li, Jing; Cao, Wei; Lv, Xiao-xing; Jiang, Li; Li, Yue-jun; Li, Wang-zhou; Chen, Shao-zong; Li, Xue-yong

2013-01-01

Aim: To examine the changes in electrolyte concentrations after addition of zeolite-based hemostat QuikClot in blood and the effects of zeolite on blood coagulation in vitro. Methods: Fresh blood was taken from healthy adult volunteers and sheep, and the electrolyte concentrations in blood were measured using a blood electrolyte analyzer. Zeolite Saline Solution (ZSS) was prepared by addition of 2 g zeolite to 0.9% NaCl solution (4, 8, or 16 mL). The electrolytes in ZSS were measured using inductively coupled plasma atomic emission spectroscopy. The prothrombin time (PT) and activated partial thromboplastin time (APTT) of blood were measured using the test tube method. The activated clotting time (ACT) and clotting rate (CR) of blood were measured with Sonoclot Coagulation and Platelet Function Analyzer. Results: Addition of zeolite (50 and 100 mg) in 2 mL human blood significantly increased Ca2+ concentration, while Na+ and K+ concentrations were significantly decreased. Addition of zeolite (50 and 100 mg) in 0.9% NaCl solution (2 mL) caused similar changes in Ca2+ and Na+ concentrations. Si4+ (0.2434 g/L) and Al3+ (0.2575 g/L) were detected in ZSS (2 g/8 mL). Addition of ZSS in sheep blood shortened APTT in a concentration dependent manner, without changing PT. ZSS or aqueous solution of CaCl2 that contained Ca2+ concentration identical to that of ZSS significantly shortened ACT in human blood without significantly changing CR, and the effect of ZSS on ACT was not significantly different from that of CaCl2. Conclusion: Zeolite releases Ca2+ into blood, thus accelerating the intrinsic pathway of blood coagulation and shortening the clot formation time. PMID:23334236

Zeolite-based hemostat QuikClot releases calcium into blood and promotes blood coagulation in vitro.

PubMed

Li, Jing; Cao, Wei; Lv, Xiao-xing; Jiang, Li; Li, Yue-jun; Li, Wang-zhou; Chen, Shao-zong; Li, Xue-yong

2013-03-01

To examine the changes in electrolyte concentrations after addition of zeolite-based hemostat QuikClot in blood and the effects of zeolite on blood coagulation in vitro. Fresh blood was taken from healthy adult volunteers and sheep, and the electrolyte concentrations in blood were measured using a blood electrolyte analyzer. Zeolite Saline Solution (ZSS) was prepared by addition of 2 g zeolite to 0.9% NaCl solution (4, 8, or 16 mL). The electrolytes in ZSS were measured using inductively coupled plasma atomic emission spectroscopy. The prothrombin time (PT) and activated partial thromboplastin time (APTT) of blood were measured using the test tube method. The activated clotting time (ACT) and clotting rate (CR) of blood were measured with Sonoclot Coagulation and Platelet Function Analyzer. Addition of zeolite (50 and 100 mg) in 2 mL human blood significantly increased Ca(2+) concentration, while Na(+) and K(+) concentrations were significantly decreased. Addition of zeolite (50 and 100 mg) in 0.9% NaCl solution (2 mL) caused similar changes in Ca(2+) and Na(+) concentrations. Si(4+) (0.2434 g/L) and Al(3+) (0.2575 g/L) were detected in ZSS (2 g/8 mL). Addition of ZSS in sheep blood shortened APTT in a concentration dependent manner, without changing PT. ZSS or aqueous solution of CaCl2 that contained Ca(2+) concentration identical to that of ZSS significantly shortened ACT in human blood without significantly changing CR, and the effect of ZSS on ACT was not significantly different from that of CaCl2. Zeolite releases Ca(2+) into blood, thus accelerating the intrinsic pathway of blood coagulation and shortening the clot formation time.

Photoacoustic monitoring of clot formation during surgery and tumor surgery

NASA Astrophysics Data System (ADS)

Juratli, Mazen A.; Galanzha, Ekaterina I.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Suen, James Y.; Zharov, Vladimir P.

2013-03-01

When a blood vessel is injured, the normal physiological response of the body is to form a clot (thrombus) to prevent blood loss. Alternatively, even without injury to the blood vessel, the pathological condition called thromboembolism may lead to the formation of circulating blood clots (CBCs), also called emboli, which can clog blood vessels throughout the body. Veins of the extremities (venous thromboembolism), lungs (pulmonary embolism ), brain (embolic stroke), heart (myocardial infarction), kidneys, and gastrointestinal tract are often affected. Emboli are also common complications of infection, inflammation, cancer, surgery, radiation and coronary artery bypass grafts. Despite the clear medical significance of CBCs, however, little progress has been made in the development of methods for real-time detection and identification of CBCs. To overcome these limitations, we developed a new modification of in vivo photoacoustic (PA) flow cytometry (PAFC) for real-time detection of white, red, and mixed clots through a transient decrease, increase or fluctuation of PA signal amplitude, respectively. In this work, using PAFC and mouse models, we present for the first time direct evidence that some medical procedures, such as conventional or cancer surgery may initiate the formation of CBCs. In conclusion, the PA diagnostic platform can be used in real-time to define risk factors for cardiovascular diseases, assist in the prognosis and potential prevention of stroke by using a well-timed therapy or as a clot count as a marker of therapy efficacy.

Inner clot diffusion and permeation during fibrinolysis.

PubMed Central

Diamond, S L; Anand, S

1993-01-01

A model of fibrinolysis was developed using multicomponent convection-diffusion equations with homogeneous reaction and heterogeneous adsorption and reaction. Fibrin is the dissolving stationary phase and plasminogen, tissue plasminogen activator (tPA), urokinase (uPA), and plasmin are the soluble mobile species. The model is based on an accurate molecular description of the fibrin fiber and protofibril structure and contains no adjustable parameters and one phenomenological parameter estimated from experiment. The model can predict lysis fronts moving across fibrin clots (fine or coarse fibers) of various densities under different administration regimes using uPA and tPA. We predict that pressure-driven permeation is the major mode of transport that allows for kinetically significant thrombolysis during clinical situations. Without permeation, clot lysis would be severely diffusion limited and would require hundreds of minutes. Adsorption of tPA to fibrin under conditions of permeation was a nonequilibrium process that tended to front load clots with tPA. Protein engineering efforts to design optimal thrombolytics will likely be affected by the permeation processes that occur during thrombolysis. PMID:8312497

Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis.

PubMed

Roessler, Florian C; Teichert, Andrea; Ohlrich, Marcus; Marxsen, Jan H; Stellmacher, Florian; Tanislav, Christian; Seidel, Günter

2014-11-30

Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations. Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10 min, 180 × g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10 min, 2570 × g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60 kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60 kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179 W/cm(2)) or 2-MHz transcranial Doppler (TCD) (0.457 W/cm(2)). With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7 ± 7.8% (rt-PA), 40.8 ± 8.6% (rt-PA+TCCS), and 40.4 ± 8.3% (rt-PA+TCD). These systemic investigations were conducted for the first time. This protocol should be used for standardized sonothrombolysis investigations. Copyright © 2014 Elsevier B.V. All rights reserved.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

PubMed

Nakatsuka, Matthew A; Barback, Christopher V; Fitch, Kirsten R; Farwell, Alexander R; Esener, Sadik C; Mattrey, Robert F; Cha, Jennifer N; Goodwin, Andrew P

2013-12-01

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Increased Plasma Clot Permeability and Susceptibility to Lysis Are Associated with Heavy Menstrual Bleeding of Unknown Cause: A Case-Control Study

PubMed Central

Szczepaniak, Piotr; Zabczyk, Michał; Undas, Anetta

2015-01-01

Background Formation of compact and poorly lysable clots has been reported in thromboembolic disorders. Little is known about clot properties in bleeding disorders. Objectives We hypothesized that more permeable and lysis-sensitive fibrin clots can be detected in women with heavy menstrual bleeding (HMB). Methods We studied 52 women with HMB of unknown cause and 52 age-matched control women. Plasma clot permeability (Ks), turbidity and efficiency of fibrinolysis, together with coagulation factors, fibrinolysis proteins, and platelet aggregation were measured. Results Women with HMB formed looser plasma fibrin clots (+16% [95%CI 7–18%] Ks) that displayed lower maximum absorbancy (-7% [95%CI -9 – -1%] ΔAbsmax), and shorter clot lysis time (-17% [95%CI -23 – -11%] CLT). The HMB patients and controls did not differ with regard to coagulation factors, fibrinogen, von Willebrand antigen, thrombin generation markers and the proportion of subjects with defective platelet aggregation. The patients had lower platelet count (-12% [95%CI -19 – -2%]), tissue plasminogen activator antigen (-39% [95%CI -41 – -29%] tPA:Ag), and plasminogen activator inhibitor-1 antigen (-28% [95%CI -38 – -18%] PAI-1:Ag) compared with the controls. Multiple regression analysis upon adjustment for age, body mass index, glucose, and fibrinogen showed that decreased tPA:Ag and shortened CLT were the independent predictors of HMB. Conclusions Increased clot permeability and susceptibility to fibrinolysis are associated with HMB, suggesting that altered plasma fibrin clot properties might contribute to bleeding disorders of unknown origin. PMID:25909989

Insights into abnormal hemostasis in the Quebec platelet disorder from analyses of clot lysis.

PubMed

Diamandis, M; Adam, F; Kahr, W H A; Wang, P; Chorneyko, K A; Arsenault, A L; Rivard, G E; Hayward, C P M

2006-05-01

The Quebec platelet disorder (QPD) is inherited and characterized by delayed-onset bleeding following hemostatic challenge. Other characteristics include increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets in the setting of normal to increased u-PA in plasma. There is also consumption of platelet plasminogen activator inhibitor-1 and increased generation of plasmin in platelets accompanied by proteolysis of stored alpha-granule proteins, including Factor V. Although fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood and platelets on clot lysis have not been evaluated. We used thromboelastography (TEG), biochemical evaluations of whole blood clot lysis, assessments of clot ultrastructure, and perfusion of blood over preformed fibrin to gain insights into the disturbed hemostasis in the QPD. Thromboelastography was not sensitive to the increased u-PA in QPD blood. However, there was abnormal plasmin generation in QPD whole blood clots, generated at low shear, with biochemical evidence of increased fibrinolysis. The incorporation of QPD platelets into a forming clot led to progressive disruption of fibrin and platelet aggregates unless drugs were added to inhibit plasmin. In whole blood perfusion studies, QPD platelets showed normal adherence to fibrin, but their adhesion was followed by accelerated fibrinolysis. The QPD is associated with "gain-of-function" abnormalities that increase the lysis of forming or preformed clots. These findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding.

Blood Clots

MedlinePlus

... sitting for long periods. If you travel by airplane, walk the aisle periodically. For long car trips, ... Your-Risk-for-Excessive-Blood-Clotting_UCM_448771_Article.jsp. Accessed April 18, 2016. What causes excessive ...

Evaluation of high resolution ultrasound as a tool for assessing the 3D volume of blood clots during in vitro thrombolysis.

PubMed

Auboire, Laurent; Escoffre, Jean-Michel; Fouan, Damien; Jacquet, Jean-René; Ossant, Frédéric; Grégoire, Jean-Marc; Bouakaz, Ayache

2017-07-24

Thrombosis is a major cause of several diseases, i.e. myocardial infarction, cerebral stroke and pulmonary embolism. Thrombolytic therapies are required to induce fast and efficient recanalization of occluded vessels. To evaluate the in vitro efficacy of these thrombolytic strategies, measuring clot dissolution is essential. This study aimed to evaluate and validate high resolution ultrasound as a tool to assess the exact volume of clots in 3D and in real time during in vitro thrombolytic drug testing. This new method was validated by measuring the effects of concentration range of recombinant tissue type plasminogen activator on a blood clot during complete occlusion or 70% stenosis of a vessel. This study shows that high resolution ultrasound imaging allows for a real-time assessment of the 3D volume of a blood clot with negligible inter- and intra-operator variabilities. The conclusions drawn from this study demonstrate the promising potential of high resolution ultrasound imaging for the in vitro assessment of new thrombolytic drugs.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma

NASA Astrophysics Data System (ADS)

Yeon, Ju Hun; Mazinani, Nima; Schlappi, Travis S.; Chan, Karen Y. T.; Baylis, James R.; Smith, Stephanie A.; Donovan, Alexander J.; Kudela, Damien; Stucky, Galen D.; Liu, Ying; Morrissey, James H.; Kastrup, Christian J.

2017-02-01

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

A comparison of the mechanical, kinetic, and biochemical properties of fibrin clots formed with two different fibrin sealants.

PubMed

Hickerson, William L; Nur, Israel; Meidler, Roberto

2011-01-01

The objective of the present study was to compare the mechanical, kinetic, and biochemical properties of fibrin clots produced using EVICEL Fibrin Sealant (Human) and TISSEEL Fibrin Sealant. The stiffness/elasticity and strength of fibrin clots formed with EVICEL and TISSEEL were assessed using applied mechanical force and thromboelastography (TEG). The factor XIII content of the fibrin clots was also evaluated. Mean Young modulus and tensile strength of the fibrin clots produced by EVICEL were significantly higher than those of clots produced by TISSEEL (P < 0.05 for both). The mean time to initial clot formation and mean time to the predefined level of clot formation were numerically shorter for EVICEL compared with TISSEEL. Furthermore, mean maximal amplitude of the clots formed with EVICEL was significantly greater than that for the clots formed with TISSEEL. Mean concentration of factor XIII for the EVICEL fibrinogen samples tested was 9 IU/ml compared with undetectable concentrations of factor XIII for the TISSEEL fibrinogen samples. Fibrin clots formed with EVICEL have a much higher resistance to stretching and tensile strength and are more capable of maintaining their structure against applied force than those formed with TISSEEL. EVICEL also allows more rapid development of fibrin clots than TISSEEL. This superior clot strength and resilience obtained with EVICEL relative to TISSEEL may be due in large part to the presence of factor XIII.

Interactions between individual ultrasound-stimulated microbubbles and fibrin clots.

PubMed

Acconcia, Christopher; Leung, Ben Y C; Manjunath, Anoop; Goertz, David E

2014-09-01

The use of ultrasound-stimulated microbubbles (USMBs) to promote thrombolysis is well established, but there remains considerable uncertainty about the mechanisms of this process. Here we examine the microscale interactions between individual USMBs and fibrin clots as a function of bubble size, exposure conditions and clot type. Microbubbles (n = 185) were placed adjacent to clot boundaries ("coarse" or "fine") using optical tweezers and exposed to 1-MHz ultrasound as a function of pressure (0.1-0.39 MPa). High-speed (10 kfps) imaging was employed, and clots were subsequently assessed with 2-photon microscopy. For fine clots, 46% of bubbles "embedded" within 10 μm of the clot boundary at pressures of 0.1 and 0.2 MPa, whereas at 0.39 MPa, 53% of bubbles penetrated and transited into the clots with an incidence inversely related to their diameter. A substantial fraction of penetrating bubbles induced fibrin network damage and promoted the uptake of nanobeads. In coarse clots, penetration occurred more readily and at lower pressures than in fine clots. The results therefore provide direct evidence of therapeutically relevant effects of USMBs and indicate their dependence on size, exposure conditions and clot properties. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Acousto-mechanical and thermal properties of clotted blooda)

PubMed Central

Nahirnyak, Volodymyr M.; Yoon, Suk Wang; Holland, Christy K.

2007-01-01

The efficacy of ultrasound-assisted thrombolysis as an adjunct treatment of ischemic stroke is being widely investigated. To determine the role of ultrasound hyperthermia in the process of blood clot disruption, the acousto-mechanical and thermal properties of clotted blood were measured in vitro, namely, density, speed of sound, frequency-dependent attenuation, specific heat, and thermal conductivity. The amplitude coefficient of attenuation of the clots was determined for 120 kHz, 1.0 MHz, and 3.5 MHz ultrasound at room temperature (20±2 °C). The attenuation coefficient ranged from 0.10 to 0.30 Np/cm in porcine clots and from 0.09 to 0.23 Np/cm in human clots. The experimentally determined values of specific heat and thermal conductivity for porcine clotted blood are (3.2±0.5)×103 J/kg·K and 0.55±0.13 W/m·K, respectively, and for human clotted blood are (3.5±0.8)×103 J/kg·K and 0.59±0.11 W/m·K, respectively. Measurements of the acousto-mechanical and thermal properties of clotted blood can be helpful in theoretical modeling of ultrasound hyperthermia in ultrasound-assisted thrombolysis and other high-intensity focused ultrasound applications. PMID:16838520

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

PubMed

Stachowicz, Aneta; Siudut, Jakub; Suski, Maciej; Olszanecki, Rafał; Korbut, Ryszard; Undas, Anetta; Wiśniewski, Jacek R

2017-01-01

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

Too much TV causes lung blood clot deaths.

PubMed

2016-08-10

Lung blood clots - also known as pulmonary embolisms - usually stem from clots in the leg or pelvis after inactivity has slowed blood flow. It is particularly dangerous if the clot travels to the lung and lodges in a small blood vessel.

Platelets are responsible for the accumulation of β-amyloid in blood clots inside and around blood vessels in mouse brain after thrombosis.

PubMed

Kucheryavykh, Lilia Y; Dávila-Rodríguez, Josué; Rivera-Aponte, David E; Zueva, Lidia V; Washington, A Valance; Sanabria, Priscilla; Inyushin, Mikhail Y

2017-01-01

Platelets contain beta-amyloid precursor protein (APP) as well as Aβ peptide (Aβ) that can be released upon activation. During thrombosis, platelets are concentrated in clots and activated. We used in vivo fluorescent analysis and electron microscopy in mice to determine to what degree platelets are concentrated in clots. We used immunostaining to visualize Aβ after photothrombosis in mouse brains. Both in vivo results and electron microscopy revealed that platelets were 300-500 times more concentrated in clots than in non-clotted blood. After thrombosis in control mice, but not in thrombocytopenic animals, Aβ immunofluorescence was present inside blood vessels in the visual cortex and around capillaries in the entorhinal cortex. The increased concentration of platelets allows enhanced release of Aβ during thrombosis, suggesting an additional source of Aβ in the brains of Alzheimer's patients that may arise if frequent micro-thrombosis events occur in their brains. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin

PubMed Central

Cines, Douglas B.; Lebedeva, Tatiana; Nagaswami, Chandrasekaran; Hayes, Vincent; Massefski, Walter; Litvinov, Rustem I.; Rauova, Lubica; Lowery, Thomas J.

2014-01-01

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract. PMID:24335500

Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin

NASA Technical Reports Server (NTRS)

Reed, G. L.; Matsueda, G. R.; Haber, E.

1992-01-01

Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.

Spatiotemporal Characterization of a Fibrin Clot Using Quantitative Phase Imaging

PubMed Central

Gannavarpu, Rajshekhar; Bhaduri, Basanta; Tangella, Krishnarao; Popescu, Gabriel

2014-01-01

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents. PMID:25386701

Fibrin-specific and effective clot lysis requires both plasminogen activators and for them to be in a sequential rather than simultaneous combination.

PubMed

Pannell, R; Li, S; Gurewich, V

2017-08-01

Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p < 0.0001) despite having significantly less tPA, as a result of the wash. A mechanism is described by which this phenomenon can be explained. The findings are believed to have significant therapeutic implications.

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels.

PubMed

Badiei, N; Sowedan, A M; Curtis, D J; Brown, M R; Lawrence, M J; Campbell, A I; Sabra, A; Evans, P A; Weisel, J W; Chernysh, I N; Nagaswami, C; Williams, P R; Hawkins, K

2015-01-01

Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

Effect of low frequency ultrasound on combined rt-PA and eptifibatide thrombolysis in human clots.

PubMed

Meunier, Jason M; Holland, Christy K; Pancioli, Arthur M; Lindsell, Christopher J; Shaw, George J

2009-01-01

Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 microL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA + Epf), with (+US) or without (-US) ultrasound for 30 minutes at 37 degrees C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. LR values for (- US) treated clots were 0.8+/-0.1(control), 1.8+/-0.3 (Epf), 1.5+/-0.2 (rt-PA), and 1.3+/-0.4 (rt-PA + Epf) (% clot width/minute) respectively. In comparison, the (+ US) group exhibited LR values of 1.6+/-0.2 (control), 4.3+/-0.4 (Epf), 6.3+/-0.4 (rt-PA), and 4.6+/-0.6 (rt-PA + Epf). For (- US) treated clots, FCL was 6.0+/-0.8 (control), 9.2+/-2.5 (Epf), 15.6+/-1.7 (rt-PA), and 28.0+/-2.2% (rt-PA + Epf) respectively. FCL for (+ US) clots was 13.5+/-2.4 (control), 20.7+/-6.4 (Epf), 44.4+/-3.6 (rt-PA) and 30.3+/-3.6% (rt-PA + Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined

Quantification of intraventricular blood clot in MR-guided focused ultrasound surgery

NASA Astrophysics Data System (ADS)

Hess, Maggie; Looi, Thomas; Lasso, Andras; Fichtinger, Gabor; Drake, James

2015-03-01

Intraventricular hemorrhage (IVH) affects nearly 15% of preterm infants. It can lead to ventricular dilation and cognitive impairment. To ablate IVH clots, MR-guided focused ultrasound surgery (MRgFUS) is investigated. This procedure requires accurate, fast and consistent quantification of ventricle and clot volumes. We developed a semi-autonomous segmentation (SAS) algorithm for measuring changes in the ventricle and clot volumes. Images are normalized, and then ventricle and clot masks are registered to the images. Voxels of the registered masks and voxels obtained by thresholding the normalized images are used as seed points for competitive region growing, which provides the final segmentation. The user selects the areas of interest for correspondence after thresholding and these selections are the final seeds for region growing. SAS was evaluated on an IVH porcine model. SAS was compared to ground truth manual segmentation (MS) for accuracy, efficiency, and consistency. Accuracy was determined by comparing clot and ventricle volumes produced by SAS and MS, and comparing contours by calculating 95% Hausdorff distances between the two labels. In Two-One-Sided Test, SAS and MS were found to be significantly equivalent (p < 0.01). SAS on average was found to be 15 times faster than MS (p < 0.01). Consistency was determined by repeated segmentation of the same image by both SAS and manual methods, SAS being significantly more consistent than MS (p < 0.05). SAS is a viable method to quantify the IVH clot and the lateral brain ventricles and it is serving in a large-scale porcine study of MRgFUS treatment of IVH clot lysis.

Characterisation of clot microstructure properties in stable coronary artery disease.

PubMed

Sabra, Ahmed; Lawrence, Matthew James; Aubrey, Robert; Obaid, Daniel; Chase, Alexander; Smith, Dave; Thomas, Phillip; Storton, Sharon; Davies, Gareth R; Hawkins, Karl; Williams, Phylip Rhodri; Morris, Keith; Evans, Phillip Adrian

2017-01-01

Coronary artery disease (CAD) is associated with an increased prothrombotic tendency and is also linked to unfavourably altered clot microstructure. We have previously described a biomarker of clot microstructure (d f ) that is unfavourably altered in acute myocardial infarction. The d f biomarker assesses whether the blood will form denser or looser microstructures when it clots. In this study we assessed in patients with stable chest pain whether d f can differentiate between obstructed and unobstructed CAD. A blood sample prior to angiography was obtained from 251 consecutive patients undergoing diagnostic coronary angiography. Patients were categorised based on angiographic findings as presence or absence of obstructive CAD (stenosis ≥50%). The blood sample was assessed using the d f biomarker, standard laboratory markers and platelet aggregometry (Multiplate). A significant difference (p=0.028) in d f was observed between obstructive CAD (1.748±0.057, n=83) and unobstructive CAD (1.732±0.052, n=168), where patients with significant CAD produce denser, more tightly packed clots. d f was also raised in men with obstructive CAD compared with women (1.745±0.055 vs 1.723±0.052, p=0.007). Additionally d f significantly correlated with the platelets response to arachidonic acid as measured by the ASPItest area under the curve readings from platelet aggregometry (correlation coefficient=0.166, p=0.008), a low value of the ASPItest indicating effective aspirin use was associated with looser, less dense clots. For the first time, we characterise clot microstructure, as measured by d f , in patients with stable CAD. d f can potentially be used to risk-stratify patients with stable CAD and assess the efficacy of therapeutic interventions by measuring changes in clot microstructure.

Combination of ultrasound and rtPA enhances fibrinolysis in an In Vitro clot system

PubMed Central

Winter, Philipp; Müller-Werkmeister, Hendrik; Strand, Susanne; König, Jochem; Kempski, Oliver; Ringel, Florian; Kantelhardt, Sven R.; Keric, Naureen

2017-01-01

Background Catheter-based lysis with recombinant tissue plasminogen activator (rtPA) is a well-established therapy for spontaneous intracerebral hemorrhage (ICH). The effectiveness of this therapy can be increased with ultrasound, but the optimal conditions are not yet clearly established. Using a novel in vitro system of blood clots previously developed by our group, we investigated various parameters of intralesional sonothrombolysis using an endosonography catheter in combination with rtPA. Methods Standardized human blood clots were equipped with a drainage catheter and weighed before and after 4 treatments: control (drainage only), rtPA only, ultrasound only and the combination of rtPA+ultrasound. The effectiveness of ultrasound was further analysed in terms of optimal frequency, duration and distance to the probe. Temperature and acoustic peak rarefaction pressure (APRP) were assessed to analyse potential adverse effects and quantify lysis. Histo-morphological analysis of the treated clots was performed by H&E staining and confocal laser scanning microscopy using fluorescent fibrinogen. Results The combined treatment rtPA+ultrasound achieved the highest lysis rates with a relative weight of 30.3%±5.5% (p≤0.0001) compared to all other groups. Similar results were observed when treating aged clots. Confocal fluorescent microscopy of the treated clots revealed a rarefied fibrin mesh without cavitations. No relevant temperature increase occurred (0.53±0.75°C). The optimal insonation treatment time was 1 hour. APRP measurements showed a lysis threshold of 515.5±113.4 kPa. Application of 10 MHz achieved optimal lysis and lysis radius, while simultaneously proving to be the best frequency for morphologic imaging of the clot and surrounding tissue. Conclusions These promising data provide the basis for an individualized minimal invasive ICH therapy by rtPA and sonothrombolysis independent of ICH age. PMID:29145482

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Milk-clotting enzymes, microbial. 173.150 Section... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting enzyme produced by pure-culture fermentation process may be safely used in the production...

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Milk-clotting enzymes, microbial. 173.150 Section... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting enzyme produced by pure-culture fermentation process may be safely used in the production...

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Milk-clotting enzymes, microbial. 173.150 Section... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting enzyme produced by pure-culture...

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Milk-clotting enzymes, microbial. 173.150 Section... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.150 Milk-clotting enzymes, microbial. Milk-clotting enzyme produced by pure-culture fermentation process may be safely used in the production...

Diastolic timed Vibro-Percussion at 50 Hz delivered across a chest wall sized meat barrier enhances clot dissolution and remotely administered Streptokinase effectiveness in an in-vitro model of acute coronary thrombosis

PubMed Central

2012-01-01

Background Low Frequency Vibro-Percussion (LFVP) assists clearance of thrombi in catheter systems and when applied to the heart and timed to diastole is known to enhance coronary flow. However LFVP on a clotted coronary like vessel given engagement over a chest wall sized barrier (to resemble non-invasive heart attack therapy) requires study. Methods One hour old clots (n=16) were dispensed within a flexible segment of Soft-Flo catheter (4 mm lumen), weighted, interfaced with Heparinized Saline (HS), secured atop a curved dampening base, and photographed. A ~4 cm meat slab was placed over the segment and randomized to receive intermittent LFVP (engaged, - disengaged at 1 second intervals), or no LFVP for 20 minutes. HS was pulsed (~120/80 mmHg), with the diastolic phase coordinated to match LFVP delivery. The segment was then re-photographed and aspirated of fluid to determine post clot weight. The trial was then repeated with 0.5 mls of Streptokinase (15,000 IU/100 microlitre) delivered ~ 2 cm upstream from the clot. Results LFVP - HS only samples (vs. controls) showed; a) development of clot length fluid channels absent in the control group (p < 0.0002); b) enhanced dissolved clot mixing scores ( 5.0 vs. 0.8, p < 2.8 E – 6); and c) increased percent clot dissolution (23.0% vs. 1.8% respectively, p < 8.5 E-6). LFVP - SK samples had a similar comparative clot disruptive profile, however fluid channels developed faster and percent clot dissolution more than doubled (51.0% vs. 3.0%, p< 9.8 E- 6). Conclusion Diastolic timed LFVP (50 Hz) engaged across a chest wall sized barrier enhances clot disruptive effects to an underlying coronary like system. PMID:23146079

The combination of recombinant factor VIIa and fibrinogen correct clotting ex vivo in patient samples obtained following cardiopulmonary bypass surgery.

PubMed

Sørensen, Benny; Asvaldsdottir, Hanna S; Gudmundsdottir, Brynja R; Onundarson, Pall T

2009-12-01

Cardiac surgery involving cardio pulmonary bypass (CPB) may be associated with development of a coagulopathy that increases risk of bleeding. In the present ex vivo study we investigated the influence of fibrinogen and rFVIIa, alone or in combination, on whole blood coagulation thromboelastometry using pre- and postoperative blood samples from 18 consecutive adult patients undergoing CPB surgery. Dynamic thromboelastometric clotting profiles were recorded using citrated whole blood activated with trace amounts of tissue factor (Innovin, final dilution 1:17000). Blood samples were collected before surgery (control) and postoperative samples were obtained following in vivo neutralization of heparin with protamine sulphate. All blood samples were treated with heparinase to ensure neutralization of possible residual heparin effect. The post-operative blood samples were spiked with buffer, rFVIIa (2 microg/mL), fibrinogen (1 mg/mL), or the combination of rFVIIa and fibrinogen. Despite neutralization of heparin, CPB surgery left a measurable coagulopathy that was thromboelastometrically characterized by prolonged onset of clotting, reduced maximum velocity of clot formation (MaxVel), and decreased maximum clot firmness (MCF). Ex vivo spiking of the postoperative samples with rFVIIa shortened the clotting time. Fibrinogen also shortened the clotting time and, in addition, improved the MaxVel, and MCF. Finally, adding the combination of rFVIIa and fibrinogen to the postoperative samples corrected all thromboelastometric parameters to the preoperative range. In conclusion, the correction of whole blood clotting abnormalities that occurs with rFVIIa and/or fibrinogen suggests that future clinical trials on treatment of bleeding during CPB surgery should study the haemostatic effect of fibrinogen or possibly the combination of rFVIIa and fibrinogen.

The effects of QuikClot Combat Gauze on hemorrhage control in the presence of hemodilution.

PubMed

Johnson, Don; Agee, Samantha; Reed, Amanda; Gegel, Brian; Burgert, James; Gasko, John; Loughren, Michael

2012-01-01

Although hemostatic agents may be effective at stopping hemorrhage, they may fail because of hemodilution from intravenous fluids. The purpose of this study was to investigate the effects of QuikClot Combat Gauze (QCG) on rebleeding in a class II hemorrhage in the presence of hemodilution in a lethal femoral injury. This was a prospective experimental, between swine subjects design. Pigs were assigned to one of two groups: QCG (n=15) or control (n=15). Thirty percent of the pig's blood was exsanguinated and then a 3:1 ratio of ringers lactate was administered. A groin injury was created by transecting the femoral artery and vein to simulate a battlefield injury and allowed to bleed for one minute. After one minute of hemorrhage, proximal pressure was applied to the injury, and QCG was placed into the wound followed by standard wound packing. The control group underwent the same procedures with the exception of the hemostatic agent. For both groups, 5 minutes of direct pressure was applied to the wound followed by a standard pressure dressing. Dressings were removed after 30 minutes, and the amount of hemorrhage was calculated in milliliters for each group for a period of 5 minutes. An activated clotting time was used to exclude any pigs with coagulation pathology. A multivariate analysis of variance indicated that there were no significant differences in the groups relative to weight, amount of one minute hemorrhage, fluid deficit replacement, blood volume, and the activated clotting time (P>.05) indicating that the groups were equivalent on these parameters. A t test indicated that there was significantly less bleeding (P=.002) in the QCG group (36 mL±112 mL) compared to the control group (340 mL±297 ml). QCG produces a robust clot that can more effectively tolerate hemodilution compared to a control group.

High red blood cell composition in clots is associated with successful recanalization during intra-arterial thrombectomy.

PubMed

Shin, Jong Wook; Jeong, Hye Seon; Kwon, Hyon-Jo; Song, Kyu Sang; Kim, Jei

2018-01-01

We evaluated the composition of individual clots retrieved during intra-arterial thrombectomy in relation to recanalization success, stroke subtype, and the presence of clot signs on initial brain images. We analyzed clot and interventional data from 145 retrieval trials performed for 37 patients (69.5±14.0 years, 20 men, large artery atherosclerosis, n = 7; cardioembolism, n = 22; undetermined etiology, n = 8) who had undergone intra-arterial thrombectomy. Rates of clot retrieval and successful recanalization (Arterial Occlusive Lesion score of 2-3) for separate retrieval trials were evaluated. The area occupied by red blood cell (RBC), fibrin/platelets, and white blood cell (WBC) was measured from digitized images of hematoxylin-eosin stained clots. Compositional differences were compared according to recanalization success, stroke subtype, and the presence of hyperdense clot sign on initial computed tomography and/or blooming artifact on magnetic resonance image. Of the 145 total retrieval trials (3.4±2.4 times per patient), clot was retrieved in 93 trials (64%), while recanalization was successful in 73 (50%). Fibrin/platelets (63%) occupied the greatest area in retrieved clots, followed by RBCs (33%) and WBCs (4%). Clots retrieved from successful recanalization exhibited higher RBC composition (37%) than those retrieved from non-recanalization trials (20%, p = 0.001). RBC composition was higher in cardioembolic stroke (38%) rather than large artery atherosclerosis (23%) and undetermined etiology (26%, p = 0.01). Clots exhibiting clot signs (40%) had higher RBC composition than those without clot signs (19%, p = 0.001). RBC-rich clots were associated with successful recanalization of intra-arterial thrombectomy, cardioembolic stroke, and the presence of clot-signs on initial brain images.

High red blood cell composition in clots is associated with successful recanalization during intra-arterial thrombectomy

PubMed Central

Shin, Jong Wook; Jeong, Hye Seon; Kwon, Hyon-Jo; Song, Kyu Sang

2018-01-01

We evaluated the composition of individual clots retrieved during intra-arterial thrombectomy in relation to recanalization success, stroke subtype, and the presence of clot signs on initial brain images. We analyzed clot and interventional data from 145 retrieval trials performed for 37 patients (69.5±14.0 years, 20 men, large artery atherosclerosis, n = 7; cardioembolism, n = 22; undetermined etiology, n = 8) who had undergone intra-arterial thrombectomy. Rates of clot retrieval and successful recanalization (Arterial Occlusive Lesion score of 2–3) for separate retrieval trials were evaluated. The area occupied by red blood cell (RBC), fibrin/platelets, and white blood cell (WBC) was measured from digitized images of hematoxylin-eosin stained clots. Compositional differences were compared according to recanalization success, stroke subtype, and the presence of hyperdense clot sign on initial computed tomography and/or blooming artifact on magnetic resonance image. Of the 145 total retrieval trials (3.4±2.4 times per patient), clot was retrieved in 93 trials (64%), while recanalization was successful in 73 (50%). Fibrin/platelets (63%) occupied the greatest area in retrieved clots, followed by RBCs (33%) and WBCs (4%). Clots retrieved from successful recanalization exhibited higher RBC composition (37%) than those retrieved from non-recanalization trials (20%, p = 0.001). RBC composition was higher in cardioembolic stroke (38%) rather than large artery atherosclerosis (23%) and undetermined etiology (26%, p = 0.01). Clots exhibiting clot signs (40%) had higher RBC composition than those without clot signs (19%, p = 0.001). RBC-rich clots were associated with successful recanalization of intra-arterial thrombectomy, cardioembolic stroke, and the presence of clot-signs on initial brain images. PMID:29782513

A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

PubMed

Coleman, Lewis S

2007-01-01

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

A Synthetic Fibrin-Crosslinking Polymer for Modulating Clot Properties and Inducing Hemostasis

PubMed Central

Chan, Leslie W.-G.; Wang, Xu; Wei, Hua; Pozzo, Lilo D.; White, Nathan J.; Pun, Suzie H.

2015-01-01

Clotting factor replacement is the standard management of acute bleeding in congenital and acquired bleeding disorders. We present a synthetic approach to hemostasis using an engineered hemostatic polymer (PolySTAT) that circulates innocuously in the blood, identifies sites of vascular injury, and promotes clot formation to stop bleeding. PolySTAT induces hemostasis by crosslinking the fibrin matrix within clots, mimicking the function of the transglutaminase Factor XIII. Furthermore, synthetic PolySTAT binds specifically to fibrin monomers and is uniformly integrated into fibrin fibers during fibrin polymerization, resulting in a fortified, hybrid polymer network with enhanced resistance to enzymatic degradation. In vivo hemostatic activity was confirmed in a rat model of trauma and fluid resuscitation in which intravenous administration of PolySTAT improved survival by reducing blood loss and resuscitation fluid requirements. PolySTAT-induced fibrin crosslinking is a novel approach to hemostasis utilizing synthetic polymers for non-invasive modulation of clot architecture with potentially wide-ranging therapeutic applications. PMID:25739763

EMBOLIC MIDDLE CEREBRAL ARTERY OCCLUSION MODEL USING THROMBIN AND FIBRINOGEN COMPOSED CLOTS IN RAT

PubMed Central

Ren, Ming; Lin, Zi-Jing; Qian, Hai; Gourav, Choudhury Roy; liu, Ran; Liu, Hanli; Yang, Shao-Hua

2012-01-01

Ischemic stroke accounts for over 80% in total human stroke which mostly affect middle cerebral artery (MCA) territory. Embolic stroke models induced by injection of homologous clots into the internal carotid artery and MCA closely mimic human stroke and have been commonly used in stroke research. Studies indicate that the size and composition of clots are critical for the reproducibility of the stroke model. In the present study, we modified the homologous clots formation by addition of thrombin and fibrinogen which produced even distribution of fibrin with tight cross linkage of red blood cells. We optimized the embolic MCA occlusion model in rats using different size of the mixed clots. A precise lodgment of the clots at the MCA bifurcation and highly reproducible ischemic lesion in the MCA territory were demonstrated in the embolic MCA occlusion model induced by injection of 10 pieces of 1-mm long mixed clots made in PE-60 catheter. We further tested the effect of recombinant tissue plasminogen activator (rtPA) in this embolic MCA occlusion model. rtPA induced thrombolysis, improved neurological outcome, and significantly reduced ischemic lesion volume when administered at 1 hour after embolism as compared with control. In summary, we have established a reproducible embolic MCA occlusion model using clots made of homologous blood, thrombin and fibrinogen. The mixed clots enable precise lodgment at the MCA bifurcation which is responsive to thrombolytic therapy of rtPA. PMID:22985597

Embolic middle cerebral artery occlusion model using thrombin and fibrinogen composed clots in rat.

PubMed

Ren, Ming; Lin, Zi-Jing; Qian, Hai; Choudhury, Gourav Roy; Liu, Ran; Liu, Hanli; Yang, Shao-Hua

2012-11-15

Ischemic stroke accounts for over 80% in total human stroke which mostly affect middle cerebral artery (MCA) territory. Embolic stroke models induced by injection of homologous clots into the internal carotid artery and MCA closely mimic human stroke and have been commonly used in stroke research. Studies indicate that the size and composition of clots are critical for the reproducibility of the stroke model. In the present study, we modified the homologous clots formation by addition of thrombin and fibrinogen which produced even distribution of fibrin with tight cross linkage of red blood cells. We optimized the embolic MCA occlusion model in rats using different size of the mixed clots. A precise lodgment of the clots at the MCA bifurcation and highly reproducible ischemic lesion in the MCA territory were demonstrated in the embolic MCA occlusion model induced by injection of 10 pieces of 1-mm long mixed clots made in PE-60 catheter. We further tested the effect of recombinant tissue plasminogen activator (rtPA) in this embolic MCA occlusion model. rtPA induced thrombolysis, improved neurological outcome, and significantly reduced ischemic lesion volume when administered at 1h after embolism as compared with control. In summary, we have established a reproducible embolic MCA occlusion model using clots made of homologous blood, thrombin and fibrinogen. The mixed clots enable precise lodgment at the MCA bifurcation which is responsive to thrombolytic therapy of rtPA. Copyright © 2012 Elsevier B.V. All rights reserved.

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

PubMed Central

Fan, Natalie K.; Keegan, Philip M.; Platt, Manu O.; Averett, Rodney D.

2015-01-01

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease. PMID:25867016

A French National Survey on Clotting Disorders in Mastocytosis.

PubMed

Carvalhosa, Ana B; Aouba, Achille; Damaj, Gandhi; Canioni, Danielle; Brouzes, Chantal; Gyan, Emmanuel; Durupt, Stéphane; Durieu, Isabelle; Cathebras, Pascal; Costédoat-Chalumeau, Nathalie; Launay, David; Pilmis, Benoit; Barete, Stephane; Frenzel, Laurent; Lortholary, Olivier; Hermine, Olivier; Hermans, Cedric; Chandesris, Marie-Olivia

2015-10-01

Mastocytosis is characterized by a clonal mast cell proliferation with organ infiltration and uncontrolled degranulation. Although not characteristic and poorly explained, some patients develop clotting abnormalities. We retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the national French Reference Centre for Mastocytosis database. From our cohort of 14 adult patients with clotting abnormalities (median age 46 years [range 26-75]), 4 had a presentation suggestive of a primary hemostasis disorder alone (by their symptoms and/or abnormal clotting tests [PFA, von Willebrand's disease [vWD] screening]) and 10 had a laboratory impairment of secondary hemostasis. Among these, 7 had bleeds characteristic of a coagulation cascade disorder (severe/life-threatening in 5 and mild in 2 patients). Clotting abnormalities were of variable severity, typically related to intense crisis of degranulation, such as anaphylactic reactions, and/or to severe organ infiltration by mast cells. Importantly, classical hemostatic management with platelet transfusion, fresh frozen plasma, or vitamin K infusions was unsuccessful, as opposed to the use of agents inhibiting mast cell activity, particularly steroids. This illustrates the crucial role of mast cell mediators such as tryptase and heparin, which interfere both with primary (mainly via inhibition of von Willebrand factor) and secondary hemostasis. There was interestingly an unusually high number of aggressive mastocytosis (particularly mast cell leukemia) and increased mortality in the group with secondary hemostasis disorders (n = 5, 36% of the whole cohort). Mast cell degranulation and/or high tumoral burden induce both specific biologic antiaggregant and anticoagulant states with a wide clinical spectrum ranging from asymptomatic to life-threatening bleeds. Hemostatic control is achieved by mast cell inhibitors such as steroids.

Factor XIII stiffens fibrin clots by causing fiber compaction.

PubMed

Kurniawan, N A; Grimbergen, J; Koopman, J; Koenderink, G H

2014-10-01

Factor XIII-induced cross-linking has long been associated with the ability of fibrin blood clots to resist mechanical deformation, but how FXIII can directly modulate clot stiffness is unknown. We hypothesized that FXIII affects the self-assembly of fibrin fibers by altering the lateral association between protofibrils. To test this hypothesis, we studied the cross-linking kinetics and the structural evolution of the fibers and clots during the formation of plasma-derived and recombinant fibrins by using light scattering, and the response of the clots to mechanical stresses by using rheology. We show that the lateral aggregation of fibrin protofibrils initially results in the formation of floppy fibril bundles, which then compact to form tight and more rigid fibers. The first stage is reflected in a fast (10 min) increase in clot stiffness, whereas the compaction phase is characterized by a slow (hours) development of clot stiffness. Inhibition of FXIII completely abrogates the slow compaction. FXIII strongly increases the linear elastic modulus of the clots, but does not affect the non-linear response at large deformations. We propose a multiscale structural model whereby FXIII-mediated cross-linking tightens the coupling between the protofibrils within a fibrin fiber, thus making the fiber stiffer and less porous. At small strains, fiber stiffening enhances clot stiffness, because the clot response is governed by the entropic elasticity of the fibers, but once the clot is sufficiently stressed, the modulus is independent of protofibril coupling, because clot stiffness is governed by individual protofibril stretching. © 2014 International Society on Thrombosis and Haemostasis.

Impaired thrombin generation and fibrin clot formation in patients with dilutional coagulopathy during major surgery.

PubMed

Schols, S E M; Lancé, M D; Feijge, M A H; Damoiseaux, J; Marcus, M A; Hamulyák, K; Ten Cate, H; Heemskerk, J W M; van Pampus, E C M

2010-02-01

Patients subjected to haemodilution during surgery are at increased risk of bleeding. We hypothesised that, in the acquired dilutional coagulopathy, insufficient haemostasis is due to either insufficient thrombin generation or insufficient fibrin clot formation. In tissue factor-activated plasmas from patients with coagulation deficiency, we measured time curves of thrombin generation and fibrin clot formation (thromboelastography). Investigated were in study A: 10 patients treated with vitamin K antagonist and five healthy subjects; in study B: 30 patients undergoing cardiopulmonary bypass (CPB) surgery and infused with on average 2,000 ml crystalloids and colloids (no major bleeding); in study C: 58 patients undergoing major general surgery, and transfused with >5,000 ml crystalloids, colloids and red cell concentrates, who experienced major bleeding and were post-transfused with fresh frozen plasma. The treatment with vitamin K antagonist led to a progressive reduction in thrombin generation but not fibrin clot formation. In CPB patients, plasma factor levels post-surgery were 53-60% of normal. This was accompanied by moderate reduction in both haemostatic processes. In plasmas from patients undergoing major surgery, factor levels were 38-41% of normal, and these levels increased after plasma transfusion. Taking preset thresholds for normal thrombin generation and fibrin clot formation, at least one of these processes was low in 88-93% of the patients with (persistent) bleeding, but only in 40-53% of the patients without bleeding. In conclusion, the ability of thrombin generation and fibrin clot formation is independently reduced in acquired dilutional coagulopathy, while minimal levels of both are required for adequate haemostasis.

Effect of Low Frequency Ultrasound on Combined rt-PA and Eptifibatide Thrombolysis in Human Clots

PubMed Central

Meunier, Jason M.; Holland, Christy K.; Pancioli, Arthur M.; Lindsell, Christopher J.; Shaw, George J.

2009-01-01

Introduction Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. Materials and Methods Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 μL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA+Epf), with or without ultrasound for 30 minutes at 37°C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. Results and Conclusions LR values for (−US) treated clots were 0.8±0.1(control), 1.8±0.3 (Epf), 1.5±0.2 (rt-PA), and 1.3±0.4 (rt-PA+Epf) (% clot width/minute) respectively. In comparison, the (+US) group exhibited LR values of 1.6±0.2 (control), 4.3±0.4 (Epf), 6.3±0.4 (rt-PA), and 4.6±0.6 (rt-PA+Epf). For (−US) treated clots, FCL was 6.0±0.8 (control), 9.2±2.5 (Epf), 15.6±1.7 (rt-PA), and 28.0±2.2% (rt-PA+Epf) respectively. FCL for (+US) clots was 13.5±2.4 (control), 20.7±6.4 (Epf), 44.4±3.6 (rt-PA) and 30.3±3.6% (rt-PA+Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of

Developing Mesoscale Model of Fibrin-Platelet Network Representing Blood Clotting =

NASA Astrophysics Data System (ADS)

Sun, Yueyi; Nikolov, Svetoslav; Bowie, Sam; Alexeev, Alexander; Lam, Wilbur; Myers, David

Blood clotting disorders which prevent the body's natural ability to achieve hemostasis can lead to a variety of life threatening conditions such as, excessive bleeding, stroke, or heart attack. Treatment of these disorders is highly dependent on understanding the underlying physics behind the clotting process. Since clotting is a highly complex multi scale mechanism developing a fully atomistic model is currently not possible. We develop a mesoscale model based on dissipative particle dynamics (DPD) to gain fundamental understanding of the underlying principles controlling the clotting process. In our study, we examine experimental data on clot contraction using stacks of confocal microscopy images to estimate the crosslink density in the fibrin networks and platelet location. Using this data we reconstruct the platelet rich fibrin network and study how platelet-fibrin interactions affect clotting. Furthermore, we probe how different system parameters affect clot contraction. ANSF CAREER Award DMR-1255288.

The effect of intravenous safflower oil emulsion on the clotting mechanism.

PubMed

Van Way, C W; Dunn, E L; Hamstra, R D

1983-08-01

A new fat emulsion for intravenous use, derived from safflower oil (Abbott Laboratories), was studied. The clotting mechanism was compared with a battery of tests performed during the infusion of total parenteral nutrition (TPN) using glucose alone and during infusion of TPN using both glucose and fat. Five adult surgical patients underwent TPN with 7.0 per cent amino acid solution for ten days, receiving glucose as their only nonprotein calorie source on days one, two, nine, and ten (40 kcal/kg/day). On days three through eight, 10 per cent fat emulsion (600-900 ml/day) was given each day to provide one-third of the nonprotein calories. Simplate bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen (Biuret method), platelet count, platelet aggregation, serum functional antithrombin, and viscoelastic curves were measured on days one, three, six, and ten. Some of these studies were abnormal at baseline and during the study. No significant changes were seen with fat emulsion infusion. The patients did not exhibit any evidence of clinical bleeding. This new intravenous fat emulsion did not appear to be associated with alterations in the clotting mechanism. However, two of five patients showed increases in serum triglycerides and one patient died during the study.

A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

PubMed Central

Hao, YouJin; Balasubramanian, Natesan; Jing, Yingjun; Montiel, Rafael; Faria, Tiago Q.; Brito, Rui M.; Simões, Nelson

2013-01-01

Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42×10−7 M and 1.22×10−7 M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction. PMID:23874900

Capture of lipopolysaccharide (endotoxin) by the blood clot: a comparative study.

PubMed

Armstrong, Margaret T; Rickles, Frederick R; Armstrong, Peter B

2013-01-01

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

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

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

PubMed

Galanzha, Ekaterina I; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A; Keyrouz, Salah G; Mehta, Jawahar L; Zharov, Vladimir P

2011-10-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

In vivo flow cytometry of circulating clots using negative phototothermal and photoacoustic contrasts

PubMed Central

Galanzha, Ekaterina I.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Keyrouz, Salah G.; Mehta, Jawahar L.; Zharov, Vladimir P.

2012-01-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of severe thromboembolisms including ischemia at strokes and myocardial dysfunction at heart attack. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 µm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting them by negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. PMID:21976458

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis

PubMed Central

Höök, Peter; Brito-Robinson, Teresa; Kim, Oleg; Narciso, Cody; Goodson, Holly V.; Weisel, John W.; Alber, Mark S.; Zartman, Jeremiah J.

2017-01-01

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions. PMID:28856043

Chronologic echogenicity changes and histologic correlates in experimental blood clots: a serial sonographic study.

PubMed

Watanabe, Masaki; Majidi, Shahram; Chaudhry, Saqib A; Qureshi, Adnan I

2012-12-01

We performed this study to identify echogenicity changes in clots and their correlations with histologic characteristics over time. B-mode sonography was performed at 2, 6, 24, and 48 hours after clot formation in venous blood from healthy donors. Analysis of echogenicity was performed by grayscale median measurement with normalization. We classified clots into 3 groups: dense, intermediate, and loose according to red blood cell (RBC) density on hematoxylin-eosin staining. Statistically significant chronologic changes in the grayscale median measurements were shown, with a trough at 6 hours and a peak at 24 hours (mean ± SD: 2 hours, 46.8 ± 4.1; 6 hours, 32.2 ± 13.1; 24 hours, 55.4 ± 9.9; 48 hours, 49.0 ± 13.9; P = .027). The grayscale median value was higher in dense RBC clots and lower in loose RBC clots (dense, 61.0 ± 10.6; intermediate, 49.4 ± 13.1; loose, 34.0 ± 12.1; P < .0353). Sonography using the grayscale median showed chronologic echogenicity changes in clots over a 48-hour period, and the grayscale median correlated with RBC density.

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear.

PubMed

Govindarajan, Vijay; Zhu, Shu; Li, Ruizhi; Lu, Yichen; Diamond, Scott L; Reifman, Jaques; Mitrophanov, Alexander Y

2018-02-27

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ∼0.1 and ∼2 molecules/μm 2 . Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot's structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot's occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow. Copyright © 2018 Biophysical Society. All rights reserved.

Plasma clots gelled by different amounts of calcium for stem cell delivery.

PubMed

Gessmann, Jan; Seybold, Dominik; Peter, Elvira; Schildhauer, Thomas Armin; Köller, Manfred

2013-01-01

Freshly prepared autologous plasma clots may serve as a carrier matrix for expanded multipotent mesenchymal stromal cells (MSCs) or bone marrow cells. By varying the calcium concentration, plasma clots with different properties can be produced. The purpose of this in vitro study was to determine the optimal calcium concentrations for the clotting process, intra-clot cell viability, and clot lysis. Different plasma clots were prepared by adding an equal volume of RPMI1640 (with or without MSCs) to citrate plasma (either containing platelets or platelet-free). Clotting was initiated by the addition of CaCl(2) (10 g/100 ml H(2)O, 10 % solution). The final concentration of CaCl(2) ranged from 1 to 10 % by volume of plasma. Viability and distribution of the MSCs were analysed by calcein-AM/propidium iodide staining. MSC-embedded plasma clots were dissolved with trypsin (0.25 %), and recovered cells were further incubated for 1 week under cell culture conditions. The viability of MSCs embedded in clots formed by the addition of 1-8 % by volume CaCl2 was not affected by incubation of up to 1 week. In contrast, clots produced by higher volumes of CaCl(2) solutions (9-10 % by volume of plasma) showed decreased numbers of viable cells. Intra-clot cell proliferation was highest in clots produced by addition of 5 % CaCl(2) by plasma volume. Osteocalcin release was not influenced in platelet-free plasma but decreased in platelet-containing plasma. Morphological analysis of stained recovered MSCs revealed that lysis of the plasma clot did not affect cell morphology or subsequent spontaneous proliferation. Clot formation and clot stability can be controlled by changing the concentration of CaCl(2) added to plasma. The addition of 5 % CaCl(2) produced a plasma clot with optimal results for stem cell delivery.

A constitutive model for developing blood clots with various compositions and their nonlinear viscoelastic behavior.

PubMed

van Kempen, Thomas H S; Donders, Wouter P; van de Vosse, Frans N; Peters, Gerrit W M

2016-04-01

The mechanical properties determine to a large extent the functioning of a blood clot. These properties depend on the composition of the clot and have been related to many diseases. However, the various involved components and their complex interactions make it difficult at this stage to fully understand and predict properties as a function of the components. Therefore, in this study, a constitutive model is developed that describes the viscoelastic behavior of blood clots with various compositions. Hereto, clots are formed from whole blood, platelet-rich plasma and platelet-poor plasma to study the influence of red blood cells, platelets and fibrin, respectively. Rheological experiments are performed to probe the mechanical behavior of the clots during their formation. The nonlinear viscoelastic behavior of the mature clots is characterized using a large amplitude oscillatory shear deformation. The model is based on a generalized Maxwell model that accurately describes the results for the different rheological experiments by making the moduli and viscosities a function of time and the past and current deformation. Using the same model with different parameter values enables a description of clots with different compositions. A sensitivity analysis is applied to study the influence of parameter variations on the model output. The relative simplicity and flexibility make the model suitable for numerical simulations of blood clots and other materials showing similar behavior.

Identification of quantitative trait loci for fibrin clot phenotypes: The EuroCLOT study

PubMed Central

Williams, Frances MK; Carter, Angela M; Kato, Bernet; Falchi, Mario; Bathum, Lise; Surdulescu, Gabriela; Kyvik, Kirsten Ohm; Palotie, Aarno; Spector, Tim D; Grant, Peter J

2012-01-01

Objectives Fibrin makes up the structural basis of an occlusive arterial thrombus and variability in fibrin phenotype relates to cardiovascular risk. The aims of the current study from the EU consortium EuroCLOT were to 1) determine the heritability of fibrin phenotypes and 2) identify QTLs associated with fibrin phenotypes. Methods 447 dizygotic (DZ) and 460 monozygotic (MZ) pairs of healthy UK Caucasian female twins and 199 DZ twin pairs from Denmark were studied. D-dimer, an indicator of fibrin turnover, was measured by ELISA and measures of clot formation, morphology and lysis were determined by turbidimetric assays. Heritability estimates and genome-wide linkage analysis were performed. Results Estimates of heritability for d-dimer and turbidometric variables were in the range 17 - 46%, with highest levels for maximal absorbance which provides an estimate of clot density. Genome-wide linkage analysis revealed 6 significant regions with LOD>3 on 5 chromosomes (5, 6, 9, 16 and 17). Conclusions The results indicate a significant genetic contribution to variability in fibrin phenotypes and highlight regions in the human genome which warrant further investigation in relation to ischaemic cardiovascular disorders and their therapy. PMID:19150881

Thrombin generation, ProC(®)Global, prothrombin time and activated partial thromboplastin time in thawed plasma stored for seven days and after methylene blue/light pathogen inactivation.

PubMed

Thiele, Thomas; Hron, Gregor; Kellner, Sarah; Wasner, Christina; Westphal, Antje; Warkentin, Theodore E; Greinacher, Andreas; Selleng, Kathleen

2016-01-01

Methylene blue pathogen inactivation and storage of thawed plasma both lead to changes in the activity of several clotting factors. We investigated how this translates into a global loss of thrombin generation potential and alterations in the protein C pathway. Fifty apheresis plasma samples were thawed and each divided into three subunits. One subunit was stored for 7 days at 4 °C, one was stored for 7 days at 22 °C and one was stored at 4 °C after methylene blue/light treatment. Thrombin generation parameters, ProC(®)Global-NR, prothrombin time and activated partial thromboplastin time were assessed on days 0 and 7. The velocity of thrombin generation increased significantly after methylene blue treatment (increased thrombin generation rate; time to peak decreased) and decreased after storage (decreased thrombin generation rate and peak thrombin; increased lag time and time to peak). The endogenous thrombin generation potential remained stable after methylene blue treatment and storage at 4 °C. Methylene blue treatment and 7 days of storage at 4 °C activated the protein C pathway, whereas storage at room temperature and storage after methylene blue treatment decreased the functional capacity of the protein C pathway. Prothrombin time and activated partial thromboplastin time showed only modest alterations. The global clotting capacity of thawed plasma is maintained at 4 °C for 7 days and directly after methylene blue treatment of thawed plasma. Thrombin generation and ProC(®)Global are useful tools for investigating the impact of pathogen inactivation and storage on the clotting capacity of therapeutic plasma preparations.

Ischemic Strokes (Clots)

MedlinePlus

... Month Infographic Stroke Hero F.A.S.T. Quiz Ischemic Strokes (Clots) Updated:May 21,2018 Ischemic stroke accounts for about 87 percent of all cases. View a detailed animation of ischemic stroke . Ischemic strokes occur as a result of an ...

Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

PubMed Central

Zamolodchikov, Daria

2012-01-01

Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ42-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ42, and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ42. Our results indicate the existence of 2 mechanisms of Aβ42 involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)–fibrin binding. PMID:22238323

Mesoscopic Modeling of Blood Clotting: Coagulation Cascade and Platelets Adhesion

NASA Astrophysics Data System (ADS)

Yazdani, Alireza; Li, Zhen; Karniadakis, George

2015-11-01

The process of clot formation and growth at a site on a blood vessel wall involve a number of multi-scale simultaneous processes including: multiple chemical reactions in the coagulation cascade, species transport and flow. To model these processes we have incorporated advection-diffusion-reaction (ADR) of multiple species into an extended version of Dissipative Particle Dynamics (DPD) method which is considered as a coarse-grained Molecular Dynamics method. At the continuum level this is equivalent to the Navier-Stokes equation plus one advection-diffusion equation for each specie. The chemistry of clot formation is now understood to be determined by mechanisms involving reactions among many species in dilute solution, where reaction rate constants and species diffusion coefficients in plasma are known. The role of blood particulates, i.e. red cells and platelets, in the clotting process is studied by including them separately and together in the simulations. An agonist-induced platelet activation mechanism is presented, while platelets adhesive dynamics based on a stochastic bond formation/dissociation process is included in the model.

Monitoring of unfractionated heparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent (PiCT®).

PubMed

Schaden, E; Jilch, S; Hacker, S; Schober, A; Kozek-Langenecker, S

2012-12-24

To achieve sufficient and safe anticoagulation with unfractionated heparin (UFH) a close and reliable drug monitoring is necessary. In general, the activated partial thromboplastin time (APTT) is used for this purpose. In acute phase response, however, the APTT test procedure might be unreliable e.g. with false low results in the presence of elevated factor VIII. In this so called heparin resistance, measurement of anti-Xa activity is recommended over APTT to avoid potentially harmful dose escalation. A combination of anti-Xa measurement and global hemostatic testing with ROTEM® employing the anti-Xa sensitive PiCT® reagent showed high correlation with enoxaparin levels. This test modification could also be suitable for monitoring UFH. Aim of the study was to evaluate the correlation between PiCT®-ROTEM® and levels of UFH. In this in-vitro study blood samples from healthy volunteers were spiked with UFH and subjected to different ROTEM® tests. There was a linear correlation between UFH level and clotting time (CT) in the PiCT®-ROTEM® test with an excellent correlation coefficient of 0.92. Additional endpoints showed similar results (PiCT®-ROTEM® MaxVel r = -0.85 and PiCT®-ROTEM® t_MaxVel r = 0.88). As a point-of-care applicable tool ROTEM® is immediately at hand. If further clinical studies confirm sensitivity in heparin resistance, PiCT®-ROTEM® could permit rapid UFH dose adjustments especially required in critical illness with acute phase response. Copyright © 2012 Elsevier B.V. All rights reserved.

Elevated NT-proBNP is associated with unfavorably altered plasma fibrin clot properties in atrial fibrillation.

PubMed

Matusik, Paweł T; Matusik, Patrycja S; Kornacewicz-Jach, Zdzisława; Małecka, Barbara; Ząbek, Andrzej; Undas, Anetta

2017-09-15

Dense fibrin clot formation and hypofibrinolysis have been reported in atrial fibrillation (AF). It is unclear which factors affect fibrin clot properties in AF. We investigated plasma fibrin clot permeability (K s ), clot lysis time (CLT), endogenous thrombin potential (ETP) as well as other coagulation and fibrinolysis parameters along with N-terminal pro-B-type natriuretic peptide (NT-proBNP) in 160 AF patients (median age, 70.5years). Previous stroke (n=15; 9.4%) was associated with decreased K s (P=0.04) and longer CLT (P=0.005), together with higher antiplasmin (P=0.03) and lower tissue-type plasminogen activator (P=0.01). Lower K s (P=0.04) and tendency towards longer CLT (P=0.10) were observed in patients with a left atrium diameter>40mm. Patients with a CHA 2 DS 2 -VASc score of 3 or more (82.5%) were characterized by higher thrombin-activatable fibrinolysis inhibitor antigen (P=0.009). K s was inversely correlated with log NT-proBNP (r=-0.34, P<0.0001), plasminogen activator inhibitor-1 (PAI-1) antigen (r=-0.24, P=0.002) and C-reactive protein (r=-0.18, P=0.02), while CLT was positively correlated with log NT-proBNP (R=0.61, P<0.0001) and ETP (r=0.37, P<0.0001), which were interrelated (r=0.59, P<0.0001). After adjustment for potential confounders, PAI-1 (odds ratio [OR]: 1.14; 95% confidence interval [CI]: 1.02-1.26) was the only independent predictor of low K s (the lowest quartile,≤6×10 -9 cm 2 ), while NT-proBNP (OR: 1.21; 95% CI: 1.12-1.31) and PAI-1 (OR: 1.30; 95% CI: 1.12-1.51) both predicted prolonged CLT (the top quartile,≥109min). In AF patients prothrombotic fibrin clot properties assessed ex vivo are determined by PAI-1 and NT-proBNP and this phenotype is associated with prior ischemic stroke. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular mechanisms of the effect of ultrasound on the fibrinolysis of clots

PubMed Central

Chernysh, Irina N.; Everbach, E. Carr; Purohit, Prashant K.; Weisel, John W.

2016-01-01

Summary Background Ultrasound accelerates tissue-type plasminogen activator (t-PA)-induced fibrinolysis of clots in vitro and in vivo. Objective To identify mechanisms for the enhancement of t-PA-induced fibrinolysis of clots. Methods Turbidity is an accurate and convenient method, not previously used, to follow the effects of ultrasound. Deconvolution microscopy was used to determine changes in structure, while fluorescence recovery after photobleaching was used to characterize the kinetics of binding/unbinding and transport. Results The ultrasound pulse repetition frequency affected clot lysis times, but there were no thermal effects. Ultrasound in the absence of t-PA produced a slight but consistent decrease in turbidity, suggesting a decrease in fibrin diameter due solely to the action of the ultrasound, likely caused by an increase in protofibril tension because of vibration from ultrasound. Changes in fibrin network structure during lysis with ultrasound were visualized in real time by deconvolution microscopy, revealing that the network becomes unstable when 30–40% of the protein in the network was digested, whereas without ultrasound, the fibrin network was digested gradually and retained structural integrity. Fluorescence recovery after photobleaching during lysis revealed that the off-rate of oligomers from digesting fibers was not much affected but the number of binding/unbinding sites was increased. Conclusions Ultrasound causes a decrease in the diameter of the fibers due to tension as a result of vibration, leading to increased binding sites for plasmin(ogen)/t-PA. The positive feedback of this structural change together with increased mixing/transport of t-PA/plasmin(ogen) is likely to account for the observed enhancement of fibrinolysis by ultrasound. PMID:25619618

Investigating Interactions between Ultrasound Stimulated Microbubbles and the Fibrin Network of Blood Clots

NASA Astrophysics Data System (ADS)

Acconcia, Christopher N.

The occlusion of blood vessels by thrombus is a major cause of mortality and morbidity in cardiovascular diseases such as deep vein thrombosis, myocardial infarction and ischemic stroke. In these contexts, prompt restoration of blood flow is of the utmost importance and is poorly addressed by current methods in many cases. For example, the treatment standard for ischemic stroke is administration of the thrombolytic agent tissue plasminogen activator, which is only minimally effective and has associated safety issues. There is, therefore, a need for the development of alternative recanalization strategies and amongst these, bubble mediated sonothrombolysis (thrombolysis by ultrasound) has emerged as a promising approach. Though it is well established that ultrasound stimulated microbubbles can potentiate the lysis of blood clots, the mechanisms are not well understood and this lack of understanding is a hindrance to the development of improved ultrasound exposure strategies. This thesis has revealed insights into the mechanisms of bubble mediated sonothrombolysis which can be used to guide the development of improved exposure strategies and contrast agents (i.e. bubble sizes) for sonothrombolysis treatments. The experimental approach involved fast frame optical imaging of ultrasound stimulated microbubbles interacting with clots, and two-photon fluorescence imaging of clots following ultrasound exposure. It was demonstrated that bubbles can penetrate fibrin clots, disrupt the fibrin network, generate patent tunnels, enhance the transport of fluid into the clot and induce clot boundary displacements. Furthermore, the occurrence and extent of these therapeutically relevant effects were shown to be highly dependent on pulse length and bubble size: longer pulses and larger bubbles were associated with greater disruption of fibrin networks and greater fluid transport distances. Finally, it was shown that bubbles can induce the ejection of erythrocytes from blood clots

Blood Thinners: Can I Still Get Blood Clots?

MedlinePlus

... get blood clots? If you're taking a blood thinner, is it still possible to get a blood clot? Answers from Rekha Mankad, M.D. Yes. Medications that are commonly called blood thinners — such as aspirin, warfarin (Coumadin, Jantoven), dabigatran ( ...

Viscoelasticity and structure of blood clots generated in-vitro by rheometry: A comparison between human, horse, rat, and camel.

PubMed

Dibiasi, Christoph; Plewka, Jacek; Ploszczanski, Leon; Glanz, Veronika; Lichtenegger, Helga; Windberger, Ursula

2018-04-14

Although the coagulation system is evolutionary well preserved, profound species differences exist in viscoelastic as well as in common laboratory tests of coagulation. Evaluating differences in clot formation and material characterisation of clots of four mammalian species on macro-, micro- and nanoscales by the means of rheometry, scanning electron microscopy (SEM) and small angle x-ray scattering (SAXS). Blood samples were collected from healthy human volunteers, laboratory rats (HL/LE inbred strain), warmblood horses and dromedary camels. Clot formation was observed by oscillating shear rheometry until plateau formation of the shear storage modulus G', at which point selected clots were prepared for scanning electron microscopy. SEM images were analysed for fibre diameter and fractal dimension. Additionally, scattering profiles for plasma and whole blood samples were obtained with SAXS. Viscoelasticity of clots showed great interspecies variation: clots of rats and horses exhibited shorter clotting times and higher G' plateau values, when compared to human clots. Camel clots showed unique clotting characteristics with no G' plateau formation in the timeframe observed. Less differentiating features were found with SEM and SAXS, although the rat fibre network appears to be more convoluted and dense, which resulted in a higher fractal dimension. Clotting kinetic differs between the species, which is not only of clinical interest, but could also be an important finding for animal models of blood coagulation.

Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome): Association with Thrombin Generation and Eosinophilia.

PubMed

Mastalerz, Lucyna; Celińska-Lӧwenhoff, Magdalena; Krawiec, Piotr; Batko, Bogdan; Tłustochowicz, Witold; Undas, Anetta

2015-01-01

Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome), we investigated whether fibrin clot properties are unfavorably altered in EGPA. Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male), aged 48 (range, 21-80) years. The control group comprised 34 age- and sex- matched volunteers. Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10-9 cm2), faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s), thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07), higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L), and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min); all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%. This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease.

Unfavorably Altered Fibrin Clot Properties in Patients with Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss Syndrome): Association with Thrombin Generation and Eosinophilia

PubMed Central

Mastalerz, Lucyna; Celińska-Lӧwenhoff, Magdalena; Krawiec, Piotr; Batko, Bogdan; Tłustochowicz, Witold; Undas, Anetta

2015-01-01

Objectives Given reports on the increased prevalence of thromboembolic incidents in patients with eosinophilic granulomatosis with polyangiitis (EGPA; Churg-Strauss syndrome), we investigated whether fibrin clot properties are unfavorably altered in EGPA. Methods Ex vivo plasma fibrin clot characteristics, including clot permeability, turbidimetry and efficiency of fibrinolysis using two assays, were investigated in 34 consecutive patients with remission in EGPA according to the Birmingham Vasculitis Activity Score version 3 (23 female, 11 male), aged 48 (range, 21–80) years. The control group comprised 34 age- and sex- matched volunteers. Results Compared with controls, patients with EGPA were characterized by denser fiber clots (estimated pore size, Ks, 7.30±0.93 vs 10.14±1.07 10−9 cm2), faster fibrin polymerization (lag phase in a turbidimetric curve, 41.8±3.6 vs 47.4±2.9 s), thicker fibrin fibers (maximum absorbance, ΔAbs, 0.87±0.09 vs 0.72±0.07), higher maximum levels of D-dimer released from clots (DDmax 4.10±0.46 vs 3.54±0.35 mg/L), and prolonged clot lysis time (t50%; 9.50±1.45 vs 7.56±0.87 min); all p<0.0001. Scanning electron microscopy images confirmed denser plasma fibrin networks composed of thinner fibers formed in EGPA. Antineutrophil cytoplasmic antibody status and C-reactive protein did not affect clot variables. Multivariate analysis adjusted for fibrinogen showed that Ks was predicted by eosinophil count, peak thrombin generation, factor VIII, and soluble CD40 ligand, whereas eosinophil count, peak thrombin generation and antiplasmin predicted t50%. Conclusion This study is the first to show that EGPA is associated with prothrombotic plasma fibrin clot phenotype, which may contribute to thromboembolic manifestations reported in this disease. PMID:26540111

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination.

PubMed

Kitamura, Yutaka; Watanabe, Taisuke; Nakamura, Masayuki; Isobe, Kazushige; Kawabata, Hideo; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

2018-01-01

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl 2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

PubMed Central

Kitamura, Yutaka; Watanabe, Taisuke; Nakamura, Masayuki; Isobe, Kazushige; Kawabata, Hideo; Uematsu, Kohya; Okuda, Kazuhiro; Nakata, Koh; Tanaka, Takaaki; Kawase, Tomoyuki

2018-01-01

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix. PMID:29450197

Moringa oleifera Lam.: Protease activity against blood coagulation cascade.

PubMed

Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

2012-01-01

The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. These findings support the traditional usage of M. oleifera extracts for wound healing.

Clot retraction affects the extent of ultrasound-enhanced thrombolysis in an ex vivo porcine thrombosis model.

PubMed

Sutton, Jonathan T; Ivancevich, Nikolas M; Perrin, Stephen R; Vela, Deborah C; Holland, Christy K

2013-05-01

We investigated ultrasound-enhanced thrombolysis in two whole-blood clot models using a Food and Drug Administration-approved contrast agent (Definity, Lantheus Medical Imaging; Billerica, MA USA) and thrombolytic drug (recombinant tissue-type plasminogen activator [rt-PA]) (Genentech; South San Francisco, CA USA). Porcine venous blood was collected from donor hogs and coagulated in vials made of two different materials. This method produced clots with differing compositional properties, as determined by routine scanning electron microscopy and histology. Clots were deployed in an ex vivo porcine thrombosis model, and exposed to an intermittent ultrasound scheme previously developed to maximize stable cavitation while acoustic emissions were detected. Exposure to 3.15 μg/mL rt-PA promoted lysis in both clot models, compared with exposure to plasma alone. However, only unretracted clots experienced significant enhancement of thrombolysis in the presence of rt-PA, Definity, and ultrasound, compared with treatment with rt-PA. In these clots, microscopy revealed loose erythrocyte aggregates, a significantly less extensive fibrin network and a higher porosity, which may facilitate increased penetration of thrombolytics by cavitation. Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Blood Clots and Travel: What You Need to Know

MedlinePlus

... Icon View public health webinars on blood disorders Blood Clots and Travel: What You Need to Know Language: ... trip. Understand What Can Increase Your Risk for Blood Clots Even if you travel a long distance, the ...

A simple clot based assay for detection of procoagulant cell-derived microparticles.

PubMed

Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

2016-05-01

Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

The application of large amplitude oscillatory stress in a study of fully formed fibrin clots

NASA Astrophysics Data System (ADS)

Lamer, T. F.; Thomas, B. R.; Curtis, D. J.; Badiei, N.; Williams, P. R.; Hawkins, K.

2017-12-01

The suitability of controlled stress large amplitude oscillatory shear (LAOStress) for the characterisation of the nonlinear viscoelastic properties of fully formed fibrin clots is investigated. Capturing the rich nonlinear viscoelastic behaviour of the fibrin network is important for understanding the structural behaviour of clots formed in blood vessels which are exposed to a wide range of shear stresses. We report, for the first time, that artefacts due to ringing exist in both the sample stress and strain waveforms of a LAOStress measurement which will lead to errors in the calculation of nonlinear viscoelastic properties. The process of smoothing the waveforms eliminates these artefacts whilst retaining essential rheological information. Furthermore, we demonstrate the potential of LAOStress for characterising the nonlinear viscoelastic properties of fibrin clots in response to incremental increases of applied stress up to the point of fracture. Alternating LAOStress and small amplitude oscillatory shear measurements provide detailed information of reversible and irreversible structural changes of the fibrin clot as a consequence of elevated levels of stress. We relate these findings to previous studies involving large scale deformations of fibrin clots. The LAOStress technique may provide useful information to help understand why some blood clots formed in vessels are stable (such as in deep vein thrombosis) and others break off (leading to a life threatening pulmonary embolism).

Removal of Chronic Intravascular Blood Clots using Liquid Plasma

NASA Astrophysics Data System (ADS)

Jung, Jae-Chul; Choi, Myeong; Koo, Il; Yu, Zengqi; Collins, George

2011-10-01

An electrical embolectomy device for removing chronic intravascular blood clots using liquid plasma under saline environment was demonstrated. We employed a proxy experimental blood clot model of deep vein thrombosis (DVT) and actual equine blood clot. Thermal damage to contiguous tissue and the collagen denaturing via the plasma irradiation were investigated by histological analysis using birefringence of the tissue and verified by FT-IR spectroscopic study, respectively, which showed the high removal rate up to 2 mm per minute at room temperature and small thermal damage less than 200 μm.

Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease.

PubMed

Esposito, Marilena; Di Pierro, Prospero; Dejonghe, Winnie; Mariniello, Loredana; Porta, Raffaele

2016-08-01

Two different milk clotting enzymes, belonging to the aspartic protease family, were extracted from both artichoke leaves and alpine thistle flowers, and the latter was covalently immobilized by using a polyacrylic support containing polar epoxy groups. Our findings showed that the alpine thistle aspartic protease was successfully immobilized at pH 7.0 on Immobeads IB-150P beads and that, under these experimental conditions, an immobilization yield of about 68% and a recovery of about 54% were obtained. Since the enzyme showed an optimal pH of 5.0, a value very similar to the one generally used for milk clotting during cheese making, and exhibited a satisfactory stability over time, the use of such immobilized vegetable rennet for the production of novel dairy products is suggested. Copyright © 2016. Published by Elsevier Ltd.

Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin.

PubMed

Perizzolo, K E; Sullivan, S; Waugh, D F

1985-03-01

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.

Moringa oleifera Lam.: Protease activity against blood coagulation cascade

PubMed Central

Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

2012-01-01

Background: The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Materials and Methods: Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results: Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions: These findings support the traditional usage of M. oleifera extracts for wound healing. PMID:22224061

Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots.

PubMed

Zubairova, Laily D; Nabiullina, Roza M; Nagaswami, Chandrasekaran; Zuev, Yuriy F; Mustafin, Ilshat G; Litvinov, Rustem I; Weisel, John W

2015-12-04

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1-0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis.

Fiber-based optic sensor for detecting human blood clot: present and future revival

NASA Astrophysics Data System (ADS)

Elshikeri, Nada; Bakhtiar, Hazri

2018-05-01

Sustaining human’s life-frame away from being impeded by the clot - ghost term, we attempt to approach a mobile fiber-based optical sensor (f-s) for detecting blood clot in a blood vessel (intra-arteries/veins). Blood vessels are the part of the circulatory system that transport blood throughout the human body, thus their significance of being protected arise to the monograph focus. MRI (magnetic resonance imaging), X-rays and other medical instruments are diagnostic immobility techniques with a slackest interval. The corer causation of fiber-based optical sensor is to detect a clump of blood in the bloodstream by providing a prompt mobile diagnostic intervals preserving last-minutes-breath of human’s life. The detector (f-s) has been etched by diluting sulphuric acid ~10% at certain zone to sensate its function. The in-vitro monograph peaks its maximal monitoring when the sensor is attached to Raman Spectroscopy (RS) setup. RS quantifies the relative intensities of fibrinogen bond, which is the first type of blood coagulation elements of blood plasma. Blood coagulation parameters are the major concern of the monograph investigation, such as total haemoglobin (tHb), clotting reaction time (t), clot progression time (t2), maximum clot amplitude (ma) and mean refractive index (r). A blood sample will be drawn from the patient and after centrifugation to separate blood plasma from its constituents, then an immediate sloshing of blood plasma in the (f-s) packet which has its plug-in to RS. Estimating the quantitative analysis of blood sample concentration, RS will determine the presence of coagulation in terms of intensity and medical procedures will dominate the treatment process. Thus, the suggestive monograph provides a definite instrument for investigating blood coagulation intra-arteries/veins promptly.

Freeze-dried plasma enhances clot formation and inhibits fibrinolysis in the presence of tissue plasminogen activator similar to pooled liquid plasma.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Sauaia, Angela; Stettler, Gregory; Dzieciatkowska, Monika; Hansen, Kirk; Banerjee, Anirban; Silliman, Christopher C

2017-08-01

Systemic hyperfibrinolysis is an integral part of trauma-induced coagulopathy associated with uncontrolled bleeding. Recent data suggest that plasma-first resuscitation attenuates hyperfibrinolysis; however, the availability, transport, storage, and administration of plasma in austere environments remain challenging and have limited its use. Freeze-dried plasma (FDP) is a potential alternative due to ease of storage, longer shelf life, and efficient reconstitution. FDP potentially enhances clot formation and resists breakdown better than normal saline (NS) and albumin and similar to liquid plasma. Healthy volunteers underwent citrated blood draw followed by 50% dilution with NS, albumin, pooled plasma (PP), or pooled freeze-dried plasma (pFDP). Citrated native and tissue plasminogen activator (t-PA)-challenge (75 ng/mL) thrombelastography were done. Proteins in PP, pFDP, and albumin were analyzed by mass spectroscopy. pFDP and PP had superior clot-formation rates (angle) and clot strength (maximum amplitude) compared with NS and albumin in t-PA-challenge thrombelastographies (angle: pFDP, 67.9 degrees; PP, 67.8 degrees; NS, 40.6 degrees; albumin, 35.8 degrees; maximum amplitude: pFDP, 62.4 mm; PP, 63.5 mm; NS, 44.8 mm; albumin, 41.1 mm). NS and albumin dilution increased susceptibility to t-PA-induced hyperfibrinolysis compared with pFDP and PP (NS, 62.4%; albumin, 62.6%; PP, 8.5%; pFDP, 6.7%). pFDP was similar to PP in the attenuation of t-PA-induced fibrinolysis. Most proteins (97%) were conserved during the freeze-dry process, with higher levels in 12% of pFDP proteins compared with PP. pFDP enhances clot formation and attenuates hyperfibrinolysis better than NS and albumin and is a potential alternative to plasma resuscitation in the treatment of hemorrhagic shock. © 2017 AABB.

Clot formation is associated with fibrinogen and platelet forces in a cohort of severely-injured Emergency Department trauma patients

PubMed Central

White, Nathan J.; Newton, Jason C.; Martin, Erika J.; Mohammed, Bassem M.; Contaifer, Daniel; Bostic, Jessica L.; Brophy, Gretchen M.; Spiess, Bruce D.; Pusateri, Anthony E.; Ward, Kevin R.; Brophy, Donald F.

2015-01-01

Introduction Anticoagulation, fibrinogen consumption, fibrinolytic activation, and platelet dysfunction all interact to produce different clot formation responses after trauma. However, the relative contributions of these coagulation components to overall clot formation remains poorly defined. We examined for sources of heterogeneity in clot formation responses after trauma. Methods Blood was sampled in the Emergency Department from patients meeting trauma team activation criteria at an urban trauma center. Plasma prothrombin time (PT) ≥ 18 sec was used to define traumatic coagulopathy. Mean kaolin-activated thrombelastography (TEG) parameters were calculated and tested for heterogeneity using Analysis of Means (ANOM). Discriminant analysis and forward stepwise variable selection with linear regression were used to determine if PT, fibrinogen, platelet contractile force (PCF), and D-Dimer concentration, representing key mechanistic components of coagulopathy, each contribute to heterogeneous TEG responses after trauma. Results Of 95 subjects, 16% met criteria for coagulopathy. Coagulopathic subjects were more severely injured with greater shock, and received more blood products in the first 8 hours compared to non-coagulopathic subjects. Mean (SD) TEG maximal amplitude (MA) was significantly decreased in the coagulopathic group=57.5 (4.7) mm, vs. 62.7 (4.7), T test p<0.001. The MA also exceeded the ANOM predicted upper decision limit for the non-coagulopathic group and the lower decision limit for the coagulopathic group at alpha=0.05, suggesting significant heterogeneity from the overall cohort mean. Fibrinogen and PCF best discriminated TEG MA using discriminant analysis. Fibrinogen, PCF, and D-Dimer were primary covariates for TEG MA using regression analysis. Conclusion Heterogeneity in TEG-based clot formation in Emergency Department trauma patients was linked to changes in MA. Individual parameters representing fibrin polymerization, platelet contractile

Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

NASA Astrophysics Data System (ADS)

Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

2012-11-01

Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that there is turnover of fibrin in an uncrosslinked clot. A peptide representing the knobs involved in fibrin polymerization can compete for the holes and dissolve a preformed fibrin clot, or increase the fraction of soluble oligomers, with striking rearrangements in clot structure. These results imply that in vivo clots or thrombi are more dynamic structures than previously believed that may be remodeled as a result of local environmental conditions, may account for some embolization, and suggest a target for therapeutic intervention.

Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots

PubMed Central

Zubairova, Laily D.; Nabiullina, Roza M.; Nagaswami, Chandrasekaran; Zuev, Yuriy F.; Mustafin, Ilshat G.; Litvinov, Rustem I.; Weisel, John W.

2015-01-01

Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1–0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis. PMID:26635081

The in vivo effect of fibrinogen and factor XIII on clot formation and fibrinolysis in Glanzmann's thrombasthenia.

PubMed

Shenkman, Boris; Livnat, Tami; Misgav, Mudi; Budnik, Ivan; Einav, Yulia; Martinowitz, Uriel

2012-01-01

Glanzmann's thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl(2) and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.

Common agents used to unblock blood clots within tympanostomy tubes: an ex vivo study and review of literature.

PubMed

Burke, Emily L; Walvekar, Rohan R; Lin, James; Hagan, Joseph; Kluka, Evelyn A

2009-12-01

To determine the efficacy of common solutions used to dissolve blood clots blocking tympanostomy tubes (TTs) of differing lengths and diameters. An ex vivo experimental study. Ear models were built by the study investigator. Tympanostomy tubes were inserted into the models and blocked with blood clots. Test solutions were applied to the blood clots, and time for clearance was recorded via microscopic visual confirmation. Richards T-tube had higher odds of unclogging than collar button tubes (odds ratio: 2.37, 95% confidence intervals 1.02-5.54, p=0.042). Vinegar and 3% hydrogen peroxide were most effective for Richards T-tubes and collar button tubes, respectively. Common solutions (vinegar and hydrogen peroxide) were more effective than antibiotic drops in clearing blood clot blocking TTs.

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

PubMed

Jiménez-Aristizabal, Román F; López, Catalina; Álvarez, María E; Giraldo, Carlos; Prades, Marta; Carmona, Jorge U

2017-09-01

To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β 1 ) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-β 1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fibrin clot structure remains unaffected in young, healthy individuals after transient exposure to diesel exhaust

PubMed Central

2010-01-01

Exposure to urban particulate matter has been associated with an increased risk of cardiovascular disease and thrombosis. We studied the effects of transient exposure to diesel particles on fibrin clot structure of 16 healthy individuals (age 21- 44). The subjects were randomly exposed to diesel exhaust and filtered air on two separate occasions. Blood samples were collected before exposure, and 2 and 6 hours after exposure. There were no significant changes on clot permeability, maximum turbidity, lag time, fibre diameter, fibre density and fibrinogen level between samples taken after diesel exhaust exposure and samples taken after filtered air exposure. These data show that there are no prothrombotic changes in fibrin clot structure in young, healthy individuals exposed to diesel exhaust. PMID:20565709

QuikClot masquerading as Glaser shot: a new forensic pathology artifact?

PubMed

Moeng, Shirley Faith Angela Portia; Moar, Joshua J

2012-09-01

Although in the forensic sphere, Glaser ammunition or handgun shot cartridges represent one of the most significant advances in firearms ballistics in the past 100 years, the mineral-based zeolite hemostatic agent QuikClot represents a no less significant development in the surgical and clinical context of the trauma-induced management of battlefield wounding and hemorrhage. Because of the essential structural configuration of both Glaser ammunition and QuikClot, consisting as they do of metallic beads on the one hand and mineral-based spherules on the other, the possibility exists that a medicolegal autopsy on a victim of gunshot wounding and on whom surgery has been performed with introduction of QuikClot in an effort to stem bleeding, the spherules of QuikClot might be misinterpreted as the pellets of handgun shot cartridges.We present a case of fatal wounding by a 9-mm handgun in which the discovery of QuikClot in the peritoneal cavity at autopsy initially raised the possibility of wounding by Glaser ammunition.

14-Day thawed plasma retains clot enhancing properties and inhibits tPA-induced fibrinolysis.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Shepherd-Singh, Raymond; Sauaia, Angela; Stettler, Gregory R; Nunns, Geoffrey R; Silliman, Christopher C

2017-11-01

Plasma-first resuscitation attenuates trauma-induced coagulopathy (TIC); however, the logistics of plasma-first resuscitation require thawed plasma (TP) be readily available due to the obligatory thawing time of fresh frozen plasma (FFP). The current standard is storage of TP for up to 5 days at 4°C, based on factor levels at outdate, for use in patients at risk for TIC, but there remains a 2.2% outdated wastage rate. However, the multitude of plasma proteins in attenuating TIC remains unknown. We hypothesize that TP retains the ability to enhance clotting and reduce tPA-induced fibrinolysis at 14-day storage. FFP was thawed and stored at 4°C at the following intervals: 14, 10, 7, 5, 3, and 1-day prior to the experiment. Healthy volunteers underwent blood draws followed by 50% dilution with TP stored at previously mentioned intervals as well as FFP, normal saline (NS), albumin, and whole blood (WB) control. Samples underwent tPA-modified (75 ng/mL) thrombelastography (TEG) with analysis of R-time, angle, maximum amplitude (MA), and LY30. TEG properties did not change significantly over the thawed storage. 14-day TP retained the ability to inhibit tPA-induced hyperfibrinolysis (median LY30% 9.6%) similar to FFP (5.6%), WB (14.6%), and superior to albumin (59.3%) and NS (58.1%). 14-day TP also retained faster clot formation (median angle, 66.2°) and superior clot strength (MA, 61.5 mm) to albumin (34.8°, 21.6 mm) and NS (41.6°, 32.2 mm). TP plasma stored for 14 days retains clot-enhancing ability and resistance to clot degradation similar to FFP. A clinical trial is needed to validate these in vitro results. Copyright © 2017 Elsevier Inc. All rights reserved.

Erythrocyte-Derived Nanoparticles as a Theranostic Agent for Near-Infrared Fluorescence Imaging and Thrombolysis of Blood Clots.

PubMed

Vankayala, Raviraj; Corber, Samantha R; Mac, Jenny T; Rao, Masaru P; Shafie, Mohammad; Anvari, Bahman

2018-04-01

Ischemic stroke occurs when a blood clot obstructs or narrows the arteries that supply blood to the brain. Currently, tissue plasminogen activator (tPA), a thrombolytic agent, is the only United States Food and Drug Administration (FDA)-approved pharmacologic treatment for ischemic stroke. Despite its effective usage, the major limitation of tPA that stems from its short half-life in plasma (≈5 min) is the potential for increased risk of hemorrhagic complications. To circumvent these limitations, herein, the first proof-of-principle demonstration of a theranostic nanoconstruct system derived from erythrocytes doped with the FDA-approved near-infrared (NIR) imaging agent, indocyanine green, and surface-functionalized with tPA is reported. Using a clot model, the dual functionality of these nanoconstructs in NIR fluorescence imaging and clot lysis is demonstrated. These biomimetic theranostic nanoconstructs may ultimately be effective in imaging and treatment of blood clots involved in ischemic stroke. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Delayed double reading of whole blood clotting test (WBCT) results at 20 and 30 minutes enhances diagnosis and treatment of viper envenomation.

PubMed

Benjamin, Jordan Max; Chippaux, Jean-Philippe; Sambo, Bio Tamou; Massougbodji, Achille

2018-01-01

The whole blood clotting test (WBCT) is a simple test of coagulation that is often used in the assessment, diagnosis, and therapeutic monitoring of snakebite patients in sub-Saharan Africa. WBCT requires only a clean glass tube and several milliliters of venous blood and is ideal for use in poorly equipped health centers throughout the rural areas where 95% of snakebites occur. However, questions surrounding the accuracy and reliability of the test remain unanswered due to variations in testing conditions and a lack of comparative research with which to validate them. This is the first study to evaluate WBCT results at both 20-min (WBCT20) and 30-min (WBCT30) reading times in the same group of snakebite patients. In order to define the best reading time, the authors compared the results of serial WBCT evaluation at both 20 and 30 min after collection in 23 patients treated for snake envenomation in Bembèrèkè, northern Benin. WBCT results were identical at both reading times in patients without coagulopathy or when coagulation was restored permanently following a single dose of antivenom. Out of 17 patients with coagulopathy, 14 showed discrepancies between WBCT20 and WBCT30 results in at least one pair of serial evaluations. These could be completely contradictory results (e.g. normal clot at WBCT20 and no clot at WBCT30) or a marked difference in the quality of the clot (e.g. no clotting activity at WBCT20 and an unstable partial clot at WBCT30). WBCT discrepancies were encountered most frequently in three situations: initial normalization of hemostasis following antivenom therapy, detection of a secondary resumption of coagulopathy, or final restoration of hemostasis after a secondary resumption had occurred. This study suggests that the WBCT is robust and that a sequential reading should improve the diagnosis and monitoring of venom-induced coagulopathies. It also indicates the possibility of discrepancies in the sensitivity of WBCT20 and WBCT30 for

The hydraulic permeability of blood clots as a function of fibrin and platelet density.

PubMed

Wufsus, A R; Macera, N E; Neeves, K B

2013-04-16

Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02-0.54 had permeabilities of 1.2 × 10(-1)-1.5 × 10(-4)μm(2). Platelet-rich clots with a platelet volume fraction of 0.01-0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10(-2)-1.5 × 10(-5)μm(2). The permeability of fibrin gels and of clots with platelet volume fraction of <0.2 were modeled as an array of disordered cylinders with uniform diameters. Clots with a platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The Hydraulic Permeability of Blood Clots as a Function of Fibrin and Platelet Density

PubMed Central

Wufsus, A.R.; Macera, N.E.; Neeves, K.B.

2013-01-01

Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02–0.54 had permeabilities of 1.2 × 10−1–1.5 × 10−4μm2. Platelet-rich clots with a platelet volume fraction of 0.01–0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10−2–1.5 × 10−5μm2. The permeability of fibrin gels and of clots with platelet volume fraction of <0.2 were modeled as an array of disordered cylinders with uniform diameters. Clots with a platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies. PMID:23601328

Plasma fibrin clot properties in the G20210A prothrombin mutation carriers following venous thromboembolism: the effect of rivaroxaban.

PubMed

Janion-Sadowska, Agnieszka; Natorska, Joanna; Siudut, Jakub; Ząbczyk, Michal; Stanisz, Andrzej; Undas, Anetta

2017-08-30

We sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (K s ) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2-6 hours (h) after and 20-25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (K s -12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for K s and CLT before rivaroxaban intake. At 2-6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (K s +38 %, and +37 %, CLT -25 % and -25 %, CLT-TAFI -20 % and -24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (K s -12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20-25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

Effect of chitosan and coagulation factors on the wound repair phenotype of bioengineered blood clots.

PubMed

Hoemann, Caroline D; Marchand, Catherine; Rivard, Georges-Etienne; El-Gabalawy, Hani; Poubelle, Patrice E

2017-11-01

Controlling the blood clot phenotype in a surgically prepared wound is an evolving concept in scaffold-guided tissue engineering. Here, we investigated the effect of added chitosan (80% or 95% Degree of Deacetylation, DDA) or coagulation factors (recombinant human Factor VIIa, Tissue Factor, thrombin) on inflammatory factors released by blood clots. We tested the hypothesis that 80% DDA chitosan specifically enhances leukotriene B 4 (LTB 4 ) production. Human or rabbit whole blood was combined with isotonic chitosan solutions, coagulation factors, or lipopolysaccharide, cultured in vitro at 37°C, and after 4hours the serum was assayed for LTB 4 or inflammatory factors. Only 80% DDA chitosan clots produced around 15-fold more LTB 4 over other clots including 95% DDA chitosan clots. All serum contained high levels of PDGF-BB and CXCL8. Normal clots produced very low type I cytokines compared to lipopolysaccharide clots, with even lower IL-6 and IL-12 and more CCL3/CCL4 produced by chitosan clots. Coagulation factors had no detectable effect on clot phenotype. Conclusion In blood clots from healthy individuals, 80% DDA chitosan has a unique influence of inducing more LTB 4 , a potent neutrophil chemoattractant, with similar production of PDGF-BB and CXCL8, and lower type I cytokines, compared to whole blood clots. Copyright © 2017 Elsevier B.V. All rights reserved.

Relationship between activated clotting time and ischemic or hemorrhagic complications: analysis of 4 recent randomized clinical trials of percutaneous coronary intervention.

PubMed

Brener, Sorin J; Moliterno, David J; Lincoff, A Michael; Steinhubl, Steven R; Wolski, Kathy E; Topol, Eric J

2004-08-24

Unfractionated heparin (UFH) is the most widely used antithrombin during percutaneous coronary intervention (PCI). Despite significant pharmacological and mechanical advancements in PCI, uncertainty remains about the optimal activated clotting time (ACT) for prevention of ischemic or hemorrhagic complications. We analyzed the outcome of all UFH-treated patients enrolled in 4 large, contemporary PCI trials with independent adjudication of ischemic and bleeding events. Of 9974 eligible patients, maximum ACT was available in 8369 (84%). The median ACT was 297 seconds (interquartile range 256 to 348 seconds). The incidence of death, myocardial infarction, or revascularization at 48 hours, by ACT quartile, was 6.2%, 6.8%, 6.0%, and 5.7%, respectively (P=0.40 for trend). Covariate-adjusted rate of ischemic complications was not correlated with maximal procedural ACT (continuous value, P=0.29). Higher doses of UFH (>5000 U, or up to 90 U/kg) were independently associated with higher rates of events. The incidence of major or minor bleeding at 48 hours, by ACT quartile, was 2.9%, 3.5%, 3.8%, and 4.0%, respectively (P=0.04 for trend). In a multivariable logistic model with a spline transformation for ACT, there was a linear increase in risk of bleeding as the ACT approached 365 seconds (P=0.01), which leveled off beyond that value. Increasing UFH weight-indexed dose was independently associated with higher bleeding rates (OR 1.04 [1.02 to 1.07] for each 10 U/kg, P=0.001). In patients undergoing PCI with frequent stent and potent platelet inhibition use, ACT does not correlate with ischemic complications and has a modest association with bleeding complications, driven mainly by minor bleeding. Lower values do not appear to compromise efficacy while increasing safety.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

PubMed

Kasahara, Kohji; Kaneda, Mizuho; Miki, Toshiaki; Iida, Kazuko; Sekino-Suzuki, Naoko; Kawashima, Ikuo; Suzuki, Hidenori; Shimonaka, Motoyuki; Arai, Morio; Ohno-Iwashita, Yoshiko; Kojima, Soichi; Abe, Mitsuhiro; Kobayashi, Toshihide; Okazaki, Toshiro; Souri, Masayoshi; Ichinose, Akitada; Yamamoto, Naomasa

2013-11-07

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-β-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbβ3-myosin complex is formed as a primary axis to promote platelet contraction.

In vitro investigations into enhancement of tPA bioavailability in whole blood clots using pulsed-high intensity focused ultrasound exposures.

PubMed

Jones, Guy; Hunter, Finnie; Hancock, Hilary A; Kapoor, Ankur; Stone, Michael J; Wood, Bradford J; Xie, Jianwu; Dreher, Matthew R; Frenkel, Victor

2010-01-01

Investigations were carried out on the manner by which pulsed-high intensity focused ultrasound (HIFU) enhances the effectiveness of tissue plasminogen activator (tPA) in whole blood clots, in vitro. Scanning electronic microscope (SEM) of the surface of the clots showed that the exposures increased exposed fibrin, as well as the number of openings to more interior regions. These findings were supported by fluorescent antibody labeling of tPA in frozen sections of clots treated post-HIFU. Here, improved accumulation at the surface and penetration of the tPA into the clots were observed in those treated with HIFU. Fluorescence recovery after photobleaching was also performed, indicating that the diffusion coefficient increased 6.3-fold for fluorescently labeled dextrans, comparable in size to tPA, in the HIFU-treated clots. Improved understanding of the manner by which pulsed--HIFU exposures can improve the effectiveness of thrombolytics will help optimize the exposures for this application and potentially facilitate translation to the clinic.

In Vitro Investigations Into Enhancement of tPA Bioavailability in Whole Blood Clots Using Pulsed–High Intensity Focused Ultrasound Exposures

PubMed Central

Jones, Guy; Hunter, Finnie; Hancock, Hilary A.; Kapoor, Ankur; Stone, Michael J.; Wood, Bradford J.; Xie, Jianwu; Dreher, Matthew R.

2012-01-01

Investigations were carried out on the manner by which pulsed–high intensity focused ultrasound (HIFU) enhances the effectiveness of tissue plasminogen activator (tPA) in whole blood clots, in vitro. Scanning electronic microscope (SEM) of the surface of the clots showed that the exposures increased exposed fibrin, as well as the number of openings to more interior regions. These findings were supported by fluorescent antibody labeling of tPA in frozen sections of clots treated post-HIFU. Here, improved accumulation at the surface and penetration of the tPA into the clots were observed in those treated with HIFU. Fluorescence recovery after photobleaching was also performed, indicating that the diffusion coefficient increased 6.3-fold for fluorescently labeled dextrans, comparable in size to tPA, in the HIFU-treated clots. Improved understanding of the manner by which pulsed–HIFU exposures can improve the effectiveness of thrombolytics will help optimize the exposures for this application and potentially facilitate translation to the clinic. PMID:20064753

Quantitative photoacoustic characterization of blood clot in blood: A mechanobiological assessment through spectral information

NASA Astrophysics Data System (ADS)

Biswas, Deblina; Vasudevan, Srivathsan; Chen, George C. K.; Sharma, Norman

2017-02-01

Formation of blood clots, called thrombus, can happen due to hyper-coagulation of blood. Thrombi, while moving through blood vessels can impede blood flow, an important criterion for many critical diseases like deep vein thrombosis and heart attacks. Understanding mechanical properties of clot formation is vital for assessment of severity of thrombosis and proper treatment. However, biomechanics of thrombus is less known to clinicians and not very well investigated. Photoacoustic (PA) spectral response, a non-invasive technique, is proposed to investigate the mechanism of formation of blood clots through elasticity and also differentiate clots from blood. Distinct shift (increase in frequency) of the PA response dominant frequency during clot formation is reported. In addition, quantitative differentiation of blood clots from blood has been achieved through parameters like dominant frequency and spectral energy of PA spectral response. Nearly twofold increases in dominant frequency in blood clots compared to blood were found in the PA spectral response. Significant changes in energy also help in quantitatively differentiating clots from blood, in the blood. Our results reveal that increase in density during clot formation is reflected in the PA spectral response, a significant step towards understanding the mechanobiology of thrombus formation. Hence, the proposed tool, in addition to detecting thrombus formation, could reveal mechanical properties of the sample through quantitative photoacoustic spectral parameters.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.

PubMed

Lundblom, Klara; Macharia, Alex; Lebbad, Marianne; Mohammed, Adan; Färnert, Anna

2011-08-08

Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Prolonged Activated Clotting Time after Protamine Administration Does Not Indicate Residual Heparinization after Cardiopulmonary Bypass in Pediatric Open Heart Surgery.

PubMed

Yamamoto, Tomohiro; Wolf, Hans-Gerd; Sinzobahamvya, Nicodème; Asfour, Boulos; Hraska, Victor; Schindler, Ehrenfried

2015-08-01

In open heart surgery, heparinization is commonly neutralized using an empirical heparin:protamine ratio ranging between 1:1 and 1:1.5. However, these ratios may result in protamine overdose that should be avoided for its negative side effects on the coagulation system. This study aimed to indicate the appropriate treatment for prolonged activated clotting time (ACT) after protamine administration following cardiopulmonary bypass (CPB) in pediatric open heart surgery by investigating the underlying reasons for it. Twenty-seven children (<10 kg) undergoing open heart surgery were included. Heparin was administered only before CPB (400 IU/kg) and in the pump priming volume for CPB (2,000 IU) and was neutralized by 1:1 protamine after CPB. The blood heparin concentration was measured using anti-Xa assay. ACT and blood concentrations of heparin, coagulation factors, thrombin-antithrombin complex, and prothrombin fragment 1 + 2 were assessed. A rotational thromboelastometry (ROTEM; Tem International GmbH, München, Bayern, Germany) was used to confirm the coagulation status and residual heparin after protamine administration. Anti-Xa assay showed that there is no residual heparin in the blood after 1:1 protamine administration. Nevertheless, ACT (128.89 ± 3.09 seconds before heparin administration) remained prolonged (177.14 ± 5.43 seconds at 10 minutes after protamine, 182.00 ± 5.90 seconds at 30 minutes after protamine). The blood concentrations of coagulation factors were significantly lower than those before heparin administration (p < 0.01). The low FIBTEM MCF of ROTEM (4.43 ± 0.32 mm) at 10 minutes after protamine indicated low fibrinogen concentration. Prolonged ACT after heparin neutralization by 1:1 protamine administration does not necessarily indicate residual heparin, but low blood concentrations of coagulation factors should be considered as a reason as well. Accordingly, supply of coagulation factors instead of additional

Differences in lupus anticoagulant final conclusion through clotting time or Rosner index for mixing test interpretation.

PubMed

Depreter, Barbara; Devreese, Katrien M J

2016-09-01

Lupus anticoagulant (LAC) testing includes a screening, mixing and confirmation step. Although recently published guidelines on LAC testing are a useful step towards standardization, a lack of consensus remains whether to express mixing tests in clotting time (CT) or index of circulating anticoagulant (ICA). The influence of anticoagulant therapy, e.g. vitamin K antagonists (VKA) or direct oral anticoagulants (DOAC) on both methods of interpretation remains to be investigated. The objective of this study was to contribute to a simplification and standardization of the LAC three-step interpretation on the level of the mixing test. Samples from 148 consecutive patients with LAC request and prolonged screening step, and 77 samples from patients non-suspicious for LAC treated with VKA (n=37) or DOAC (n=30) were retrospectively evaluated. An activated partial thromboplastin time (aPTT) and dilute Russell's viper venom time (dRVVT) were used for routine LAC testing. The supplemental anticoagulant samples were tested with dRVVT only. We focused on the interpretation differences for mixing tests expressed as CT or ICA and compared the final LAC conclusion within each distinct group of concordant and discordant mixing test results. Mixing test interpretation by CT resulted in 10 (dRVVT) and 16 (aPTT) more LAC positive patients compared to interpretation with ICA. Isolated prolonged dRVVT screen mix ICA results were exclusively observed in samples from VKA-treated patients without suspicion for LAC. We recommend using CT in respect to the 99th percentile cut-off for interpretation of mixing steps in order to reach the highest sensitivity and specificity in LAC detection.

Blood Clots That Kill: Preventing DVT | NIH MedlinePlus the Magazine

MedlinePlus

... please turn Javascript on. Feature: Deep Vein Thrombosis Blood Clots That Kill: Preventing DVT Past Issues / Spring 2011 ... Contents Deep vein thrombosis, or DVT, is a blood clot that forms in a vein deep in the ...

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

PubMed Central

2011-01-01

Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

Profound effects of cardiopulmonary bypass priming solutions on the fibrin part of clot formation: an ex vivo evaluation using rotation thromboelastometry.

PubMed

Brinkman, Arinda C M; Romijn, Johannes W A; van Barneveld, Lerau J M; Greuters, Sjoerd; Veerhoek, Dennis; Vonk, Alexander B A; Boer, Christa

2010-06-01

Dilutional coagulopathy as a consequence of cardiopulmonary bypass (CPB) system priming may also be affected by the composition of the priming solution. The direct effects of distinct priming solutions on fibrinogen, one of the foremost limiting factors during dilutional coagulopathy, have been minimally evaluated. Therefore, the authors investigated whether hemodilution with different priming solutions distinctly affects the fibrinogen-mediated step in whole blood clot formation. Prospective observational laboratory study. University hospital laboratory. Eight male healthy volunteers. Blood samples diluted with gelatin-, albumin-, or hydroxyethyl starch (HES)-based priming solutions were ex-vivo evaluated for clot formation by rotational thromboelastometry. The intrinsic pathway (INTEM) coagulation time increased from 186 +/- 19 seconds to 205 +/- 16, 220 +/- 17, and 223 +/- 18 seconds after dilution with gelatin-, albumin-, or HES-containing prime solutions (all p < 0.05 v baseline). The extrinsic pathway (EXTEM) coagulation time was only minimally affected by hemodilution. Moreover, all 3 priming solutions significantly reduced the INTEM and EXTEM maximum clot firmness. The HES-containing priming solution induced the largest decrease in the maximum clot firmness attributed to fibrinogen, from 13 +/- 1 mm (baseline) to 6 +/- 1 mm (p < 0.01 v baseline). All studied priming solutions prolonged coagulation time and decreased clot formation, but the fibrinogen-limiting effect was the most profound for the HES-containing priming solution. These results suggest that the composition of priming solutions may distinctly affect blood clot formation, in particular with respect to the fibrinogen component in hemostasis. Copyright 2010 Elsevier Inc. All rights reserved.

In vitro evaluation of clot capture efficiency of an absorbable vena cava filter.

PubMed

Dria, Stephen J; Eggers, Mitchell D

2016-10-01

The purpose of this study was to determine the in vitro clot capture efficiency (CCE) of an investigational absorbable inferior vena cava filter (IVCF) vs the Greenfield IVCF. Investigational absorbable and Greenfield filters were challenged with polyacrylamide clot surrogates ranging from 3 × 5 to 10 × 24 mm (diameter × length) in a flow loop simulating the venous system. Filters were challenged with clots until CCE standard error of 5% or less was achieved under binomial statistics. Pressure gradients across the filters were measured for the largest size clot, enabling calculation of forces on the filter. The in vitro CCE of the absorbable IVCF was statistically similar to that of the Greenfield filter for all clot sizes apart from the 3 × 10-mm clot, for which there was statistically significant difference between filter CCEs (absorbable filter, 59%; Greenfield filter, 31%; P = .0001). CCE ranged from an average 32% for the 3 × 5-mm clot to 100% for 7 × 10-mm and larger clots for the absorbable IVCF. Pressure gradient across the absorbable filter with 10 × 24-mm clot averaged 0.14 mm Hg, corresponding to a net force on the filter of 2.1 × 10(-3) N, compared with 0.39 mm Hg or 5.8 × 10(-3) N (P < .001) for the Greenfield filter. CCE of the absorbable filter was statistically similar to or an improvement on that of the Greenfield stainless steel filter for all clot sizes tested. CCE of the Greenfield filter in this study aligned with data from previous studies. Given the efficacy of the Greenfield filter in attenuating the risk of pulmonary embolism, the current study suggests that the absorbable filter may be a viable candidate for subsequent human testing. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

High-quality and -quantity DNA extraction from frozen archival blood clots for genotyping of single-nucleotide polymorphisms.

PubMed

Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke; Vogel, Ulla; Andersen, Paal Skytt

2013-06-01

The recovery of biological samples for genetic epidemiological studies can be cumbersome. Blood clots are routinely collected for serological examinations. However, the extraction of DNA from blood clots can be difficult and often results in low yields. The aim was to compare the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Serum tubes with clotted blood were stored at -20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp DNA Micro kit (Qiagen), and the Nucleospin 96 Blood kit (Macherey-Nagel). Furthermore, blood clots from 1055 inflammatory bowel disease patients were purified using the Maxwell 16 Blood purification kit (Promega). The DNA was extracted according to the manufacturers` instructions and real-time PCR and the A(260)/A(280) ratio were used to evaluate the quality of the extracted DNA. The highest DNA yield was obtained by the Maxwell 16 Blood purification kit (Promega) with a median of 4.90 μg (range 0.8-25 μg) pr 300 μL total blood. PureGene with glycogen (Qiagen) had the second highest yield with a median of 0.65 μg (range 0.5-2.6 μg) pr 300 μL total blood. The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots, even after prolonged storage. The recovered DNA served as a reliable PCR template for single-nucleotide polymorphism assays.

Antiplatelet Usage Impacts Clot Density in Acute Anterior Circulation Ischemic Stroke

PubMed Central

Pikija, Slaven; Magdic, Jozef; Lukic, Anita; Schreiber, Catharina; Mutzenbach, Johannes Sebastian; McCoy, Mark R.; Sellner, Johann

2016-01-01

We explored whether clot density in middle cerebral artery (MCA) occlusion is related to clinical variables, stroke etiology, blood constituents, and prestroke medication. We performed a retrospective chart review of patients with acute ischemic stroke of the anterior circulation admitted to two Central European stroke centers. The acquisition of non-contrast enhanced CT (NECT) and CT angiography (CTA) within 4.5 h of symptom onset was obligatory. We assessed the site of MCA occlusion as well as density, area, and length of the clot in 150 patients. The Hounsfield unit values for the clot were divided with contralateral MCA segment to yield relative Hounsfield Unit ratio (rHU). The site of the vessel occlusion (M1 vs. M2) and antiplatelet usage, but not stroke etiology, significantly influenced rHU. We found an inverse correlation of rHU with erythrocyte count (p < 0.001). The multivariate analysis revealed that a higher rHU (i.e., clot being more hyperdense) was more likely with the use of antiplatelets (OR 4.24, CI 1.10–16.31, p = 0.036). Erythrocyte (OR 0.18, CI 0.05–0.55, p = 0.003), and thrombocyte counts (OR 0.99, CI 0.98–0.99, p = 0.029) were associated with odds for more hypodense clots (lower rHU). Our study disclosed that antiplatelet therapy impacts the composition of intracranial clots of the anterior circulation. PMID:27563874

Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

PubMed

Gagaoua, Mohammed; Hoggas, Naouel; Hafid, Kahina

2015-02-01

The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

Invadopodia formation in blood clots: Not so SLUGgish after all.

PubMed

Knowles, Lynn M; Maranchie, Jodi K; Pilch, Jan

2014-01-01

Blood clotting specifically supports the metastatic dissemination of malignant cells to the lung. We have recently demonstrated that 2 tumor types that are prone to form lung metastases, renal cell carcinoma and soft tissue sarcoma, share specific adhesive mechanisms that support the invasion and colonization of blood clots in the pulmonary vasculature.

[Blood clots in erythrocyte concentrates during transfusion].

PubMed

Wagner, T; Drexler, C; Kröll, W; Jüngling, G; Lanzer, G; Gabriel, C

2008-12-01

The presence of multiple blood clots in leucocyte-depleted erythrocyte concentrates during a transfusion gave rise to an investigation to find the exact cause. Determination of the various blood group systems was carried out using the gel centrifugation method and also the polymerase chain reaction (PCR) using sequence-specific primers. In addition the human leucocyte antigens (HLA) class 1 and class 2 markers were determined with molecular biological methods. The erythrocytes in the blood bags containing the blood clots showed a mixed-field agglutination in each blood group where the donor and recipient had different phenotypes. The HLA groups, however, could be solely attributed to the patient, since during the preparation of erythrocyte concentrates all leucocytes are removed and only very few residual cells containing DNA are present. To the best of our knowledge, this is the first detailed report on blood clots from patient blood in erythrocyte concentrates, which occurred during a transfusion. The retrograde filling of the blood bag with patient blood during the transfusion led to coagulation in the bag. Therefore, careful attention must be taken when dealing with stored blood and corresponding training must be regularly carried out.

Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components.

PubMed

Rottenberger, Z; Komorowicz, E; Szabó, L; Bóta, A; Varga, Z; Machovich, R; Longstaff, C; Kolev, K

2013-03-01

Proteases expressed in atherosclerotic plaque lesions generate collagen fragments, release glycosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and expose extracellular matrix (ECM) proteins (e.g. decorin) at sites of fibrin formation. Here we address the effect of these vessel wall components on the lysis of fibrin by the tissue plasminogen activator (tPA)/plasminogen system and on the mechanical stability of clots. MMP-8-digested collagen fragments, isolated CS, DS, glycosylated decorin and its core protein were used to prepare mixed matrices with fibrin (additives present at a 50-fold lower mass concentration than fibrinogen). Scanning electron microscopy (SEM) showed that the presence of ECM components resulted in a coarse fibrin structure, most pronounced for glycosylated decorin causing an increase in the median fiber diameter from 85 to 187 nm. Rheological measurements indicated that these structural alterations were coupled to decreased shear resistance (1.8-fold lower shear stress needed for gel/fluid transition of the clots containing glycosylated decorin) and rigidity (reduction of the storage modulus from 54.3 to 33.2 Pa). The lytic susceptibility of the modified fibrin structures was increased. The time to 50% lysis by plasmin was reduced approximately 2-fold for all investigated ECM components (apart from the core protein of decorin which produced a moderate reduction of the lysis time by 25%), whereas fibrin-dependent plasminogen activation by tPA was inhibited by up to 30%. ECM components compromise the chemical and mechanical stability of fibrin as a result of changes in its ultrastructure. © 2012 International Society on Thrombosis and Haemostasis.

In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis.

PubMed

Ageitos, José Manuel; Vallejo, Juan Andrés; Serrat, Manuel; Sánchez-Pérez, Angeles; Villa, Tomás G

2013-06-01

The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.

Current status of clot dissolution therapy.

DOT National Transportation Integrated Search

1962-11-01

Clot dissolution using fibrinolytic agents is a promising approach to the treatment of thromboembolic disease, particularly peripheral venous and arterial occlusion. In their present state of development, these agents are not recommended for general ...

Activation of platelet-rich plasma using soluble type I collagen.

PubMed

Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M

2008-04-01

Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important to oral tissue healing. But application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation through the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this work, our hypothesis was that soluble type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and stimulating growth factor release from the platelets and granulocytes. PRP from human donors was clotted using type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of platelet-derived growth factor (PDGF)-AB, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor (VEGF) from both types of clots was measured over 10 days using enzyme-linked immunosorbent assasy. Clots formed using type I collagen exhibited far less retraction than those formed with bovine thrombin. Bovine thrombin and type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-beta1 during the first 5 days after activation. The use of type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF compared with currently available methods of clot activation.

ACTIVATION OF PLATELET-RICH PLASMA USING SOLUBLE TYPE I COLLAGEN

PubMed Central

Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M.

2008-01-01

PURPOSE Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important in oral tissue healing. However, application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation via the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this paper, our hypothesis was that soluble Type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and of stimulating growth factor release from the platelets and granulocytes. MATERIALS AND METHODS PRP from human donors was clotted using Type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of PDGF-AB, TGF-β1 and VEGF from both types of clots was measured over 10 days using ELISA. RESULTS Clots formed using Type I collagen had far less retraction than those formed with bovine thrombin. Bovine thrombin and Type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-β1 during the first five days after activation. CONCLUSIONS The use of Type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF when compared to currently available methods of clot activation. PMID:18355591

Thermal Blood Clot Formation and use in Microfluidic Device Valving Applications

NASA Technical Reports Server (NTRS)

Tai, Yu-Chong (Inventor); Shi, Wendian (Inventor); Guo, Luke (Inventor)

2014-01-01

The present invention provides a method of forming a blood-clot microvalve by heating blood in a capillary tube of a microfluidic device. Also described are methods of modulating liquid flow in a capillary tube by forming and removing a blood-clot microvalve.

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa.

PubMed

Sheffield, William P; Eltringham-Smith, Louise J

2011-12-20

The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α(2)-antiplasmin (α(2)AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α(2)AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α(2)AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H(6)NQEQVSPLTLLAG(4)Y (designated XL1); H(6)DQMMLPWAVTLG(4)Y (XL2); H(6)WQHKIDLPYNGAG(4)Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α(2)AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α(2)AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA. Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved

Prolonged Prothrombin Time After Recombinant Activated Factor VII Therapy in Critically Bleeding Trauma Patients Is Associated With Adverse Outcomes

DTIC Science & Technology

2010-07-01

using the FVII coagulant activity (FVII:C) assay, a one- stage assay using thromboplastin tissue factor , which quantifies FVII clotting activity in...and the resultant production of dysfunctional factors II, VII, and X. This study focused on PT specifically because this measure examines the TF...ORIGINAL ARTICLE Prolonged Prothrombin Time After Recombinant Activated Factor VII Therapy in Critically Bleeding Trauma Patients Is Associated With

Some Important Milestones in the Field of Blood Clotting.

PubMed

Doolittle, Russell F

2016-01-01

Several different kinds of 'milestone' in the field of blood coagulation are described from the middle decades of the 20th century. Although viewed from the standpoint of clotting per se, attention is also given to implications for innate immunity. The first milestone considered is the protracted saga of clotting dependence on vitamin K, an adventure that spanned more than five decades beginning in the 1920s. The second has to do with the discovery of a half-dozen 'new' clotting factors during the period immediately following World War II. A third pursues a narrower focus and examines the once mysterious transformation of fibrinogen into fibrin. Finally, the clinical treatment of classical hemophilia had a remarkable turning point in the 1960s as the result of simple but sensible measures. © 2015 S. Karger AG, Basel.

Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indication for a blood clot formation supporting process.

PubMed

Kaestner, Lars; Tabellion, Wiebke; Lipp, Peter; Bernhardt, Ingolf

2004-12-01

Prostaglandin E(2) (PGE(2)) is released from platelets when they are activated. Using fluorescence imaging and the patch-clamp technique, we provide evidence that PGE(2) at physiological concentrations (10(-10) M) activates calcium rises mediated by calcium influx through a non-selective cation-channel in human red blood cells. The extent of calcium increase varied between cells with a total of 45% of the cells responding. It is well known that calcium increases elicited the calcium-activated potassium channel (Gardos channel) in the red cell membrane. Previously, it was shown that the Gardos channel activation results in potassium efflux and shrinkage of the cells. Therefore, we conclude that the PGE(2) responses of red blood cells described here reveal a direct and active participation of erythrocytes in blood clot formation.

Googling Service Boundaries for Endovascular Clot Retrieval Hub Hospitals in a Metropolitan Setting: Proof-of-Concept Study.

PubMed

Phan, Thanh G; Beare, Richard; Chen, Jian; Clissold, Benjamin; Ly, John; Singhal, Shaloo; Ma, Henry; Srikanth, Velandai

2017-05-01

There is great interest in how endovascular clot retrieval hubs provide services to a population. We applied a computational method to objectively generate service boundaries for such endovascular clot retrieval hubs, defined by traveling time to hub. Stroke incidence data merged with population census to estimate numbers of stroke in metropolitan Melbourne, Australia. Traveling time from randomly generated addresses to 4 endovascular clot retrieval-capable hubs (Royal Melbourne Hospital [RMH], Monash Medical Center [MMC], Alfred Hospital [ALF], and Austin Hospital [AUS]) estimated using Google Map application program interface. Boundary maps generated based on traveling time at various times of day for combinations of hubs. In a 2-hub model, catchment was best distributed when RMH was paired with MMC (model 1a, RMH 1765 km 2 and MMC 1164 km 2 ) or with AUS (model 1c, RMH 1244 km 2 and AUS 1685 km 2 ), with no statistical difference between models ( P =0.20). Catchment was poorly distributed when RMH was paired with ALF (model 1b, RMH 2252 km 2 and ALF 676 km 2 ), significantly different from both models 1a and 1c (both P <0.05). Model 1a had the greatest proportion of patients arriving within ideal time of 30 minutes followed by model 1c ( P <0.001). In a 3-hub model, the combination of RMH, MMC, and AUS was superior to that of RMH, MMC, and ALF in catchment distribution and travel time. The method was also successfully applied to the city of Adelaide demonstrating wider applicability. We provide proof of concept for a novel computational method to objectively designate service boundaries for endovascular clot retrieval hubs. © 2017 American Heart Association, Inc.

Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

PubMed Central

Wang, Caroline W.; Perez, Matthew J.; Helmke, Brian P.; Viola, Francesco; Lawrence, Michael B.

2015-01-01

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties. PMID:26042775

Integration of acoustic radiation force and optical imaging for blood plasma clot stiffness measurement.

PubMed

Wang, Caroline W; Perez, Matthew J; Helmke, Brian P; Viola, Francesco; Lawrence, Michael B

2015-01-01

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood's transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Micro-elastometry on whole blood clots using actuated surface-attached posts (ASAPs).

PubMed

Judith, Robert M; Fisher, Jay K; Spero, Richard Chasen; Fiser, Briana L; Turner, Adam; Oberhardt, Bruce; Taylor, R M; Falvo, Michael R; Superfine, Richard

2015-03-07

We present a novel technology for microfluidic elastometry and demonstrate its ability to measure stiffness of blood clots as they form. A disposable micro-capillary strip draws small volumes (20 μL) of whole blood into a chamber containing a surface-mounted micropost array. The posts are magnetically actuated, thereby applying a shear stress to the blood clot. The posts' response to magnetic field changes as the blood clot forms; this response is measured by optical transmission. We show that a quasi-static model correctly predicts the torque applied to the microposts. We experimentally validate the ability of the system to measure clot stiffness by correlating our system with a commercial thromboelastograph. We conclude that actuated surface-attached post (ASAP) technology addresses a clinical need for point-of-care and small-volume elastic haemostatic assays.

Blood clots in the cumulus-oocyte complex predict poor oocyte quality and post-fertilization development.

PubMed

Ebner, T; Moser, M; Shebl, O; Sommergruber, M; Yaman, C; Tews, G

2008-06-01

Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship between the cumulus-oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices (P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage. Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo.

Effects of Chitosan Derivative N-[(2-Hydroxy-3-Trimethylammonium)Propyl]Chloride on Anticoagulant Activity of Guinea Pig Plasma.

PubMed

Drozd, N N; Shagdarova, B Ts; Il'ina, A V; Varlamov, V P

2017-07-01

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

What is this chocolate milk in my circuit? A cause of acute clotting of a continuous renal replacement circuit: Questions.

PubMed

Kakajiwala, Aadil; Chiotos, Kathleen; Brothers, Julie; Lederman, April; Amaral, Sandra

2016-12-01

One of the greatest problems associated with continuous renal replacement therapy (CRRT) is the early clotting of filters. A literature search revealed three case reports of lipemic blood causing recurrent clotting and reduced CRRT circuit survival time in adult patients, but no reports of cases in children. A 23-month-old male infant with Martinez-Frias syndrome and multivisceral transplant was admitted to the hospital with severe sepsis and hemolytic anemia. He developed acute kidney injury, fluid overload and electrolyte imbalances requiring CRRT and was also administered total parenteral nutrition (TPN) and fat emulsion. The first circuit lasted 60 h before routine change was required. The second circuit showed acute clotting after only 18 h, and brownish-milky fluid was found in the circuit tubing layered between the clotted blood. The patient's serum triglyceride levels were elevated at 988 mg/dL. The lipid infusion was stopped and CRRT restarted. Serum triglyceride levels improved to 363 mg/dL. The new circuit lasted 63 h before routine change was required. Clotting of CRRT circuits due to elevated triglyceride levels is rare and has not been reported in the pediatric population. Physicians should be mindful of this risk in patients receiving TPN who have unexpected clotting of CRRT circuits.

Reduced clot debris size using standing waves formed via high intensity focused ultrasound

NASA Astrophysics Data System (ADS)

Guo, Shifang; Du, Xuan; Wang, Xin; Lu, Shukuan; Shi, Aiwei; Xu, Shanshan; Bouakaz, Ayache; Wan, Mingxi

2017-09-01

The feasibility of utilizing high intensity focused ultrasound (HIFU) to induce thrombolysis has been demonstrated previously. However, clinical concerns still remain related to the clot debris produced via fragmentation of the original clot potentially being too large and hence occluding downstream vessels, causing hazardous emboli. This study investigates the use of standing wave fields formed via HIFU to disintegrate the thrombus while achieving a reduced clot debris size in vitro. The results showed that the average diameter of the clot debris calculated by volume percentage was smaller in the standing wave mode than in the travelling wave mode at identical ultrasound thrombolysis settings. Furthermore, the inertial cavitation dose was shown to be lower in the standing wave mode, while the estimated cavitation bubble size distribution was similar in both modes. These results show that a reduction of the clot debris size with standing waves may be attributed to the particle trapping of the acoustic potential well which contributed to particle fragmentation.

Laboratory heterogeneity of the lupus anticoagulant: a multicentre study using different clotting assays on a panel of 78 samples. Hemostasis Committee of the "Société Française de Biologie Clinique".

PubMed

1992-05-15

The laboratory heterogeneity of the lupus anticoagulant (LA) was investigated in a multicentre study using a panel of 78 plasma samples diagnosed as containing a LA. Consecutive samples were collected by 12 participants using various screening tests, and sent to 7 laboratories which performed one or more clotting assays among the following: activated partial thromboplastin time (APTT), dilute Russell viper venom time, kaolin clotting time (KCT), dilute tissue thromboplastin time (dTTI) and a platelet neutralization test. For APTT and dTTI, 10 versions of these tests including standard and mixing procedures were carried out. They varied by reagents, phospholipid concentration or methodology. Cut-off times were determined for each test by comparing the results of the panel to those of a control population. When the data of all clotting assays were pooled, 70 of the 78 selected plasmas were considered to contain LA, 15 of them having a low-titer inhibitor. Sensitivity, defined as the proportion of positive results among LA-containing plasmas, varied from 62 to 100% and was positively related to responsiveness (defined as the mean ratio of clotting time to cut-off time). Laboratory heterogeneity of LA-containing plasma was illustrated by a star symbol plot analysis. Different populations of samples, with LA preferentially recognized by one assay (or group of assays) irrespective of the overall sensitivity of this assay, were identified. Multiple component analysis demonstrated the heterogeneity of low-titer inhibitors, which complicates their recognition in routine laboratory investigation.

Mechanical properties and fibrin characteristics of endovascular coil–clot complexes: relevance to endovascular cerebral aneurysm repair paradigms

PubMed Central

Haworth, Kevin J; Weidner, Christopher R; Abruzzo, Todd A; Shearn, Jason T; Holland, Christy K

2015-01-01

Background Although coil embolization is known to prevent rebleeding from acutely ruptured cerebral aneurysms, the underlying biological and mechanical mechanisms have not been characterized. We sought to determine if microcoil-dependent interactions with thrombus induce structural and mechanical changes in the adjacent fibrin network. Such changes could play an important role in the prevention of aneurysm rebleeding. Methods The stiffness of in vitro human blood clots and coil–clot complexes implanted into aneurysm phantoms were measured immediately after formation and after retraction for 3 days using unconfined uniaxial compression assays. Scanning electron microscopy of the coil–clot complexes showed the effect of coiling on clot structure. Results The coil packing densities achieved were in the range of clinical practice. Bare platinum coils increased clot stiffness relative to clot alone (Young’s modulus 6.9 kPa and 0.83 kPa, respectively) but did not affect fibrin structure. Hydrogel-coated coils prevented formation of a clot and had no significant effect on clot stiffness (Young’s modulus 2 kPa) relative to clot alone. Clot age decreased fiber density by 0.2 fibers/µm2 but not the stiffness of the bare platinum coil–clot complex. Conclusions The stiffness of coil–clot complexes is related to the summative stiffness of the fibrin network and associated microcoils. Hydrogel-coated coils exhibit significantly less stiffness due to the mechanical properties of the hydrogel and the inhibition of fibrin network formation by the hydrogel. These findings have important implications for the design and engineering of aneurysm occlusion devices. PMID:24668257

Clot retraction affects the extent of ultrasound-enhanced thrombolysis in an ex vivo porcine thrombosis model

PubMed Central

Sutton, Jonathan T.; Ivancevich, Nikolas M.; Perrin, Stephen R.; Vela, Deborah C.; Holland, Christy K.

2013-01-01

Using an FDA-approved contrast agent (Definity®) and thrombolytic drug (rt-PA), we investigated ultrasound-enhanced thrombolysis in two whole-blood clot models. Porcine venous blood was collected from donor hogs and coagulated in two different materials. This method produced clots with differing compositional properties, as determined by routine scanning electron microscopy and histology. Clots were deployed in an ex vivo porcine thrombolysis model, while an intermittent ultrasound scheme previously developed to maximize stable cavitation was applied and acoustic emissions were detected. Exposure of clots to 3.15 μg/mL rt-PA promoted lysis in both clot models, compared to exposure to plasma alone. However, in the presence of rt-PA, Definity®, and ultrasound, only unretracted clots experienced significant enhancement of thrombolysis compared to treatment with rt-PA. In these clots, microscopy studies revealed loose erythrocyte aggregates, a significantly less extensive fibrin network, and a higher porosity, which may facilitate increase penetration of thrombolytics by cavitation. PMID:23453629

Blood Clotting Inspired Polymer Physics

NASA Astrophysics Data System (ADS)

Sing, Charles Edward

The blood clotting process is one of the human body's masterpieces in targeted molecular manipulation, as it requires the activation of the clotting cascade at a specific place and a specific time. Recent research in the biological sciences have discovered that one of the protein molecules involved in the initial stages of the clotting response, von Willebrand Factor (vWF), exhibits counterintuitive and technologically useful properties that are driven in part by the physical environment in the bloodstream at the site of a wound. In this thesis, we take inspiration from initial observations of the vWF in experiments, and aim to describe the behaviors observed in this process within the context of polymer physics. By understanding these physical principles, we hope to harness nature's ability to both direct molecules in both spatial and conformational coordinates. This thesis is presented in three complementary sections. After an initial introduction describing the systems of interest, we first describe the behavior of collapsed Lennard-Jones polymers in the presence of an infinite medium. It has been shown that simple bead-spring homopolymer models describe vWF quite well in vitro. We build upon this previous work to first describe the behavior of a collapsed homopolymer in an elongational fluid flow. Through a nucleation-protrusion mechanism, scaling relationships can be developed to provide a clear picture of a first-order globule-stretch transition and its ramifications in dilute-solution rheology. The implications of this behavior and its relation to the current literature provides qualitative explanations for the physiological process of vasoconstriction. In an effort to generalize these observations, we present an entire theory on the behavior of polymer globules under influence of any local fluid flow. Finally, we investigate the internal dynamics of these globules by probing their pulling response in an analogous fashion to force spectroscopy. We elucidate

Adhesion of Blood Clots Can Be Enhanced When Copolymerized with a Macromer That Is Crosslinked by Coagulation Factor XIIIa.

PubMed

Chan, Karen Y T; Zhao, Chunyi; Siren, Erika M J; Chan, Jeanne C Y; Boschman, Jeffrey; Kastrup, Christian J

2016-06-13

The adhesion of blood clots to blood vessels, such as through the adhesion of fibrin, is essential in hemostasis. While numerous strategies for initiating clot formation and preventing clot lysis are being developed to create improved hemostatic agents, strategies for enhancing clot adhesion have not been widely explored. Here, we show that adhesion of blood clots can be increased by adding a previously characterized synthetic polymer that is crosslinked by coagulation factor XIIIa during clotting. Addition of the polymer to normal plasma increased the adhesive strength of clots by 2-fold. It also recovered the adhesive strength of nonadhesive fibrinogen-deficient whole blood clots from <0.06 kPa to 1.9 ± 0.14 kPa, which is similar to the adhesive strength of a fibrinogen-rich clot (1.8 ± 0.64 kPa). The polymer also enabled plasma clots to remain adhered under fibrinolytic conditions. By demonstrating that the adhesive strength of clots can be increased with a synthetic material, this provides a potential strategy for creating advanced hemostatic materials, such as treatments for fibrinogen deficiency in trauma-induced coagulopathy.

Activation of platelet-rich plasma using thrombin receptor agonist peptide.

PubMed

Landesberg, Regina; Burke, Andrea; Pinsky, David; Katz, Ronald; Vo, Jennifer; Eisig, Sidney B; Lu, Helen H

2005-04-01

This study proposes an alternative preparation method of platelet-rich plasma (PRP). Specifically, we compare the use of thrombin receptor agonist peptide-6 (TRAP) and bovine thrombin as a clotting agent in the preparation of PRP. PRP was prepared by centrifugation and clotted with thrombin or TRAP. In vitro clotting times were monitored as a function of TRAP concentration, and clot retraction was determined by measuring clot diameter over time. Following the optimization of TRAP concentration, experiments were repeated with the addition of several commercially available bone substitutes. The release of PRP-relevant growth factors as a function of PRP preparation was also determined. The most rapid polymerization of PRP takes place with the addition of thrombin, followed by TRAP/Allogro (Ceramed, Lakewood, CO), TRAP/BioGlass (Mo-Sci, Rolla, MN), TRAP/BioOss (Osteohealth, Shirley, NY), and TRAP alone. Thrombin caused considerable clot retraction (43%), whereas TRAP alone resulted in only 15% retraction. TRAP/Allogro, TRAP/BioOss, and TRAP/BioGlass all exhibited minimal retraction (8%). The use of TRAP to activate clot formation in the preparation of PRP may be a safe alternative to bovine thrombin. It results in an excellent working time and significantly less clot retraction than the currently available methods of PRP production.

In Black South Africans from Rural and Urban Communities, the 4G/5G PAI-1 Polymorphism Influences PAI-1 Activity, but Not Plasma Clot Lysis Time

PubMed Central

de Lange, Zelda; Rijken, Dingeman C.; Hoekstra, Tiny; Conradie, Karin R.; Jerling, Johann C.; Pieters, Marlien

2013-01-01

Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT. PMID:24386152

In black South Africans from rural and urban communities, the 4G/5G PAI-1 polymorphism influences PAI-1 activity, but not plasma clot lysis time.

PubMed

de Lange, Zelda; Rijken, Dingeman C; Hoekstra, Tiny; Conradie, Karin R; Jerling, Johann C; Pieters, Marlien

2013-01-01

Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT.

A Fictitious Domain Method for Resolving the Interaction of Blood Flow with Clot Growth

NASA Astrophysics Data System (ADS)

Mukherjee, Debanjan; Shadden, Shawn

2016-11-01

Thrombosis and thrombo-embolism cause a range of diseases including heart attack and stroke. Closer understanding of clot and blood flow mechanics provides valuable insights on the etiology, diagnosis, and treatment of thrombotic diseases. Such mechanics are complicated, however, by the discrete and multi-scale phenomena underlying thrombosis, and the complex interactions of unsteady, pulsatile hemodynamics with a clot of arbitrary shape and microstructure. We have developed a computational technique, based on a fictitious domain based finite element method, to study these interactions. The method can resolve arbitrary clot geometries, and dynamically couple fluid flow with static or growing clot boundaries. Macroscopic thrombus-hemodynamics interactions were investigated within idealized vessel geometries representative of the common carotid artery, with realistic unsteady flow profiles as inputs. The method was also employed successfully to resolve micro-scale interactions using a model driven by in-vivo morphology data. The results provide insights into the flow structures and hemodynamic loading around an arbitrarily grown clot at arterial length-scales, as well as flow and transport within the interstices of platelet aggregates composing the clot. The work was supported by AHA Award No: 16POST27500023.

Artificial Stroke Clots: How Wide is the Gap to the Real World?

PubMed

Berndt, Maria; Prothmann, Sascha; Maegerlein, Christian; Oberdieck, Paul; Zimmer, Claus; Hegge, Barbara; Pelisek, Jaroslav; Schirmer, Lucas; Poppert, Holger; Boeckh-Behrens, Tobias

2018-02-01

Especially since the establishment of mechanical thrombectomy as part of standard stroke therapy, artificial thrombi have become important in the training of interventionalists as well as for the development and testing of devices. So far, these in vitro clots have lacked direct comparisons with ex vivo thrombi. We therefore compared the histologic appearance of dynamically produced clots with that of stroke thrombi acquired during mechanical recanalization. Thrombi of 145 consecutive patients with stroke with large-vessel occlusions were histologically compared with 10 artificial clots, dynamically created from human blood and pig's blood using a Chandler loop system. Quantified FP/RBC ratios (fibrin/platelets divided by red blood cell fraction) and white blood cell (WBC) fractions were identified and compared between artificial (human and pig) and ex vivo thrombi obtained from patients with various stroke causes. There were no significant differences in the analysis of FP/RBC ratios between artificial thrombi and ex vivo thrombi (P = 0.42) or in the more precise analyses considering etiologic subgroups. Distinct differences were observed for the WBC fraction, with lower values in artificial thrombi (median, 1.34%) than in ex vivo thrombi (median, 5%) (P < 0.001). The main clot components, FP and RBC, are presumably the most influential factors for clot stability and mechanical resistance. Similarities between artificially generated and ex vivo stroke clots (and when considering different stroke subgroups) support the usefulness of these artificial thrombi in the pre-evaluation of thrombus extraction devices and as appropriate training material. Copyright © 2017 Elsevier Inc. All rights reserved.

Histologic Analysis of Retrieved Clots in Acute Ischemic Stroke: Correlation with Stroke Etiology and Gradient-Echo MRI.

PubMed

Kim, S K; Yoon, W; Kim, T S; Kim, H S; Heo, T W; Park, M S

2015-09-01

It is unclear whether clot composition analysis is helpful to predict a stroke mechanism in acute large vessel occlusion. In addition, the relationship between early vessel signs on imaging studies and clot compositions has been poorly understood. The purpose of this study was to elucidate the relationship between clot composition and stroke etiology following mechanical thrombectomy and to investigate the effect of varied clot compositions on gradient-echo MR imaging of clots. Histopathologic analysis of retrieved clots from 37 patients with acute MCA occlusion was performed. Patients underwent gradient-echo imaging before endovascular therapy. Retrieved clots underwent semiquantitative proportion analysis to quantify red blood cells, fibrin, platelets, and white blood cells by area. Correlations between clot compositions and stroke subtypes and susceptibility vessel signs on gradient-echo imaging were assessed. Stroke etiology was classified as cardioembolism in 22 patients (59.4%), large-artery atherosclerosis in 8 (21.6%), and undetermined in 7 (18.9%). The clots from cardioembolism had a significantly higher proportion of red blood cells (37.8% versus 16.9%, P = .031) and a lower proportion of fibrin (32.3% versus 48.5%, P = .044) compared with those from large-artery atherosclerosis. The proportion of red blood cells was significantly higher in clots with a susceptibility vessel sign than in those without it (48.0% versus 1.9%, P < .001), whereas the proportions of fibrin (26.4% versus 57.0%, P < .001) and platelets (22.6% versus 36.9%, P = .011) were significantly higher in clots without a susceptibility vessel sign than those with it. The histologic composition of clots retrieved from cerebral arteries in patients with acute stroke differs between those with cardioembolism and large-artery atherosclerosis. In addition, a susceptibility vessel sign on gradient-echo imaging is strongly associated with a high proportion of red blood cells and a low proportion

Ultrastructure of hybrid chitosan-glycerol phosphate blood clots by environmental scanning electron microscopy.

PubMed

Iliescu, M; Hoemann, C D; Shive, M S; Chenite, A; Buschmann, M D

2008-03-01

Chitosan-based polymers have been extensively studied for biomedical applications. Recently, liquid solutions of chitosan in a glycerol phosphate buffer (chitosan-GP) with physiological pH and osmolality were mixed with autologous blood to form hybrid chitosan-GP/blood implants that improved the repair of articular cartilage lesions in a large animal model. The mixture of chitosan-GP and blood forms a viscous liquid, which solidifies in minutes via normal blood coagulation as well as chitosan-mediated mechanisms. Here we have examined the ultrastructure of these chitosan-GP/blood clots as well as regular blood clots and chitosan-GP gels, the latter produced by heating. Both unfixed and fixed samples of chitosan-GP/blood clots, regular blood clots, and chitosan-GP gels were investigated by environmental scanning electron microscopy (ESEM) in conjunction with energy dispersive X-ray spectrometry (EDS), the former permitting direct observation of the ultrastructure in hydrated conditions simulating the natural state. By examination of unfixed specimens using ESEM we found that chitosan formed a network structure in both chitosan-GP gels and chitosan-GP/blood clots; however this structure was altered by aldehyde fixation to produce artifactual aggregates of chitosan microparticles. We were also able to identify chitosan in chitosan-GP/blood clots by washing samples in low concentration NaCl solutions followed by local EDS analyses to identify excess chloride versus sodium, and thus presence of cationic chitosan in analyzed features. Additional results indicated that the majority of glycerol phosphate diffuses freely from chitosan-GP gels (by EDS of phosphorus) and that hyperosmotic paraformaldehyde-based fixatives (i.e. 4% w/v) significantly disturb erythrocyte morphology in fixed whole blood clots. (c) 2007 Wiley-Liss, Inc.

An optical approach for non-invasive blood clot testing

NASA Astrophysics Data System (ADS)

Kalchenko, Vyacheslav; Brill, Alexander; Fine, Ilya; Harmelin, Alon

2007-02-01

Physiological blood coagulation is an essential biological process. Current tests for plasma coagulation (clotting) need to be performed ex vivo and require fresh blood sampling for every test. A recently published work describes a new, noninvasive, in vivo approach to assess blood coagulation status during mechanical occlusion1. For this purpose, we have tested this approach and applied a controlled laser beam to blood micro-vessels of the mouse ear during mechanical occlusion. Standard setup for intravital transillumination videomicroscopy and laser based imaging techniques were used for monitoring the blood clotting process. Temporal mechanical occlusion of blood vessels in the observed area was applied to ensure blood flow cessation. Subsequently, laser irradiation was used to induce vascular micro-injury. Changes in the vessel wall, as well as in the pattern of blood flow, predispose the area to vascular thrombosis, according to the paradigm of Virchow's triad. In our experiments, two elements of Virchow's triad were used to induce the process of clotting in vivo, and to assess it optically. We identified several parameters that can serve as markers of the blood clotting process in vivo. These include changes in light absorption in the area of illumination, as well as changes in the pattern of the red blood cells' micro-movement in the vessels where blood flow is completely arrested. Thus, our results indicate that blood coagulation status can be characterized by non-invasive, in vivo methodologies.

Numerical investigation into blood clotting at the bone-dental implant interface in the presence of an electrical stimulus.

PubMed

Vanegas-Acosta, J C; Garzón-Alvarado, D A; Lancellotti, V

2013-12-01

The insertion of a dental implant activates a sequence of wound healing events ending with bone formation and implant osseointegration. This sequence starts with the blood coagulation process and the formation of a fibrin network that detains spilt blood. Fibrin formation can be simplified as the kinetic reaction between thrombin and fibrinogen preceding the conversion of fibrinogen into fibrin. Based on experimental observations of the electrical properties of these molecules, we present a hypothesis for the mechanism of a static electrical stimulus in controlling the formation of the blood clot. Specifically, the electrical stimulus increases the fibrin network formation in such a way that a preferential region of higher fibrin density is obtained. This hypothesis is validated by means of a numerical model for the blood clot formation at the bone-dental implant interface. Numerical results compare favorably to experimental observations for blood clotting with and without the static electrical stimulus. It is concluded that the density of the fibrin network depends on the strength of the static electrical stimulus, and that the blood clot formation has a preferential direction of formation in the presence of the electrical signal. © 2013 Published by Elsevier Ltd. All rights reserved.

Clot accumulation characteristics of plasminogen-bearing liposomes in a flow-system. Groningen Utrecht Institute for Drug Exploration.

PubMed

Heeremans, J L; Prevost, R; Feitsma, H; Kluft, C; Crommelin, D J

1998-01-01

In this study, the clot accumulation properties of liposome-coupled plasminogen were compared to those of free (non-liposomal) plasminogen in an in vitro, closed-loop, flow-system. After introduction of a clot into the closed system, double-radiolabelled plasminogen-liposomes were administered and the accumulation of radiolabel on the entire clot was measured. Liposomal plasminogen showed improved accumulation over free plasminogen, on both a fibrin clot and a whole blood clot. Moreover, once liposomal plasminogen was fibrin associated, it could not be washed away with buffer, in contrast to free plasminogen. Liposomal plasminogen was able to compete successfully with an excess of free plasminogen. The plateau levels for the accumulated amount of plasminogen depended on the incubated amount of plasminogen and were influenced by partial degradation of the clot. Furthermore, it was shown that a threshold liposomal plasminogen surface-density was needed for optimum clot accumulation.

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions... under § 410.10, subject to the specified conditions: (a) Hepatitis B vaccine: Conditions. Effective...

Deep Vein Thrombosis (DVT) / Pulmonary Embolism (PE) - Blood Clot Forming in a Vein

MedlinePlus

... Controls Cancel Submit Search the CDC Venous Thromboembolism (Blood Clots) Note: Javascript is disabled or is not supported ... Challenge HA-VTE Data & Statistics HA-VTE Resources Blood Clots and Travel Research and Treatment Centers Data & Statistics ...

The Effect of a Simulated Commercial Flight Environment with Hypoxia and Low Humidity on Clotting, Platelet, and Endothelial Function in Participants with Type 2 Diabetes - A Cross-over Study.

PubMed

Konya, Judit; Al Qaissi, Ahmed; Sathyapalan, Thozhukat; Ajjan, Ramzi; Madden, Leigh; Naseem, Khalid M; Garrett, Andrew Thomas; Kilpatrick, Eric; Atkin, Stephen L

2018-01-01

To determine if clotting, platelet, and endothelial function were affected by simulated short-haul commercial air flight conditions (SF) in participants with type 2 diabetes (T2DM) compared to controls. 10 participants with T2DM (7 females, 3 males) and 10 controls (3 females, 7 males) completed the study. Participants were randomized to either spend 2 h in an environmental chamber at sea level conditions (temperature: 23°C, oxygen concentration 21%, humidity 45%), or subject to a simulated 2-h simulated flight (SF: temperature: 23°C, oxygen concentration 15%, humidity 15%), and crossed over 7 days later. Main outcome measures: clot formation and clot lysis parameters, functional platelet activation markers, and endothelial function measured by reactive hyperemia index (RHI) by EndoPAT and serum microparticles. Comparing baseline with SF conditions, clot maximal absorption was increased in controls (0.375 ± 0.05 vs. 0.39 ± 0.05, p  < 0.05) and participants with T2DM (0.378 ± 0.089 vs. 0.397 ± 0.089, p  < 0.01), while increased basal platelet activation for both fibrinogen binding and P-selectin expression ( p  < 0.05) was seen in participants with T2DM. Parameters of clot formation and clot lysis, stimulated platelet function (stimulated platelet response to ADP and sensitivity to prostacyclin), and endothelial function were unchanged. While SF resulted in the potential of denser clot formation with enhanced basal platelet activation in T2DM, the dynamic clotting, platelet, and endothelial markers were not affected, suggesting that short-haul commercial flying adds no additional hazard for venous thromboembolism for participants with T2DM compared to controls.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526.

PubMed

Hang, Feng; Liu, Peiyi; Wang, Qinbo; Han, Jin; Wu, Zhengjun; Gao, Caixia; Liu, Zhenmin; Zhang, Hao; Chen, Wei

2016-01-12

Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL(-1)) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis.

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 2 2011-10-01 2011-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions. Notwithstanding the exclusion from coverage of vaccines (see § 405.310 of this chapter) and self-administered...

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 2 2014-10-01 2014-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions. Notwithstanding the exclusion from coverage of vaccines (see § 405.310 of this chapter) and self-administered...

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 2 2013-10-01 2013-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions. Notwithstanding the exclusion from coverage of vaccines (see § 405.310 of this chapter) and self-administered...

42 CFR 410.63 - Hepatitis B vaccine and blood clotting factors: Conditions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 2 2012-10-01 2012-10-01 false Hepatitis B vaccine and blood clotting factors... Other Health Services § 410.63 Hepatitis B vaccine and blood clotting factors: Conditions. Notwithstanding the exclusion from coverage of vaccines (see § 405.310 of this chapter) and self-administered...

Pericellular plasma clot negates the influence of scaffold stiffness on chondrogenic differentiation.

PubMed

Arora, Aditya; Kothari, Anjaney; Katti, Dhirendra S

2016-12-01

Matrix stiffness is known to play a pivotal role in cellular differentiation. Studies have shown that soft scaffolds (<2-3kPa) promote cellular aggregation and chondrogenesis, whereas, stiffer ones (>10kPa) show poor chondrogenesis in vitro. In this work we investigated if fibrin matrix from clotted blood can act as a soft surrogate which nullifies the influence of the underlying stiff scaffold, thus promoting chondrogenesis irrespective of bulk scale scaffold stiffness. For this we performed in vitro chondrogenesis on soft (∼1.5kPa) and stiff (∼40kPa) gelatin scaffolds in the presence and absence of pericellular plasma clot. Our results demonstrated that in absence of pericellular plasma clot, chondrocytes showed efficient condensation and cartilaginous matrix secretion only on soft scaffolds, whereas, in presence of pericellular plasma clot, cell rounding and cartilaginous matrix secretion was observed in both soft and stiff scaffolds. More specifically, significantly higher collagen II, chondroitin sulfate and aggrecan deposition was observed in soft scaffolds, and soft and stiff scaffolds with pericellular plasma clot as compared to stiff scaffolds without pericellular plasma clot. Moreover, collagen type I, a fibrocartilage/bone marker was significantly higher only in stiff scaffolds without plasma clot. Therefore, it can be concluded that chondrocytes surrounded by a soft fibrin network were unable to sense the stiffness of the underlying scaffold/substrate and hence facilitate chondrogenesis even on stiff scaffolds. This understanding can have significant implications in the design of scaffolds for cartilage tissue engineering. Cell fate is influenced by the mechanical properties of cell culture substrates. Outside the body, cartilage progenitor cells express significant amounts of cartilage-specific markers on soft scaffolds but not on stiff scaffolds. However, when implanted in joints, stiff scaffolds show equivalent expression of markers as seen in

Successful removal of endobronchial blood clots using bronchoscopic cryotherapy at bedside in the intensive care unit.

PubMed

Lee, Hongyeul; Leem, Cho Sun; Lee, Jae Ho; Lee, Choon-Taek; Cho, Young-Jae

2014-10-01

Acute airway obstruction after hemoptysis occurs due to the presence of blood clots. These conditions may result in life-threatening ventilation impairment. We report a case of obstruction of the large airway by endobronchial blood clots which were removed using bronchoscopic cryotherapy at the bedside of intensive care unit. A 66-year-old female with endometrial cancer who had undergone chemotherapy, was admitted to the intensive care unit due to neutropenic fever. During mechanical ventilation, the minute ventilation dropped to inadequately low levels and chest radiography showed complete opacification of the left hemithorax. Flexible bronchoscopy revealed large blood clots obstructing the proximal left main bronchus. After unsuccessful attempts to remove the clots with bronchial lavage and forceps extraction, blood clots were removed using bronchoscopic cryotherapy. This report shows that cryotherapy via flexible bronchoscopy at the bedside in the intensive of intensive care unit is a simple and effective alternative for the removal of endobronchial blood clots.

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

PubMed

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Emergency endoscopic variceal ligation in cirrhotic patients with blood clots in the stomach but no active bleeding or stigmata increases the risk of rebleeding.

PubMed

Kim, Su Jin; Choi, Cheol Woong; Kang, Dae Hwan; Kim, Hyung Wook; Park, Su Bum; Hong, Young Mi; Yoon, Ki Tae; Cho, Mong; Nam, Hyung Seok; Islam, Sm Bakhtiar Ui

2016-12-01

This study aimed to evaluate the efficacy and safety of emergency variceal ligation for the prevention of rebleeding in cirrhotic patients who are found on initial endoscopy to have blood clots in the stomach but no actively bleeding esophageal and gastric varices or stigmata. This study included 28 cirrhotic patients who underwent emergency prophylactic EVL and 41 who underwent an elective intervention between January 2009 and June 2014. Clinical outcomes were analyzed, including the rebleeding, 6-week mortality, and rebleeding-free survival rates. The rebleeding rate was higher in the emergency than in the elective group (28.6% vs. 7.3%, P =0.041). Multivariate analysis showed that emergency prophylactic EVL (odds ratio [OR] = 7.4, 95% confidence interval [CI]=1.634.8, P =0.012) and Child-Pugh score C (OR=10.6, 95% CI=1.4-80.8, P =0.022) were associated with rebleeding. In the emergency group, the gastric varices were associated with rebleeding (OR=12.0, 95% CI=1.7-83.5, P =0.012). Emergency EVL may be associated with variceal rebleeding when blood clots are present in the stomach without active esophageal and gastric variceal bleeding or stigmata. Elective intervention should be considered as a safer strategy for preventing variceal rebleeding in this situation.

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

PubMed

Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S

2009-05-04

To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Utility of single-energy and dual-energy computed tomography in clot characterization: An in-vitro study

PubMed Central

Michalak, Gregory; Kadirvel, Ramanathan; Dai, Daying; Gilvarry, Michael; Duffy, Sharon; Kallmes, David F; McCollough, Cynthia; Leng, Shuai

2017-01-01

Background and purpose Because computed tomography (CT) is the most commonly used imaging modality for the evaluation of acute ischemic stroke patients, developing CT-based techniques for improving clot characterization could prove useful. The purpose of this in-vitro study was to determine which single-energy or dual-energy CT techniques provided optimum discrimination between red blood cell (RBC) and fibrin-rich clots. Materials and methods Seven clot types with varying fibrin and RBC densities were made (90% RBC, 99% RBC, 63% RBC, 36% RBC, 18% RBC and 0% RBC with high and low fibrin density) and their composition was verified histologically. Ten of each clot type were created and scanned with a second generation dual source scanner using three single (80 kV, 100 kV, 120 kV) and two dual-energy protocols (80/Sn 140 kV and 100/Sn 140 kV). A region of interest (ROI) was placed over each clot and mean attenuation was measured. Receiver operating characteristic curves were calculated at each energy level to determine the accuracy at differentiating RBC-rich clots from fibrin-rich clots. Results Clot attenuation increased with RBC content at all energy levels. Single-energy at 80 kV and 120 kV and dual-energy 80/Sn 140 kV protocols allowed for distinguishing between all clot types, with the exception of 36% RBC and 18% RBC. On receiver operating characteristic curve analysis, the 80/Sn 140 kV dual-energy protocol had the highest area under the curve for distinguishing between fibrin-rich and RBC-rich clots (area under the curve 0.99). Conclusions Dual-energy CT with 80/Sn 140 kV had the highest accuracy for differentiating RBC-rich and fibrin-rich in-vitro thrombi. Further studies are needed to study the utility of non-contrast dual-energy CT in thrombus characterization in acute ischemic stroke. PMID:28604189

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

PubMed

Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C

2017-01-01

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

Correlation between clot density and recanalization success or stroke etiology in acute ischemic stroke patients

PubMed Central

Kallmes, David F; Brinjikji, Waleed

2017-01-01

Background Predicting recanalization success for patients undergoing endovascular treatment for acute ischemic stroke is of significant interest. Studies have previously correlated the success of recanalization with the density of the clot. We evaluated clot density and its relationship to revascularization success and stroke etiology. Methods We conducted a retrospective review of 118 patients undergoing intra-arterial therapy for acute ischemic stroke. Mean and maximum thrombus density was measured by drawing a circular region of interest on an axial slice of a non-contrast computed tomography scan. T-tests were used to compare clot density to recanalization success or to stroke etiology, namely large artery atherosclerosis and cardioembolism. Recanalization success was compared in four device groups: aspiration, stent retriever, aspiration and stent retriever, and all other. Results There was no significant difference in the mean clot density in patients with successful (n = 80) versus unsuccessful recanalization (n = 38, 50.1 ± 7.4 Hounsfield unit (HU) vs. 53 ± 12.7 HU; P = 0.17). Comparing the large artery thromboembolism (n = 35) to the cardioembolic etiology group (n = 56), there was no significant difference in mean clot density (51.5 ± 7.7 HU vs. 49.7 ± 8.5 HU; P = 0.31). A subgroup analysis of middle cerebral artery occlusions (n = 65) showed similar, non-statistically significant differences between groups. There was no difference in the rate of recanalization success in patients with a mean clot density greater than 50 HU or less than 50 HU in each of the four device groups. Conclusions There was no relationship between clot density and revascularization success or stroke etiology in our study. More research is needed to determine if clot density can predict recanalization rates or indicate etiology. PMID:28604188

21 CFR 173.150 - Milk-clotting enzymes, microbial.

Code of Federal Regulations, 2010 CFR

2010-04-01

.... Milk-clotting enzyme produced by pure-culture fermentation process may be safely used in the production... from one of the following organisms by a pure-culture fermentation process: (1) Endothia parasitica...

Phase transitions during compression and decompression of clots from platelet-poor plasma, platelet-rich plasma and whole blood.

PubMed

Liang, Xiaojun; Chernysh, Irina; Purohit, Prashant K; Weisel, John W

2017-09-15

Blood clots are required to stem bleeding and are subject to a variety of stresses, but they can also block blood vessels and cause heart attacks and ischemic strokes. We measured the compressive response of human platelet-poor plasma (PPP) clots, platelet-rich plasma (PRP) clots and whole blood clots and correlated these measurements with confocal and scanning electron microscopy to track changes in clot structure. Stress-strain curves revealed four characteristic regions, for compression-decompression: (1) linear elastic region; (2) upper plateau or softening region; (3) non-linear elastic region or re-stretching of the network; (4) lower plateau in which dissociation of some newly made connections occurs. Our experiments revealed that compression proceeds by the passage of a phase boundary through the clot separating rarefied and densified phases. This observation motivates a model of fibrin mechanics based on the continuum theory of phase transitions, which accounts for the pre-stress caused by platelets, the adhesion of fibrin fibers in the densified phase, the compression of red blood cells (RBCs), and the pumping of liquids through the clot during compression/decompression. Our experiments and theory provide insights into the mechanical behavior of blood clots that could have implications clinically and in the design of fibrin-based biomaterials. The objective of this paper is to measure and mathematically model the compression behavior of various human blood clots. We show by a combination of confocal and scanning electron microscopy that compression proceeds by the passage of a front through the sample that separates a densified region of the clot from a rarefied region, and that the compression/decompression response is reversible with hysteresis. These observations form the basis of a model for the compression response of clots based on the continuum theory of phase transitions. Our studies may reveal how clot rheology under large compression in vivo due

A Joint Model for Vitamin K-Dependent Clotting Factors and Anticoagulation Proteins.

PubMed

Ooi, Qing Xi; Wright, Daniel F B; Tait, R Campbell; Isbister, Geoffrey K; Duffull, Stephen B

2017-12-01

Warfarin acts by inhibiting the reduction of vitamin K (VK) to its active form, thereby decreasing the production of VK-dependent coagulation proteins. The aim of this research is to develop a joint model for the VK-dependent clotting factors II, VII, IX and X, and the anticoagulation proteins, proteins C and S, during warfarin initiation. Data from 18 patients with atrial fibrillation who had warfarin therapy initiated were available for analysis. Nine blood samples were collected from each subject at baseline, and at 1-5, 8, 15 and 29 days after warfarin initiation and assayed for factors II, VII, IX and X, and proteins C and S. Warfarin concentration-time data were not available. The coagulation proteins data were modelled in a stepwise manner using NONMEM ® Version 7.2. In the first stage, each of the coagulation proteins was modelled independently using a kinetic-pharmacodynamic model. In the subsequent step, the six kinetic-pharmacodynamic models were combined into a single joint model. One patient was administered VK and was excluded from the analysis. Each kinetic-pharmacodynamic model consisted of two parts: (1) a common one-compartment pharmacokinetic model with first-order absorption and elimination for warfarin; and (2) an inhibitory E max model linked to a turnover model for coagulation proteins. In the joint model, an unexpected pharmacodynamic lag was identified and the estimated degradation half-life of VK-dependent coagulation proteins were in agreement with previously published values. The model provided an adequate fit to the observed data. The joint model represents the first work to quantify the influence of warfarin on all six VK-dependent coagulation proteins simultaneously. Future work will expand the model to predict the influence of exogenously administered VK on the time course of clotting factor concentrations after warfarin overdose and during perioperative warfarin reversal procedures.

Possible benefits of catheters with lateral holes in coronary thrombus aspiration: a computational study for different clot viscosities and vacuum pressures.

PubMed

Soleimani, Sajjad; Dubini, Gabriele; Pennati, Giancarlo

2014-10-01

According to a number of clinical studies, coronary aspiration catheters are useful tools to remove a thrombus (blood clot) blocking a coronary artery. However, these thrombectomy devices may fail to remove the blood clot entirely. Few studies have been devoted to a systematic analysis of factors affecting clot aspiration. The geometric characteristics of the aspiration catheter, the physical properties of the thrombus, and the applied vacuum pressure are crucial parameters. In this study, the aspiration of a blood clot blocking a coronary bifurcation is computationally simulated. The clot is modeled as a highly viscous fluid, and a two-phase (blood and clot) problem is solved. The effects of geometric variations in the tip of the coronary catheter, including lateral hole size and location, are investigated considering different aspiration pressures and clot viscosities. A Bird-Carreau model is adopted for blood viscosity, while a power law model is used to describe the clot rheology. Computational results for blood clot aspiration show that the presence of holes in the lateral part of the tip of the catheter can be beneficial depending on clot viscosity, hole features, and applied aspiration pressure. In general, the holes are beneficial when the clot viscosity is low, while aspiration catheters without any extra lateral holes exhibit better performance for higher clot viscosity. However, when higher aspiration pressures are applied, the catheters tend to behave relatively similarly in removing clots with various viscosities, reducing the role of the clot viscosity. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 3 2013-01-01 2013-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 3 2014-01-01 2014-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 3 2012-01-01 2012-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

7 CFR 58.436 - Rennet, pepsin, other milk clotting enzymes and flavor enzymes.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 3 2011-01-01 2011-01-01 false Rennet, pepsin, other milk clotting enzymes and flavor enzymes. 58.436 Section 58.436 Agriculture Regulations of the Department of Agriculture (Continued... clotting enzymes and flavor enzymes. Enzyme preparations used in the manufacture of cheese shall be safe...

Building and validation of a prognostic model for predicting extracorporeal circuit clotting in patients with continuous renal replacement therapy.

PubMed

Fu, Xia; Liang, Xinling; Song, Li; Huang, Huigen; Wang, Jing; Chen, Yuanhan; Zhang, Li; Quan, Zilin; Shi, Wei

2014-04-01

To develop a predictive model for circuit clotting in patients with continuous renal replacement therapy (CRRT). A total of 425 cases were selected. 302 cases were used to develop a predictive model of extracorporeal circuit life span during CRRT without citrate anticoagulation in 24 h, and 123 cases were used to validate the model. The prediction formula was developed using multivariate Cox proportional-hazards regression analysis, from which a risk score was assigned. The mean survival time of the circuit was 15.0 ± 1.3 h, and the rate of circuit clotting was 66.6 % during 24 h of CRRT. Five significant variables were assigned a predicting score according to the regression coefficient: insufficient blood flow, no anticoagulation, hematocrit ≥0.37, lactic acid of arterial blood gas analysis ≤3 mmol/L and APTT < 44.2 s. The Hosmer-Lemeshow test showed no significant difference between the predicted and actual circuit clotting (R (2) = 0.232; P = 0.301). A risk score that includes the five above-mentioned variables can be used to predict the likelihood of extracorporeal circuit clotting in patients undergoing CRRT.

Segmentation of blood clot from CT pulmonary angiographic images using a modified seeded region growing algorithm method

NASA Astrophysics Data System (ADS)

Park, Bumwoo; Furlan, Alessandro; Patil, Amol; Bae, Kyongtae T.

2010-03-01

Pulmonary embolism (PE) is a medical condition defined as the obstruction of pulmonary arteries by a blood clot, usually originating in the deep veins of the lower limbs. PE is a common but elusive illness that can cause significant disability and death if not promptly diagnosed and effectively treated. CT Pulmonary Angiography (CTPA) is the first line imaging study for the diagnosis of PE. While clinical prediction rules have been recently developed to associate short-term risks and stratify patients with acute PE, there is a dearth of objective biomarkers associated with the long-term prognosis of the disease. Clot (embolus) burden is a promising biomarker for the prognosis and recurrence of PE and can be quantified from CTPA images. However, to our knowledge, no study has reported a method for segmentation and measurement of clot from CTPA images. Thus, the purpose of this study was to develop a semi-automated method for segmentation and measurement of clot from CTPA images. Our method was based on Modified Seeded Region Growing (MSRG) algorithm which consisted of two steps: (1) the observer identifies a clot of interest on CTPA images and places a spherical seed over the clot; and (2) a region grows around the seed on the basis of a rolling-ball process that clusters the neighboring voxels whose CT attenuation values are within the range of the mean +/- two standard deviations of the initial seed voxels. The rollingball propagates iteratively until the clot is completely clustered and segmented. Our experimental results revealed that the performance of the MSRG was superior to that of the conventional SRG for segmenting clots, as evidenced by reduced degrees of over- or under-segmentation from adjacent anatomical structures. To assess the clinical value of clot burden for the prognosis of PE, we are currently applying the MSRG for the segmentation and volume measurement of clots from CTPA images that are acquired in a large cohort of patients with PE in an on

[THROMBIN-MEDIATED EFFECTS OF BLOOD MICROPARTICLES ON FORMATION, STRUCTURE, AND STABILITY OF FIBRIN CLOTS].

PubMed

Nabiullina, R M; Mustafin, I G; Ataullakhanov, F I; Litvinov, R I; Zubairova, L D

2015-07-01

The effects of blood microparticles (MPs) on the dynamics of fibrin polymerization, clot structure and susceptibility to fibrinolysis were studied. Kinetics of fibrin polymerization, fibrinolysis, thrombin generation in platelet-free, microparticle-depleted and microparticle-depleted plasma replenished with cephalin, from healthy donors were analyzed in parallel. MPs have profound effects on all stages of fibrin formation, decrease its turbidity. All parameters obtained in the absence of MPs were recovered after reconstitution of phospholipids. Thrombin generation rates were reduced in the absence of MPs. In the presence of MPs the fibrin networks had less poro us structures with thinner fibers, while clots formed in the absence of MPs had larger pores and were built of thicker fibers. Clots formed in the presence of MPs were significantly more resistant to fibrinolysis. Results show that normally circulating MPs can support the formation of stable clots at the sites of vascular injury.

Time-dependent hardening of blood clots quantitatively measured in vivo with shear-wave ultrasound imaging in a rabbit model of venous thrombosis.

PubMed

Mfoumou, Etienne; Tripette, Julien; Blostein, Mark; Cloutier, Guy

2014-02-01

Provide in vivo blood clot hardening evolution with ultrasound using supersonic imaging of shear waves. We conducted a prospective study in flow stasis-induced venous thrombosis within jugular veins of white female New Zealand rabbits. Blood clot elasticity was noninvasively measured in vivo using the Young's modulus (in kilopascals), on a 2-hour and a 2-week periods after thrombus induction. Monitoring was followed by a necropsy and ex vivo mechanical characterization to validate the existence and elasticity of explanted thrombi. Stagnant blood in the region of interest underwent clotting and progressive hardening with thrombus aging. The mean Young's moduli varied from 1.0 ± 0.6 kPa (at 10 min) to 5.3 ± 1.6 kPa (at 2 hours), then to 25.0 ± 6.8 kPa (at 14 days) post-surgery. Mean ex vivo moduli of 6.2 ± 0.7 kPa at 2 hours and 29.0 ± 2.4 kPa at 2 weeks agreed with in vivo measures. Supersonic imaging of shear waves provides consistent quantitative non-invasive elasticity measurements not available with standard compression ultrasound imaging for diagnosing and following venous thromboembolism. This information translatable to humans could aid in determining whether continued anticoagulant treatment is necessary, especially in the setting of unprovoked venous thromboembolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets

PubMed Central

Oldenburg, Amy L.; Wu, Gongting; Spivak, Dmitry; Tsui, Frank; Wolberg, Alisa S.; Fischer, Thomas H.

2013-01-01

Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots. PMID:23833549

Imaging and Elastometry of Blood Clots Using Magnetomotive Optical Coherence Tomography and Labeled Platelets.

PubMed

Oldenburg, Amy L; Wu, Gongting; Spivak, Dmitry; Tsui, Frank; Wolberg, Alisa S; Fischer, Thomas H

2011-07-21

Improved methods for imaging and assessment of vascular defects are needed for directing treatment of cardiovascular pathologies. In this paper, we employ magnetomotive optical coherence tomography (MMOCT) as a platform both to detect and to measure the elasticity of blood clots. Detection is enabled through the use of rehydrated, lyophilized platelets loaded with superparamagnetic iron oxides (SPIO-RL platelets) that are functional infusion agents that adhere to sites of vascular endothelial damage. Evidence suggests that the sensitivity for detection is improved over threefold by magnetic interactions between SPIOs inside RL platelets. Using the same MMOCT system, we show how elastometry of simulated clots, using resonant acoustic spectroscopy, is correlated with the fibrin content of the clot. Both methods are based upon magnetic actuation and phase-sensitive optical monitoring of nanoscale displacements using MMOCT, underscoring its utility as a broad-based platform to detect and measure the molecular structure and composition of blood clots.

Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood.

PubMed

Tutwiler, Valerie; Litvinov, Rustem I; Lozhkin, Andrey P; Peshkova, Alina D; Lebedeva, Tatiana; Ataullakhanov, Fazoil I; Spiller, Kara L; Cines, Douglas B; Weisel, John W

2016-01-07

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle myosin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca(2+), the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders. © 2016 by The American Society of Hematology.

Kinetics and mechanics of clot contraction are governed by the molecular and cellular composition of the blood

PubMed Central

Tutwiler, Valerie; Litvinov, Rustem I.; Lozhkin, Andrey P.; Peshkova, Alina D.; Lebedeva, Tatiana; Ataullakhanov, Fazoil I.; Spiller, Kara L.; Cines, Douglas B.

2016-01-01

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack of methodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmuscle myosin II, red blood cells (RBCs), fibrin(ogen), factor XIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca2+, the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the platelet-fibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contraction may generate novel approaches to reveal and to manage thrombosis and hemostatic disorders. PMID:26603837

Plasma clot lysis time and its association with cardiovascular risk factors in black Africans.

PubMed

de Lange, Zelda; Pieters, Marlien; Jerling, Johann C; Kruger, Annamarie; Rijken, Dingeman C

2012-01-01

Studies in populations of European descent show longer plasma clot lysis times (CLT) in patients with cardiovascular disease (CVD) than in controls. No data are available on the association between CVD risk factors and fibrinolytic potential in black Africans, a group undergoing rapid urbanisation with increased CVD prevalence. We investigated associations between known CVD risk factors and CLT in black Africans and whether CLTs differ between rural and urban participants in light of differences in CVD risk.Data from 1000 rural and 1000 urban apparently healthy black South Africans (35-60 years) were cross-sectionally analysed.Increased PAI-1(act), BMI, HbA1c, triglycerides, the metabolic syndrome, fibrinogen concentration, CRP, female sex and positive HIV status were associated with increased CLTs, while habitual alcohol consumption associated with decreased CLT. No differences in CLT were found between age and smoking categories, contraceptive use or hyper- and normotensive participants. Urban women had longer CLT than rural women while no differences were observed for men.CLT was associated with many known CVD risk factors in black Africans. Differences were however observed, compared to data from populations of European descent available in the literature, suggesting possible ethnic differences. The effect of urbanisation on CLT is influenced by traditional CVD risk factors and their prevalence in urban and rural communities.

Plasma Clot Lysis Time and Its Association with Cardiovascular Risk Factors in Black Africans

PubMed Central

Jerling, Johann C.; Kruger, Annamarie; Rijken, Dingeman C.

2012-01-01

Studies in populations of European descent show longer plasma clot lysis times (CLT) in patients with cardiovascular disease (CVD) than in controls. No data are available on the association between CVD risk factors and fibrinolytic potential in black Africans, a group undergoing rapid urbanisation with increased CVD prevalence. We investigated associations between known CVD risk factors and CLT in black Africans and whether CLTs differ between rural and urban participants in light of differences in CVD risk. Data from 1000 rural and 1000 urban apparently healthy black South Africans (35–60 years) were cross-sectionally analysed. Increased PAI-1act, BMI, HbA1c, triglycerides, the metabolic syndrome, fibrinogen concentration, CRP, female sex and positive HIV status were associated with increased CLTs, while habitual alcohol consumption associated with decreased CLT. No differences in CLT were found between age and smoking categories, contraceptive use or hyper- and normotensive participants. Urban women had longer CLT than rural women while no differences were observed for men. CLT was associated with many known CVD risk factors in black Africans. Differences were however observed, compared to data from populations of European descent available in the literature, suggesting possible ethnic differences. The effect of urbanisation on CLT is influenced by traditional CVD risk factors and their prevalence in urban and rural communities. PMID:23145007

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 α IIb β 3 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fibrin clot structure is affected by levels of particulate air pollution exposure in patients with venous thrombosis.

PubMed

Pan, Xiaoxi; Gong, Yun Yun; Martinelli, Ida; Angelici, Laura; Favero, Chiara; Bertazzi, Pier Alberto; Mannucci, Pier M; Ariëns, Robert A S; Routledge, Michael N

2016-01-01

Particulate air pollution is a risk factor for cardiovascular diseases and thrombosis. Long-term exposure to particulate matter with a diameter<10μm (PM10) has been associated with an increased risk of venous thrombosis. The aim of this study was to investigate whether or not particulate air pollution alters fibrin clot structure and thus modulates thrombosis risk. We investigated fibrin polymerization by turbidity (maximum absorbance mOD), clot structure by confocal microscopy (fibre number per μm) and fibrin pore size by permeability (Ks×10(-10)cm(2)) in 103 patients with deep vein thrombosis and 121 healthy controls, for whom levels of air pollution exposure had been recorded. Exposure groups were defined by mean PM10 concentrations over the 730days before the event. We found a higher average number of fibres per clot area in patients than controls, but no difference in Ks or fibre thickness. When the two groups were divided into high or low exposure to PM10, a significantly denser fibrin clot network structure with thicker fibres (higher maximum absorbance, p<0.05), decreased permeability (lower Ks value, p<0.05) and higher average fibre numbers per clot area (p<0.05) was observed in patients in the high exposure group compared to those with low exposure. There were no significant differences in fibrin clot structure between the two exposure levels in healthy subjects. PM10 levels are associated with altered fibrin clot structure in patients with deep vein thrombosis but not in controls, suggesting that air pollution may trigger differences in fibrin clot structure only in patients predisposed to thrombotic disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Shrimp alpha-2-macroglobulin prevents the bacterial escape by inhibiting fibrinolysis of blood clots.

PubMed

Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2012-01-01

Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis.

A miniaturized fibrinolytic assay for plasminogen activators

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Nachtwey, D. S.; Damron, K. L.

1991-01-01

This report describes a micro-clot lysis assay (MCLA) for evaluating fibrinolytic activity of plasminogen activators (PA). Fibrin clots were formed in wells of microtiter plates. Lysis of the clots by PA, indicated by change in turbidity (optical density, OD), was monitored with a microplate reader at five minutes intervals. Log-log plots of PA dilution versus endpoint, the time at which the OD value was halfway between the maximum and minimum value for each well, were linear over a broad range of PA concentrations (2-200 International units/ml). The MCLA is a modification and miniaturization of well established fibrinolytic methods. The significant practical advantages of the MCLA are that it is a simple, relatively sensitive, non-radioactive, quantitative, kinetic, fibrinolytic micro-technique which can be automated.

Venom Concentrations and Clotting Factor Levels in a Prospective Cohort of Russell's Viper Bites with Coagulopathy.

PubMed

Isbister, Geoffrey K; Maduwage, Kalana; Scorgie, Fiona E; Shahmy, Seyed; Mohamed, Fahim; Abeysinghe, Chandana; Karunathilake, Harendra; O'Leary, Margaret A; Gnanathasan, Christeine A; Lincz, Lisa F

2015-01-01

Russell's viper envenoming is a major problem in South Asia and causes venom induced consumption coagulopathy. This study aimed to investigate the kinetics and dynamics of venom and clotting function in Russell's viper envenoming. In a prospective cohort of 146 patients with Russell's viper envenoming, we measured venom concentrations, international normalised ratio [INR], prothrombin time (PT), activated partial thromboplastin time (aPTT), coagulation factors I, II, V, VII, VIII, IX and X, and von Willebrand factor antigen. The median age was 39 y (16-82 y) and 111 were male. The median peak INR was 6.8 (interquartile range [IQR]: 3.7 to >13), associated with low fibrinogen [median,<0.01 g/L; IQR: <0.01-0.9 g/L), low factor V levels [median,<5%; IQR: <5-4%], low factor VIII levels [median,40%; IQR: 12-79%] and low factor X levels [median, 48%; IQR: 29-67%]. There were smaller reductions in factors II, IX and VII over time. All factors recovered over 48 h post-antivenom. The median INR remained >3 at 6 h post-antivenom but had reduced to <2, by 24 h. The aPTT had also returned to close to normal (<50 sec) at 24 h. Factor VII, VIII and IX levels were unusually high pre-antivenom, median peak concentrations of 393%, 307% and 468% respectively. Pre-antivenom venom concentrations and the INR (r = 0.20, p = 0.02) and aPTT (r = 0.19, p = 0.03) were correlated (non-parametric Spearman analysis). Russell's viper coagulopathy results in prolonged aPTT, INR, low fibrinogen, factors V, VIII and X which recover over 48 h. Severity of clotting abnormalities was associated with venom concentrations.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

PubMed

Zhang, Daimeng; Wan, Weisong; Kong, Tongtong; Zhang, Ming; Aweya, Jude Juventus; Gong, Yi; Li, Shengkang

2018-05-07

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His 156 , Asp 226 , Ser 321 ). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability

In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

PubMed

Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

2013-07-01

Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hypertonic saline-epinephrine local injection therapy for post-endoscopic sphincterotomy bleeding: removal of blood clots using pure ethanol local injection.

PubMed

Sakai, Yuji; Tsuyuguchi, Toshio; Sugiyama, Harutoshi; Nishikawa, Takao; Kurosawa, Jo; Saito, Masayoshi; Tawada, Katsunobu; Mikata, Rintaro; Tada, Motohisa; Ishihara, Takeshi; Yokosuka, Osamu

2013-08-01

Bleeding following endoscopic sphincterotomy (EST) is a rare but unavoidable complication of the procedure. We routinely perform local injection of hypertonic saline-epinephrine (HSE) for the treatment of post-EST bleeding. Any blood clot is removed only by irrigation with water after local injection of pure ethanol into the blood clot to cause crusting. We evaluated the usefulness of this treatment method. Subjects were 8 patients (1.2%) with post-EST bleeding requiring hemostatic intervention among 682 patients undergoing EST. After determination of the bleeding point, local injection of HSE was performed. When an adherent blood clot was present, pure ethanol was injected into the blood clot and then irrigation with water was performed to remove the blood clot. Endoscopic hemostasis was successfully achieved in all the 8 patients (100%). In 4 patients (50%), the adherent blood clots were successfully removed only with pure ethanol local injection into the blood clot followed by irrigation with water. No complications of the hemostatic procedure occurred in any patients. This study indicated that hemostasis with HSE local injection can be safe and useful for the treatment of post-EST bleeding, and also that blood clot removal with pure ethanol local injection can be useful.

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

PubMed

Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G

2004-10-01

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

Isolation and characterization of a new clotting factor from Bothrops jararacussu (jararacuçu) venom.

PubMed

Andrião-Escarso, S H; Sampaio, S V; Cunha, O A; Marangoni, S; Oliveira, B; Giglio, J R

1997-07-01

A detailed procedure for the isolation of a new clotting enzyme from the venom of Bothrops jararacussu (common name jararacuçu) is described. The estimated mol. wt of the native protein was 30,100 but 37,500 after reduction by dithiothreitol. Two major close bands corresponding to pI 5.18 and 5.20 were detected by electrofocusing but, after methanolysis, a single band focused at pI 8.20. The mol. wt of the protein moiety of this glycoprotein was 28,500, showing V-V-G-A-D-N-C-N-F-N... as N-terminal sequence. The content of neutral sugar was 4.8% and that of total sugars 5.3%. This clotting factor degraded only the A alpha-chain of the fibrinogen molecule. The stability of the clot, when produced in the presence of aprotinin opens new uses for snake clotting enzymes in the production of fibrin glue.

Shrimp Alpha-2-Macroglobulin Prevents the Bacterial Escape by Inhibiting Fibrinolysis of Blood Clots

PubMed Central

Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2012-01-01

Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis. PMID:23082160

Examining platelet-fibrin interactions during traumatic shock in a swine model using platelet contractile force and clot elastic modulus.

PubMed

White, Nathan J; Martin, Erika J; Brophy, Donald F; Ward, Kevin R

2011-07-01

A significant proportion of severely injured patients develop early coagulopathy, characterized by abnormal clot formation, which impairs resuscitation and increases mortality. We have previously demonstrated an isolated decrease in clot strength by thrombelastography in a swine model of nonresuscitated traumatic shock. In order to more closely examine platelet-fibrin interactions in this setting, we define the observed decrease in clot strength in terms of platelet-induced clot contraction and clot elastic modulus using the Hemostasis Analysis System (HAS) (Hemodyne Inc., Richmond, Virginia, USA). Whole blood was sampled for HAS measurements, metabolic measurements, cell counts, and fibrinogen concentration at baseline prior to injury and again at a predetermined level of traumatic shock defined by oxygen debt. Male swine (N=17) received femur fracture and controlled arterial hemorrhage to achieve an oxygen debt of 80 ml/kg. Platelet counts were unchanged, but fibrinogen concentration was reduced significantly during shock (167.6 vs. 66.7 mg/dl, P=0.0007). Platelet contractile force generated during clot formation did not change during shock (11.7 vs. 10.4 kdynes, P=0.41), but clot elastic modulus was dynamically altered, resulting in a lower final value (22.9 vs. 17.3 kdynes/cm, P<0.0001). In this model of traumatic shock, platelet function was preserved, whereas terminal clot elastic modulus was reduced during shock in a manner most consistent with early changes in the mechanical properties of the developing fibrin fiber network.

The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots.

PubMed

Acconcia, Christopher; Leung, Ben Y C; Manjunath, Anoop; Goertz, David E

2015-10-01

In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3-9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process. Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Blood clots are rapidly assembled hemodynamic sensors: Flow arrest triggers intraluminal thrombus contraction

PubMed Central

Muthard, Ryan W.; Diamond, Scott L.

2012-01-01

Objective Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. Methods and Results The collagen (κcollagen = 1.98 × 10−11 cm2) supported formation of a 20-μm thick platelet layer, which unexpectedly underwent massive platelet retraction upon flow arrest. This contraction resulted in a 5.34-fold increase in permeability due to collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κplatelet = 5.45 × 10−14 cm2 and platelet-fibrin thrombi had κthrombus = 2.71 × 10−14 cm2 for ΔP = 20.7 to 23.4 mm-Hg, the first ever measurements for clots formed under arterial flow (1130 s−1 wall shear rate). Platelet sensing of flow cessation triggered a 4.6 to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane (TXA2) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA2/ADP inhibitors. As clots become occlusive or vessels rupture, flow around developed clots diminishes facilitating full platelet retraction and hemostasis. Conclusion Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow sensing mechanism to regulate clot function. PMID:23087356

Relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism: assessment with breath-hold SPECT-CT pulmonary angiography fusion images.

PubMed

Suga, Kazuyoshi; Yasuhiko, Kawakami; Iwanaga, Hideyuki; Tokuda, Osamu; Matsunaga, Naofumi

2008-09-01

The relation between lung perfusion defects and intravascular clots in acute pulmonary thromboembolism (PTE) was comprehensively assessed on deep-inspiratory breath-hold (DIBrH) perfusion SPECT-computed tomographic pulmonary angiography (CTPA) fusion images. Subjects were 34 acute PTE patients, who had successfully performed DIBrH perfusion SPECT using a dual-headed SPECT and a respiratory tracking system. Automated DIBrH SPECT-CTPA fusion images were used to assess the relation between lung perfusion defects and intravascular clots detected by CTPA. DIBrH SPECT visualized 175 lobar/segmental or subsegmental defects in 34 patients, and CTPA visualized 61 intravascular clots at variable locations in 30 (88%) patients, but no clots in four (12%) patients. In 30 patients with clots, the fusion images confirmed that 69 (41%) perfusion defects (20 segmental, 45 subsegmental and 4 lobar defects) of total 166 defects were located in lung territories without clots, although the remaining 97 (58%) defects were located in lung territories with clots. Perfusion defect was absent in lung territories with clots (one lobar branch and three segmental branches) in four (12%) of these patients. In four patients without clots, nine perfusion defects including four segmental ones were present. Because of unexpected dissociation between intravascular clots and lung perfusion defects, the present fusion images will be a useful adjunct to CTPA in the diagnosis of acute PTE.

Clotting and fibrinolytic changes after firefighting activities.

PubMed

Smith, Denise L; Horn, Gavin P; Petruzzello, Steven J; Fahey, George; Woods, Jeffrey; Fernhall, Bo

2014-03-01

Approximately 45%-50% of all duty-related deaths among firefighters are due to sudden cardiovascular events, and a disproportionate number of these fatalities occur after strenuous fire suppression activities. The objective of this study is to evaluate the effect of strenuous firefighting activities on platelets, coagulation, and fibrinolytic activity and to document the extent to which these variables recovered 2 h after completion of the firefighting activity. Firefighters performed 18 min of simulated firefighting activities in a training structure that contained live fires. After firefighting activities, firefighters were provided with fluid and allowed to cool down and then recovered for 2 h in an adjacent room. Blood samples were obtained prefirefighting, postfirefighting, and 2 h postfirefighting. Platelet number, platelet activity, and coagulatory potential increased immediately postfirefighting and many variables (platelet function, partial thromboplastin time, and factor VIII) reflected a procoagulatory state even after 2 h of recovery. Fibrinolysis, as reflected by tissue plasminogen activator, also was enhanced immediately postfirefighting but returned to baseline values by 2 h postfirefighting. In contrast, inhibition of fibrinolysis, as evidenced by a reduction in plasminogen activator inhibitor-1, was depressed at 2 h postfirefighting. Firefighting resulted in elevated coagulatory and fibrinolytic activity. However, 2 h postfirefighting, tissue plasminogen activator returned to baseline and coagulatory potential remained elevated. The procoagulatory state that exists after firefighting may provide a mechanistic link to the reports of sudden cardiac events after strenuous fire suppression activities.

The effects of acclimatization on blood clotting parameters in exertional heat stress.

PubMed

Vesić, Zoran; Vukasinović-Vesić, Milica; Dincić, Dragan; Surbatović, Maja; Radaković, Sonja S

2013-07-01

Exertional heat stress is a common problem in military services. Considering the coagulation abnormalities are of major importance in development of severe heat stroke, we wanted to examine changes in hemostatic parameters in soldiers during exertional heat stress test as well as the effects of a 10-day passive or active acclimatization in a climatic chamber. A total of 40 male soldiers with high aerobic capacity performed exertional heat stress test (EHST) either in cool [20 degrees C, 16 degrees C wet bulb globe temperature (WBGT)], or hot (40 degrees C, 29 degrees C, (WBGT) environment, unacclimatized (U) or after 10 days of passive (P) or active (A) acclimatization. Physiological strain was measured by tympanic temperatures (Tty) and heart rates (HR). Platelet count (PC), antithrombin III (AT), and prothrombin time (PT) were assessed in blood samples collected before and immediately after the EHST. EHST in hot conditions induced physiological heat stress (increase in Tty and HR), with a significant increase in prothrombin time in the groups U and A. Platelet counts were significantly higher after the EHST compared to the basic levels in all the investigated groups, regardless environmental conditions and acclimatization state. Antithrombin levels were not affected by EHST whatsoever. In the trained soldiers, physiological heat stress caused mild changes in some serum parameters of blood clotting such as prothrombin time, while others such as antithrombin levels were not affected. Platelet counts were increased after EHST in all groups. A 10-day passive or active acclimatization in climatic chamber showed no effect on parameters investigated.

Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

PubMed

da Silva, Ronivaldo Rodrigues; Souto, Tatiane Beltramini; de Oliveira, Tássio Brito; de Oliveira, Lilian Caroline Gonçalves; Karcher, Daniel; Juliano, Maria Aparecida; Juliano, Luiz; de Oliveira, Arthur H C; Rodrigues, André; Rosa, Jose C; Cabral, Hamilton

2016-08-01

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

Tilley, Derek; Levit, Irina; Samis, John A.

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

Measurement of factor v activity in human plasma using a microplate coagulation assay.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

PubMed

Sazonova, I Y; McNamee, R A; Houng, A K; King, S M; Hedstrom, L; Reed, G L

2009-08-01

Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Recurrent clot anuria following laparoscopic pyeloplasty in a solitary functioning kidney: managing with double guide wire technique

PubMed Central

Kumar, Santosh; Singh, Shivanshu; Parmar, Kalpesh Mahesh; Garg, Nitin

2014-01-01

Clot anuria in a solitary functioning kidney is an emergency situation. Haematuria with clot anuria in an early postoperative period represents a challenge, as treatment options are limited. Manipulation of the anastomotic site may lead to anastomotic disruption and urinoma while use of thrombolytic therapy poses the danger of increasing haematuria. We report a case of anuria due to clot retention in the upper tract following laparoscopic dismembered pyeloplasty in a solitary functioning kidney, managed successfully with double guide wire technique. PMID:25540210

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

PubMed

Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard

2017-12-01

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin

Determination of tumor cell procoagulant activity by Sonoclot analysis in whole blood.

PubMed

Amirkhosravi, A; Biggerstaff, J P; Warnes, G; Francis, D A; Francis, J L

1996-12-01

Coagulation activation in cancer may be due to expression of procoagulant activity (PCA) by tumor cells. Some PCA activate coagulation, while others influence platelet aggregation. Thus, clotting time assessments of PCA may not reflect the potential for hypercoagulability. We therefore studied the effect of various malignant and non-malignant cells on whole blood coagulation using the Sonoclot Analyzer. Five human (HT29 colon, J82 bladder, MCF-7 breast, BT-474 breast and A375 malignant melanoma) and three rodent (MC28, WEHI-164 and Neuro2a) cell lines were used. Rat thymocytes and peritoneal macrophages and human endotoxin-stimulated mononuclear cells and umbilical vein endothelial cells (HUVEC) were used as non-malignant controls. All tumor cells markedly shortened the recalcification time of citrated blood and the most potent (HT29 and J82) also increased clot rate (P < 0.01). The breast cancer cells MCF-7 and BT-474 expressed only weak PCA and did not affect clotting rate. None of the non-malignant cells significantly affected clotting time or rate in whole blood. J82 and HT29 cells grown in serum-rich media shortened the recalcification time of both normal and FVII-deficient plasmas. However, cells grown in serum-free conditions had significantly less PCA in FVII-deficient plasma. We conclude that the Sonoclot Analyzer is useful for determining cellular PCA; significant PCA is a feature of malignant cells and cells grown in medium containing serum supplements cannot be reliably evaluated for PCA.

21 CFR 864.7275 - Euglobulin lysis time tests.

Code of Federal Regulations, 2012 CFR

2012-04-01

... time required for the lysis (dissolution) of a clot formed from fibrinogen in the euglobulin fraction (that fraction of the plasma responsible for the formation of plasmin, a clot lysing enzyme). This test evaluates natural fibrinolysis (destruction of a blood clot after bleeding has been arrested). The test also...

21 CFR 864.7275 - Euglobulin lysis time tests.

Code of Federal Regulations, 2013 CFR

2013-04-01

... time required for the lysis (dissolution) of a clot formed from fibrinogen in the euglobulin fraction (that fraction of the plasma responsible for the formation of plasmin, a clot lysing enzyme). This test evaluates natural fibrinolysis (destruction of a blood clot after bleeding has been arrested). The test also...

21 CFR 864.7275 - Euglobulin lysis time tests.

Code of Federal Regulations, 2011 CFR

2011-04-01

... time required for the lysis (dissolution) of a clot formed from fibrinogen in the euglobulin fraction (that fraction of the plasma responsible for the formation of plasmin, a clot lysing enzyme). This test evaluates natural fibrinolysis (destruction of a blood clot after bleeding has been arrested). The test also...

21 CFR 864.7275 - Euglobulin lysis time tests.

Code of Federal Regulations, 2014 CFR

2014-04-01

... time required for the lysis (dissolution) of a clot formed from fibrinogen in the euglobulin fraction (that fraction of the plasma responsible for the formation of plasmin, a clot lysing enzyme). This test evaluates natural fibrinolysis (destruction of a blood clot after bleeding has been arrested). The test also...

Short-term supplementation with Aronia melanocarpa extract improves platelet aggregation, clotting, and fibrinolysis in patients with metabolic syndrome.

PubMed

Sikora, Joanna; Broncel, Marlena; Markowicz, Magdalena; Chałubiński, Maciej; Wojdan, Katarzyna; Mikiciuk-Olasik, Elżbieta

2012-08-01

A diet rich in berries is believed to play a distinct role in the prevention of metabolic diseases associated with obesity. So far, there have been no published clinical observations evaluating the influence of Aronia melanocarpa on hemostasis. The aim of our study was to investigate the effects of A. melanocarpa extract (AM) supplementation on platelet aggregation, clot formation, and lysis in patients with metabolic syndrome (MS). Middle-aged non-medicated subjects with MS (n = 38) and 14 healthy volunteers were included in this study. Patients with MS were treated with 100 mg of AM three times daily for 2 months. We observed a significant reduction in the concentration of TC, LDL-C, and TG after AM supplementation. Beneficial changes in coagulation parameters were also observed. After 1 month of AM administration, we noticed significant inhibition of platelet aggregation. However, this effect became less pronounced after 2 months of supplementation. In the case of coagulation induced by endogenic thrombin, a significant decrease in the overall potential for coagulation was induced after 1 or 2 months of supplementation. Moreover, after 1 month of AM extract supplementation, we observed a beneficial reduction in the overall potential for clot formation and fibrinolysis. We observed the normalization of hemostasis parameters in MS patients after both 1 and 2 months of AM administration. After 1 month of AM supplementation, we found favorable changes in regards to the overall potential for plasma clotting, clot formation, and lysis, as well as in the lipid profiles of subjects.

An Investigation Into the Effects of In Vitro Dilution With Different Colloid Resuscitation Fluids on Clot Microstructure Formation.

PubMed

Lawrence, Matthew James; Marsden, Nick; Kaczynski, Jakub; Davies, Gareth; Davies, Nia; Hawkins, Karl; Perumal, Sounder; Brown, Martin Rowan; Morris, Keith; Davidson, Simon J; Williams, Phylip Rhodri; Evans, Phillip Adrian

2016-11-01

Balancing the beneficial effects of resuscitation fluids against their detrimental effect on hemostasis is an important clinical issue. We aim to compare the in vitro effects of 3 different colloid resuscitation fluids (4.5% albumin, hydroxyethyl starch [Voluven 6%], and gelatin [Geloplasma]) on clot microstructure formation using a novel viscoelastic technique, the gel point. This novel hemorheologic technique measures the biophysical properties of the clot and provides an assessment of clot microstructure from its viscoelastic properties. Importantly, in contrast to many assays in routine clinical use, the measurement is performed using unadulterated whole blood in a near-patient setting and provides rapid assessment of coagulation. We hypothesized that different colloids will have a lesser or greater detrimental effect on clot microstructure formation when compared against each other. Healthy volunteers were recruited into the study (n = 104), and a 20-mL sample of whole blood was obtained. Each volunteer was assigned to 1 of the 3 fluids, and the sample was diluted to 1 of 5 different dilutions (baseline, 10%, 20%, 40%, and 60%). The blood was tested using the gel point technique, which measures clot mechanical strength and quantifies clot microstructure (df) at the incipient stages of fibrin formation. df and clot mechanical strength decrease with progressive dilution for all 3 fluids. A significant reduction in df from baseline was recorded at dilutions of 20% for albumin (P < .0001), 40% for starch (P < .0001), and 60% for gelatin (P < .0001). We also observed significant differences, in terms of df, when comparing the different types of colloid (P < .0001). We found that albumin dilution produced the largest changes in clot microstructure, providing the lowest values of df (= 1.41 ± 0.061 at 60% dilution) compared with starch (1.52 ± 0.081) and gelatin (1.58 ± 0.063). We show that dilution with all 3 fluids has a significant effect on coagulation at even

Synergism of aspirin and heparin with a low-frequency non-invasive ultrasound system for augmentation of in-vitro clot lysis.

PubMed

Atar, Shaul; Neuman, Yoram; Miyamoto, Takashi; Chen, Ming; Birnbaum, Yochai; Luo, Huai; Kobal, Sergio; Siegel, Robert J

2003-06-01

Aspirin, glycoprotein IIb/IIIa inhibitors and heparin are routinely used in acute coronary syndromes. Previously we showed that there is synergism between ultrasound and heparin and tirofiban in augmenting blood clot disruption. However, there is a little data on a possible synergism of low-frequency ultrasound with aspirin for in-vitro clot dissolution, and especially on the combination of aspirin with heparin and/or glycoprotein IIb/IIIa inhibitors. Human blood clots (n = 320) were incubated for 10 or 20 minutes in saline containing aspirin alone or combined with heparin and/or tirofiban and/or eptifibatide. Clots were randomly treated with low-frequency ultrasound (27.3 kHz) or incubation only. The percent clot weight loss and the incremental effect of ultrasound were calculated. The most significant incremental effect of ultrasound on clot weight reduction was detected with aspirin alone (5.2 +/- 2.3% and 5.2 +/- 2.6% after 10' and 20', p = 0.04 and p = 0.06, respectively) and in combination with heparin (8.8 +/- 2.5% and 11.5 +/- 2.7% after 10' and 20', p = 0.01 and p = 0.0001, respectively). The greatest absolute magnitude of clot weight reduction was observed with ultrasound combined with aspirin and heparin (48.5 +/- 9.5% after 20'). The addition of tirofiban or eptifibatide to aspirin, heparin and ultrasound did not increase clot lysis. However, eptifibatide had significantly better synergism than tirofiban (p = 0.025 and p = 0.015, after 10 and 20 minutes, respectively). Aspirin alone or in combination with heparin results in significant augmentation of clot lysis and is synergistic with application of low-frequency ultrasound for 10 and 20 minutes only. These results may have important implications for a possible use of low-frequency ultrasound in treatment algorithms of acute coronary syndromes.

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin*

PubMed Central

Kim, Paul Y.; Vu, Trang T.; Leslie, Beverly A.; Stafford, Alan R.; Fredenburgh, James C.; Weitz, Jeffrey I.

2014-01-01

Fibrin (Fn) clots formed from γ′-fibrinogen (γ′-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ′-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ′-Fn, and/or altered cross-linking. Clots formed from γ′-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ′-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ′-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ′-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ′-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ′-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis. PMID:25128532

An exploration of the reflow technique for the fabrication of an in vitro microvascular system to study occlusive clots.

PubMed

Li, Yang; Pan, Chuer; Li, Yunfeng; Kumacheva, Eugenia; Ramachandran, Arun

2017-09-08

Embolic ischemia and pulmonary embolism are health emergencies that arise when a particle such as a blood clot occludes a smaller blood vessel in the brain or the lungs, and restricts flow of blood downstream of the vessel. In this work, the reflow technique (Wang et al. Biomed. Microdevices 2007, 9, 657) was adapted to produce a microchannel network that mimics the occlusion process. The technique was first revisited and a simple geometrical model was developed to quantitatively explain the shapes of the resulting microchannels for different reflow parameters. A critical modification was introduced to the reflow protocol to fabricate nearly circular microchannels of different diameters from the same master, which is not possible with the traditional reflow technique. To simulate the phenomenon of occlusion by clots, a microchannel network with three generations of branches with different diameters and branching angles was fabricated, into which fibrin clots were introduced. At low constant pressure drop (ΔP), a clot blocked a branch entrance only partially, while at higher ΔP, the branch was completely blocked. Instances of simultaneous blocking of multiple channels by clots, and the consequent changes in the flow rates in the unblocked branches of the network, were also monitored. This work provides the framework for a systematic study of the distribution of clots in a network, and the rate of dissolution of embolic clots upon the introduction of a thrombolytic drug into the network.

Comparison of digoxin concentration in plastic serum tubes with clot activator and heparinized plasma tubes.

PubMed

Dukić, Lora; Simundić, Ana-Maria; Malogorski, Davorin

2014-01-01

Sample type recommended by the manufacturer for the digoxin Abbott assay is either serum collected in glass tubes or plasma (sodium heparin, lithium heparin, citrate, EDTA or oxalate as anticoagulant) collected in plastic tubes. In our hospital samples are collected in plastic tubes. Our hypothesis was that the serum sample collected in plastic serum tube can be used interchangeably with plasma sample for measurement of digoxin concentration. Our aim was verification of plastic serum tubes for determination of digoxin concentration. Concentration of digoxin was determined simultaneously in 26 venous blood plasma (plastic Vacuette, LH Lithium heparin) and serum (plastic Vacuette, Z Serum Clot activator; both Greiner Bio-One GmbH, Kremsmünster, Austria) samples, on Abbott AxSYM analyzer using the original Abbott Digoxin III assay (Abbott, Wiesbaden, Germany). Tube comparability was assessed using the Passing Bablok regression and Bland-Altman plot. Serum and plasma digoxin concentrations are comparable. Passing Bablok intercept (0.08 [95% CI = -0.10 to 0.20]) and slope (0.99 [95% CI = 0.92 to 1.11]) showed there is no constant or proportional error. Blood samples drawn in plastic serum tubes and plastic plasma tubes can be interchangeably used for determination of digoxin concentration.

Comparison of digoxin concentration in plastic serum tubes with clot activator and heparinized plasma tubes

PubMed Central

Dukić, Lora; Šimundić, Ana-Maria; Malogorski, Davorin

2014-01-01

Introduction: Sample type recommended by the manufacturer for the digoxin Abbott assay is either serum collected in glass tubes or plasma (sodium heparin, lithium heparin, citrate, EDTA or oxalate as anticoagulant) collected in plastic tubes. In our hospital samples are collected in plastic tubes. Our hypothesis was that the serum sample collected in plastic serum tube can be used interchangeably with plasma sample for measurement of digoxin concentration. Our aim was verification of plastic serum tubes for determination of digoxin concentration. Materials and methods: Concentration of digoxin was determined simultaneously in 26 venous blood plasma (plastic Vacuette, LH Lithium heparin) and serum (plastic Vacuette, Z Serum Clot activator; both Greiner Bio-One GmbH, Kremsmünster, Austria) samples, on Abbott AxSYM analyzer using the original Abbott Digoxin III assay (Abbott, Wiesbaden, Germany). Tube comparability was assessed using the Passing Bablok regression and Bland-Altman plot. Results: Serum and plasma digoxin concentrations are comparable. Passing Bablok intercept (0.08 [95% CI = −0.10 to 0.20]) and slope (0.99 [95% CI = 0.92 to 1.11]) showed there is no constant or proportional error. Conclusion: Blood samples drawn in plastic serum tubes and plastic plasma tubes can be interchangeably used for determination of digoxin concentration. PMID:24627723

Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation

PubMed Central

Bompiani, Kristin M; Lohrmann, Jens L; Pitoc, George A; Frederiksen, James W; Mackensen, George B; Sullenger, Bruce A

2014-01-01

SUMMARY Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. PMID:25065530

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator

PubMed Central

SAZONOVA, I. Y.; MCNAMEE, R. A.; HOUNG, A. K.; KING, S. M.; HEDSTROM, L.; REED, G. L.

2013-01-01

Summary Background: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA’s fibrin-targeted properties that focus plasminogen activation on the fibrin surface. Objective: We examined whether re-programming SK’s mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. Methods and Results: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKΔ1 and SKΔ59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKΔ59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by α2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel ‘humanized’ fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKΔ1 and SKΔ59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2–3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. Conclusion: These experiments suggest that reprogramming SK’s mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency. PMID:19566545

Intracameral air injection during Ahmed glaucoma valve implantation in neovascular glaucoma for the prevention of tube obstruction with blood clot: Case Report.

PubMed

Hwang, Sung Ha; Yoo, Chungkwon; Kim, Yong Yeon; Lee, Dae Young; Nam, Dong Heun; Lee, Jong Yeon

2017-12-01

Glaucoma drainage implant surgery is a treatment option for the management of neovascular glaucoma. However, tube obstruction by blood clot after Ahmed glaucoma valve (AGV) implantation is an unpredictable clinically challenging situation. We report 4 cases using intracameral air injection for the prevention of the tube obstruction of AGV by blood clot. The first case was a 57-year-old female suffering from ocular pain because of a tube obstruction with blood clot after AGV implantation in neovascular glaucoma. Surgical blood clot removal was performed. However, intractable bleeding was noted during the removal of the blood clot, and so intracameral air injection was performed to prevent a recurrent tube obstruction. After the procedure, although blood clots formed around the tube, the tube opening where air could touch remained patent. In 3 cases of neovascular glaucoma with preoperative severe intraocular hemorrhages, intracameral air injection and AGV implantation were performed simultaneously. In all 3 cases, tube openings were patent. It appears that air impeded the blood clots formation in front of the tube opening. Intracameral air injection could be a feasible option to prevent tube obstruction of AGV implant with a blood clot in neovascular glaucoma with high risk of tube obstruction. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

The association of clot lysis time with total obesity is partly independent from the association of PAI-1 with central obesity in African adults.

PubMed

Eksteen, Philna; Pieters, Marlien; de Lange, Zelda; Kruger, Herculina S

2015-08-01

Preliminary evidence indicates that the association of fibrinolytic potential, measured as clot lysis time (CLT), with body composition may differ from that of plasminogen activator inhibitor type-1 (PAI-1). We therefore investigated the association between fibrinolytic markers (plasminogen activator inhibitor type-1 activity (PAI-1act) and CLT) and body composition using detailed body composition analyses. Data from 1288 Africans were cross-sectionally analyzed. Body composition analysis included BMI, waist circumference (WC); waist to height ratio (WHtR), skinfolds and body fat percentage measured with air-displacement plethysmography and bioelectrical impedance analysis. PAI-1act and CLT were significantly higher in women than in men, despite adjustment for differences in body composition. PAI-1act and CLT showed similar linear positive relationships with body composition (BMI, WC, WHtR, skinfolds) in men. In women CLT also showed a linear relationship with body composition, while PAI-1act levels plateaued at higher BMI and did not differ across skinfold categories. PAI-1act showed stronger correlations with body composition markers in men than it did in women, while no sex differences existed for CLT. PAI-1act associated more strongly with central obesity, while CLT associated with total body fat. Observed differences may be related to differences in adipose tissue type, distribution and sequence of accumulation between sexes. PAI-1act is strongly influenced by accumulation of visceral adipose tissue, whereas CLT is associated with obesity independent of type and sequence of body fat accumulation in this African adult study population. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.

PubMed

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-03-24

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

Comparative Effect of Quercetin and Quercetin-3-O-β-d-Glucoside on Fibrin Polymers, Blood Clots, and in Rodent Models.

PubMed

Choi, Jun-Hui; Kim, Kyung-Je; Kim, Seung

2016-11-01

The present study evaluates the in vitro, in vivo, and ex vivo antithrombotic and anticoagulant effect of two flavonoids: quercetin and quercetin-3-O-β-d-glucoside (isoquercetin). The present results have shown that quercetin and isoquercetin inhibit the enzymatic activity of thrombin and FXa and suppress fibrin clot formation and blood clotting. The prolongation effect of quercetin and isoquercetin against epinephrine and collagen-induced platelet activation may have been caused by intervention in intracellular signaling pathways including coagulation cascade and aggregation response on platelets and blood. The in vivo and ex vivo anticoagulant efficacy of quercetin and isoquercetin was evaluated in thrombin-induced acute thromboembolism model and in ICR mice. Our findings showed that in vitro and in vivo inhibitory effects of quercetin were slightly higher than that of quercetin glucoside, whereas in vitro and ex vivo anticoagulant effects of quercetin were weaker than that of quercetin glucoside because of their structural characteristics. © 2016 Wiley Periodicals, Inc.

The use of alteplase for the resolution of an intravesical clot in a neonate receiving extracorporeal membrane oxygenation.

PubMed

Olarte, J L; Glover, M L; Totapally, B R

2001-01-01

We present a case of the use of alteplase for the lysis of a large urinary bladder clot. A neonate presented with respiratory failure, secondary to a left diaphragmatic hernia necessitating the need for extracorporeal membrane oxygenation (ECMO) support. On day 3 of ECMO support, hematuria was noted, and a subsequent urinary bladder ultrasound revealed a significant urinary bladder clot. Alteplase (0.5-1 mg) was instilled into the urinary bladder via a 10 French Foley catheter (Sherwood Medical, St. Louis, MO). The catheter was clamped for 1 hour, followed by irrigation with normal saline. Multiple doses of alteplase were administered, resulting in complete resolution of the bladder clot. No adverse effects were attributed to the use of the intravesical alteplase. Alteplase seems to be safe and effective for the resolution of bladder clots, thereby potentially avoiding more invasive surgical procedures.

Conservative treatment of concomitant extraperitoneal bladder rupture and intrabladder blood clot formation: Case report of application of ureteral catheterization.

PubMed

Fu, Chih-Yuan; Shih, Chun-Han; Chang, Po-Yen; Hsiao, Chi-Hao; Wang, Yu-Chun; Chen, Ray-Jade

2012-12-01

Most extraperitoneal bladder ruptures can be treated conservatively with catheter drainage only. However, in patients with concomitant intrabladder blood clot formation and extraperitoneal bladder rupture, surgery for blood clot evacuation and bladder repair are usually needed due to occlusion of the urinary catheter. In our patient, we used a ureteral catheter to bypass the clots in the bladder to provide adequate urinary drainage. This procedure serves as a valuable tool in the conservative treatment of extraperitoneal bladder injury.

Purification and characterization of a novel milk-clotting metalloproteinase from Paenibacillus spp. BD3526.

PubMed

Hang, Feng; Wang, Qinbo; Hong, Qing; Liu, Peiyi; Wu, Zhengjun; Liu, Zhenmin; Zhang, Hao; Chen, Wei

2016-04-01

In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65°C and 60°C, respectively. The enzyme was stable over a pH range of 5.0-9.0 and at temperatures below 50°C. The MCA was completely inactivated when the enzyme was heated at 60°C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55°C and 60°C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis. Copyright © 2016. Published by Elsevier B.V.

Alteration of blood clot structures by interleukin-1 beta in association with bone defects healing

PubMed Central

Wang, Xin; Friis, Thor E.; Masci, Paul P.; Crawford, Ross W.; Liao, Wenbo; Xiao, Yin

2016-01-01

The quality of hematomas are crucial for successful early bone defect healing, as the structure of fibrin clots can significantly influence the infiltration of cells, necessary for bone regeneration, from adjacent tissues into the fibrin network. This study investigated if there were structural differences between hematomas from normal and delayed healing bone defects and whether such differences were linked to changes in the expression of IL-1β. Using a bone defect model in rats, we found that the hematomas in the delayed healing model had thinner fibers and denser clot structures. Moreover, IL-1β protein levels were significantly higher in the delayed healing hematomas. The effects of IL-1β on the structural properties of human whole blood clots were evaluated by thrombelastograph (TEG), scanning electronic microscopy (SEM), compressive study, and thrombolytic assays. S-nitrosoglutathione (GSNO) was applied to modulate de novo hematoma structure and the impact on bone healing was evaluated in the delayed healing model. We found that GSNO produced more porous hematomas with thicker fibers and resulted in significantly enhanced bone healing. This study demonstrated that IL-1β and GSNO had opposing effects on clot architecture, the structure of which plays a pivotal role in early bone healing. PMID:27767056

Detection and isolation of Japanese encephalitis virus from blood clots collected during the acute phase of infection.

PubMed

Sapkal, Gajanan N; Wairagkar, Nitin S; Ayachit, Vijay M; Bondre, Vijay P; Gore, Milind M

2007-12-01

Clinical specimens from an encephalitis outbreak in the Lakhimpur area of Uttar Pradesh, India, were investigated for identification and characterization of the etiologic agent. IgM capture ELISA showed recent Japanese encephalitis virus (JEV) infection. JEV isolation was attempted from white blood cells (WBCs) separated from blood clots of 12 patients (9 IgM positive and 3 negative) by serial co-culturing with phytohemagglutinin P-stimulated peripheral blood mononuclear leukocytes (PBMCs) obtained from pre-screened JEV sero-negative healthy individuals. JEV was isolated from two IgM-positive blood clots. Isolate 014178 was detected in WBCs and in the first passage of PBMCs by ELISA and reverse transcriptase-polymerase chain reaction. Isolate 014173 was detectable only after a second passage in PBMC co-culture. Sequence analysis of 346 nt of the C-prM region showed homology with JEV strain GP78. This is the first report on isolation of JEV from patient blood clots. Our study shows that the co-cultures of PBMCs separated from patient blood clots provide an additional source for JEV isolation.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

PubMed Central

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-01-01

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma. PMID:27009516

Contributions of Antoine Barthélémy Clot (1793-1868): A historiographical reflection of public health in Ottoman Egypt.

PubMed

Aboul-Enein, Basil H; Puddy, William

2016-08-01

This paper reviews the selected historiographic and contemporary literature that discussed the medical and public health contribution of Antoine Barthélémy Clot (Clot Bey) and how these contributions shaped modern public health in Ottoman Egypt, and the major features that led to the development of the public health infrastructure of early modern Egypt based on the contributions of Clot Bey. The literature discussed the establishment of Egypt's first modern public health and medical schools under the direct administration and guidance of Clot Bey, and his major contribution in the fields of vaccination, quarantine, the development of a culturally congruent curriculum for medical students, and the public health policies and practices enacted during the reign of Muhammad Ali Pasha that addressed major communicable diseases affecting Egypt. With considerable support from the viceroy of Egypt despite popular resistance, Clot Bey significantly modernized Egyptian medicine, medical education and reformed the public health infrastructure. He became one of the preeminent medical figures of nineteenth century Ottoman Egypt. © The Author(s) 2016.

Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction.

PubMed

Muthard, Ryan W; Diamond, Scott L

2012-12-01

Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction. Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin.

PubMed

Amelot, Aymeric A; Tagzirt, Madjid; Ducouret, Guylaine; Kuen, René Lai; Le Bonniec, Bernard F

2007-01-05

Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31- and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 microm the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.

Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys.

PubMed

Doolittle, Russell F

2015-10-01

Lampreys and hagfish are the earliest diverging of extant vertebrates and are obvious targets for investigating the origins of complex biochemical systems found in mammals. Currently, the simplest approach for such inquiries is to search for the presence of relevant genes in whole genome sequence (WGS) assemblies. Unhappily, in the past a high-quality complete genome sequence has not been available for either lampreys or hagfish, precluding the possibility of proving gene absence. Recently, improved but still incomplete genome assemblies for two species of lamprey have been posted, and, taken together with an extensive collection of short sequences in the NCBI trace archive, they have made it possible to make reliable counts for specific gene families. Particularly, a multi-source tactic has been used to study the lamprey blood clotting system with regard to the presence and absence of genes known to occur in higher vertebrates. As was suggested in earlier studies, lampreys lack genes for coagulation factors VIII and IX, both of which are critical for the "intrinsic" clotting system and responsible for hemophilia in humans. On the other hand, they have three each of genes for factors VII and X, participants in the "extrinsic" clotting system. The strategy of using raw trace sequence "reads" together with partial WGS assemblies for lampreys can be used in studies on the early evolution of other biochemical systems in vertebrates.

Acute intracranial hemorrhage secondary to thrombocytopenia: CT appearances unaffected by absence of clot retraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, J.N.; Taber, K.H.; Hayman, L.A.

To describe the in vivo CT appearance of acute intracerebral blood clots formed from anemic platelet-depleted blood. Three patients with intracerebral hemorrhage secondary only to thrombocytopenia were examined with CT within 2 1/2 hours after the onset of clinical symptoms. There were no unusual CT features found in the intracerebral hemorrhages of patients with only thrombocytopenia. Specifically, a hyperdense zone(s) surrounded by areas of decreased density was identified. Clot retraction (which cannot occur in patients with severe thrombocytopenia) is not necessary for the CT appearance of acute intracerebral hemorrhage. 22 refs., 3 figs., 1 tab.

Formation of obstructing blood clot in the ureter in a patient with Glanzmann's thrombasthenia.

PubMed

Kilincaslan, Huseyin; Leblebisatan, Goksel; Tepeler, Abdulkadir; Karakus, Suleyman C

2011-12-01

Glanzman thrombasthenia is a rare hematologic disorder characterized by qualitative thrombocyte abnormality. Patients present with episodic mucocutaneous bleeding. Thrombosis is a paradox phenomenon observed in patients with Glanzman thrombasthenia and generally considered as a treatment complication. We present a 16-year-old girl referred for severe flank pain beginning after treatment of hematuria due to Glanzman thrombasthenia. The patient underwent endoscopy for further diagnosis and treatment because of the failure of radiologic evaluation. Although the resolution of the large clots was obtained with streptokinase instillation via the ureteral catheter, clot was mobilized with gentle insertion of ureteral catheter in the present case.

Establishment of a canine model of acute pulmonary embolism with definite right ventricular dysfunction through introduced autologous blood clots.

PubMed

Zhao, Lin-Bo; Jia, Zhen-Yu; Lu, Guang-Dong; Zhu, Yin-Su; Jing, Lei; Shi, Hai-Bin

2015-04-01

To establish a canine model of acute pulmonary embolism (PE) with right ventricular (RV) dysfunction using autologous blood clots and evaluate by echocardiography and contrast-enhanced Computed Tomography (CT). Autologous blood clots formed in vitro were introduced sequentially into the pulmonary arteries of eight healthy mixed-breed dogs while monitoring pulmonary and systemic hemodynamic function. Blood clots were injected until the mean pulmonary artery pressure (MPAP) reached two-three times the baseline pressure, which was maintained up to 1 hour. The RV function was assessed by echocardiography and ECG-gated dual-source contrast CT. All animals survived the imaging procedure. The post-injection pulmonary angiograms showed extensive PE, and MPAP increased from 16.50±2.45 mmHg to 43.13±4.91 mmHg (P<0.001). On echocardiography, the RV fractional area change decreased from 42.06±3.36 to 27.96±3.54 (P<0.001), and the RV myocardial performance increased from 0.20±0.05 to 0.63±0.16 (P<0.001). On CT, the RV end-systolic volume increased from 11.11±1.81 ml to 24.71±4.60 ml (P<0.001), RV end-diastolic volume from 20.73±2.83 ml to 34.63±5.76 ml (P<0.001), and the four-chamber RV/left ventricular diameter ratio from 0.38±0.07 to 0.81±0.14 (P<0.001). Acute PE with RV dysfunction was established in a large animal model through controlled injection of autologous blood clots, which may be useful for developing and evaluating new therapeutic approaches for acute PE with RV dysfunction. Copyright © 2015 Elsevier Ltd. All rights reserved.

The t-PA-encapsulated PLGA nanoparticles shelled with CS or CS-GRGD alter both permeation through and dissolving patterns of blood clots compared with t-PA solution: an in vitro thrombolysis study.

PubMed

Wang, Shoei-Shen; Chou, Nai-Kuan; Chung, Tze-Wen

2009-12-01

Accelerated thrombolysis by pressure-driven permeation has been demonstrated in in vitro and in vivo animal models by using plasminogen activators (PAs) encapsulated liposomes or PEG microparticles. Recent reports have also described acceleration of thrombolysis using tissue type PA (t-PA) encapsulated in PLGA nanoparticles (NPs) coated with chitosan (CS) or CS-GRGD by interactions between the NPs and blood clots. However, the permeation through and dissolving patterns in thrombolysis with the aforementioned microparticles or NPs, which may be clinically relevant to the recovery status of the posttreatments, have not been reported. Therefore, this work studied such phenomena in thrombolysis with t-PA encapsulated in NPs. The t-PA solution and the NPs exhibited distinctly different permeation patterns of dissolved clots. Plasma permeates through clots showed a stream flow or burst flow phenomena when lyzed with NPs shelled with CS or CS-GRGD, respectively, whereas a diffusion pattern was observed in those lyzed with t-PA solution. At the outlet position of clots, the clots dissolved with PLGA/CS and PLGA/CS-GRGD NPs revealed extremely rough surfaces to a depth of 100 mum, indicating that a cross-permeation direction of clot lysis occurred, while those dissolved with t-PA solution showed slightly rough surfaces to a depth of 12 mum. Permeation through and clot dissolution patterns of thrombolysis with t-PA encapsulated in NPs shelled with CS or CS-GRGD distinctly differed from those dissolved with t-PA solutions in this in vitro thrombolysis model, These findings may be relevant to posttreatment of patients with conventional PA thrombolysis. Copyright 2008 Wiley Periodicals, Inc.

Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane.

PubMed

Dohan Ehrenfest, David M; Del Corso, Marco; Diss, Antoine; Mouhyi, Jaafar; Charrier, Jean-Baptiste

2010-04-01

Platelet-rich fibrin (PRF; Choukroun's technique) is a second-generation platelet concentrate for surgical use. This easy protocol allows the production of leukocyte and platelet-rich fibrin clots and membranes starting from 10-ml blood samples. The purposes of this study were to determine the cell composition and three-dimensional organization of this autologous biomaterial and to evaluate the influence of different collection tubes (dry glass or glass-coated plastic tubes) and compression procedures (forcible or soft) on the final PRF-membrane architecture. After centrifugation, blood analyses were performed on the residual waste plasmatic layers after collecting PRF clots. The PRF clots and membranes were processed for examination by light microscopy and scanning electron microscopy. Approximately 97% of the platelets and >50% of the leukocytes were concentrated in the PRF clot and showed a specific three-dimensional distribution, depending on the centrifugation forces. Platelets and fibrin formed large clusters of coagulation in the first millimeters of the membrane beyond the red blood cell base. The fibrin network was very mature and dense. Moreover, there was no significant difference in the PRF architecture between groups using the different tested collection tubes and compression techniques, even if these two parameters could have influenced the growth factor content and biologic matrix properties. The PRF protocol concentrated most platelets and leukocytes from a blood harvest into a single autologous fibrin biomaterial. This protocol offers reproducible results as long as the main production principles are respected.

A model comparing how rapidly transfusion of solvent detergent plasma restores clotting factors versus infusion of albumin-saline.

PubMed

Jilma-Stohlawetz, Petra; Kursten, Friedrich W; Horvath, Michaela; Leitner, Gerda; List, Jana; Marcek, Jana; Quehenberger, Peter; Schwameis, Michael; Bartko, Johann; Jilma, Bernd

2015-12-01

A recent randomized controlled trial demonstrated the bioequivalence between universally applicable and AB0 compatible transfusion plasma in healthy volunteers. There was a limited change in coagulation factor levels and inhibitors before and after plasmapheresis and subsequent plasma transfusion. The aim of this extension trial was to investigate the true capacity of these plasma products to restore baseline levels of coagulation factors and inhibitors after plasma depletion in comparison to haemodilution induced by infusion of albumin solution. Fourteen healthy subjects, who completed both plasma transfusion periods, underwent an additional plasmapheresis (600 mL) followed by an infusion of 1200 mL albumin (3.125%) in a third period. The fibrinogen levels, as well as other clotting factors (FII, FV, FVII and FXI), decreased by 10% after plasmapheresis, and subsequent infusion of albumin solution further aggravated this drop in clotting factors to approximately 20-25%. The clotting factors with a long half-life were not even restored 24 hours after infusion of albumin solution, whereas those with a short half-life were replenished by endogenous synthesis within 24 hours. In contrast, transfusion of either plasma product rapidly restored all clotting parameters and inhibitors (protein S and plasmin inhibitor) immediately after transfusion. This study demonstrates that albumin solution induces an enhanced dilution of clotting factors and inhibitors, whereas both plasma products quickly compensated for the experimental loss of these plasma proteins. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Haptoglobin Reduces Inflammatory Cytokine INF-γ and Facilitates Clot Formation in Acute Severe Burn Rat Model.

PubMed

Koami, Hiroyuki; Sakamoto, Yuichiro; Miyasho, Taku; Noguchi, Ryo; Sato, Norio; Kai, Keita; Chris Yamada, Kosuke; Inoue, Satoshi

2017-01-01

Haptoglobin exerts renal protective function by scavenging free hemoglobin from the urine and blood stream in patients with hemolytic disorders. Recent studies elucidate the relationships between haptoglobin and inflammation. In addition, coagulopathy is often induced by systemic inflammation characterized by the presence of vascular endothelial damage. We hypothesize that haptoglobin might have an anti-inflammatory effect and affect hypercoagulability using rat burn model. Thirty anesthetized rats of six-weeks of age received over 30% full-thickness scald burn on the dorsal skin surface. All rats were injected with either haptoglobin (Hpt) or normal saline (NS) intraperitoneally. The rats were divided into three groups: 1) control group (NS 20 mL/kg); 2) low concentration of Hpt group, L-Hpt, (Hpt 4 mL (80 U) /kg+NS 16 mL/kg); and 3) high concentration of Hpt group, H-Hpt, (Hpt 20 mL (400 U) /kg). While under anesthesia, all rats were euthanized by exsanguination at 6 hours (N=5) and 24 hours (N=5). Inflammatory and anti-inflammatory cytokines were measured and whole-blood viscoelastic tests were performed by thromboelastometry (ROTEM). Haptoglobin significantly reduced free hemoglobin 24 hours after the injury. Improvement of hematuria was confirmed in the H-Hpt group. There were no differences in thrombin-antithrombin complex and plasmin-α2 plasmin inhibitor complex. The haptoglobin tended to decrease interferon-gamma (IFN-γ) in H-Hpt group. ROTEM findings of the L-Hpt group showed significantly higher clot firmness and shorter time to maximum clot formation velocity than the control group. Haptoglobin reduced INF-γ, and accelerated speed of clot formation in acute phase of severe burn.

Kinetic Model Facilitates Analysis of Fibrin Generation and Its Modulation by Clotting Factors: Implications for Hemostasis-Enhancing Therapies

DTIC Science & Technology

2014-01-01

facilitates analysis of fibrin generation and its modulation by clotting factors : implications for hemostasis-enhancing therapies† Alexander Y...investigate the ability of fibrinogen and a recently proposed prothrombin complex concentrate composition, PCC-AT (a combination of the clotting factors II...kinetics. Moreover, the model qualitatively predicted the impact of tissue factor and tPA/tenecteplase level variations on the fibrin output. In the

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer.

PubMed

Nielsen, Vance G; Matika, Ryan W; Ley, Michele L B; Waer, Amy L; Gharagozloo, Farid; Kim, Samuel; Nfonsam, Valentine N; Ong, Evan S; Jie, Tun; Warneke, James A; Steinbrenner, Evangelina B

2014-04-01

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

Genome scan of clot lysis time and its association with thrombosis in a protein C deficient kindred

PubMed Central

Meltzer, M.E.; Hasstedt, S.J.; Vossen, C.Y.; Callas, P.W.; de Groot, Ph.G.; Rosendaal, F.R.; Lisman, T.; Bovill, E.G.

2011-01-01

Summary Background Previously we found increased clot lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. Genes influencing CLT are yet unknown. Objectives and Patients/Methods We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n=346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore we tested for quantitative trait loci (QTL) for CLT using variance component linkage analysis. Results Protein C deficient family members had shorter CLT than non-deficient members (median CLT 67 versus 75 minutes). One standard deviation increase in CLT increased risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. Heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin Activatable Fibrinolysis Inhibitor genotypes did not explain the variation in CLT. Conclusion Hypofibrinolysis appears to increase thrombosis risk in this family especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTL were found on chromosome 11 and 13. PMID:21575129

Thrombin activatable fibrinolysis inhibitor and clot lysis time in pregnant patients with antiphospholipid syndrome: relationship with pregnancy outcome and thrombosis.

PubMed

Martinez-Zamora, Maria Angeles; Tassies, Dolors; Carmona, Francisco; Espinosa, Gerard; Cervera, Ricard; Reverter, Juan Carlos; Balasch, Juan

2009-12-01

Antiphospholipid syndrome (APS) pregnancies are associated with thrombotic obstetric complications, despite treatment. This study evaluated Thrombin Activatable Fibrinolysis Inhibitor (TAFI) levels, TAFI gene polymorphisms and Clot Lysis Time (CLT) in pregnant patients with APS in relation to pregnancy outcome and thrombosis. Group 1 consisted of 67 pregnant patients with APS. Group 2 included 66 pregnant patients with uneventful term pregnancies and delivery. Patients were sampled during each trimester and at baseline. TAFI antigen and CLT and two polymorphisms of the TAFI gene, Ala147Thr and +1542C/G, were determined. Significantly prolonged CLT was found at baseline in Group 1. Allele distribution of the TAFI gene polymorphisms was similar in both groups. Basal TAFI and CLT in patients with APS having an adverse or a good obstetrical outcome were similar. Comparison of TAFI and CLT baseline levels in patients with APS with or without previous thrombosis showed no statistical differences. Patients with APS have impairment in fibrinolysis evidenced by prolonged CLT at baseline. TAFI and CLT do not seem to be useful as markers of obstetric outcome or risk of thrombosis in patients with APS.

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism.

PubMed

Stubblefield, William B; Alves, Nathan J; Rondina, Matthew T; Kline, Jeffrey A

2016-01-01

We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.

Spirulan from blue-green algae inhibits fibrin and blood clots: its potent antithrombotic effects.

PubMed

Choi, Jun-Hui; Kim, Seung; Kim, Sung-Jun

2015-05-01

We investigated in vitro and in vivo fibrinolytic and antithrombotic activity of spirulan and analyzed its partial biochemical properties. Spirulan, a sulfated polysaccharide from the blue-green alga Arthrospira platensis, exhibits antithrombotic potency. Spirulan showed a strong fibrin zymogram lysis band corresponding to its molecular mass. It specifically cleaved Aα and Bβ, the major chains of fibrinogen. Spirulan directly decreased the activity of thrombin and factor X activated (FXa), procoagulant proteins. In vitro assays using human fibrin and mouse blood clots showed fibrinolytic and hemolytic activities of spirulan. Spirulan (2 mg/kg) showed antithrombotic effects in the ferric chloride (FeCl3 )-induced carotid arterial thrombus model and collagen and epinephrine-induced pulmonary thromboembolism mouse model. These results may be attributable to the prevention of thrombus formation and partial lysis of thrombus. Therefore, we suggest that spirulan may be a potential antithrombotic agent for thrombosis-related diseases. © 2015 Wiley Periodicals, Inc.

Lysability of fibrin clots is a potential new determinant of stroke risk in atrial fibrillation.

PubMed

Skov, Jane; Sidelmann, Johannes J; Bladbjerg, Else-Marie; Jespersen, Jørgen; Gram, Jørgen

2014-09-01

Atrial fibrillation increases the risk of ischemic stroke, but the risk depends on other factors as well. Present risk stratification schemes use age and co-morbidities, but not biochemical markers. We investigated the hypothesis that the formation, structure and lysability of fibrin clots are potential determinants of stroke risk in patients with atrial fibrillation. A total of 179 patients with atrial fibrillation in stable anticoagulant treatment were included. Thirty-two had a previous ischemic stroke. We measured thrombin generation, plasma concentrations of fibrinogen and C-reactive protein and analysed fibrin structure and lysability by turbidity. Fibrinolytic capacity was measured using the euglobulin fraction of plasma expressed in terms of t-PA equivalents (IU/ml). The patients with previous stroke had a slightly higher burden of co-morbidities compared with the remaining patients as indicated by the CHA2DS2-VASc score, but no significant differences were found regarding age, fibrinogen concentration, C-reactive protein, thrombin generation or fibrinolytic capacity. Furthermore, the patients with previous stroke had a higher mass/length ratio of fibrin fibers (5.5 vs. 5.1 x10(12) Da/cm, p=0.044) and an increased lysability (79.3 vs. 55.3%, p<0.01). The higher lysability of fibrin clots in atrial fibrillation patients with previous stroke is most likely a result of a difference in fibrin fiber properties. An increased lysability may increase the risk of embolization of clots formed in the atria, and therefore fibrin clot structure seems to be a determinant of stroke risk in atrial fibrillation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Random Forests Are Able to Identify Differences in Clotting Dynamics from Kinetic Models of Thrombin Generation.

PubMed

Arumugam, Jayavel; Bukkapatnam, Satish T S; Narayanan, Krishna R; Srinivasa, Arun R

2016-01-01

Current methods for distinguishing acute coronary syndromes such as heart attack from stable coronary artery disease, based on the kinetics of thrombin formation, have been limited to evaluating sensitivity of well-established chemical species (e.g., thrombin) using simple quantifiers of their concentration profiles (e.g., maximum level of thrombin concentration, area under the thrombin concentration versus time curve). In order to get an improved classifier, we use a 34-protein factor clotting cascade model and convert the simulation data into a high-dimensional representation (about 19000 features) using a piecewise cubic polynomial fit. Then, we systematically find plausible assays to effectively gauge changes in acute coronary syndrome/coronary artery disease populations by introducing a statistical learning technique called Random Forests. We find that differences associated with acute coronary syndromes emerge in combinations of a handful of features. For instance, concentrations of 3 chemical species, namely, active alpha-thrombin, tissue factor-factor VIIa-factor Xa ternary complex, and intrinsic tenase complex with factor X, at specific time windows, could be used to classify acute coronary syndromes to an accuracy of about 87.2%. Such a combination could be used to efficiently assay the coagulation system.

Application of actuator-driven pulsed water jet in aneurysmal subarachnoid hemorrhage surgery: its effectiveness for dissection around ruptured aneurysmal walls and subarachnoid clot removal.

PubMed

Endo, Hidenori; Endo, Toshiki; Nakagawa, Atsuhiro; Fujimura, Miki; Tominaga, Teiji

2017-07-01

In clipping surgery for aneurysmal subarachnoid hemorrhage (aSAH), critical steps include clot removal and dissection of aneurysms without premature rupture or brain injuries. To pursue this goal, a piezo actuator-driven pulsed water jet (ADPJ) system was introduced in this study. This study included 42 patients, who suffered aSAH and underwent clipping surgery. Eleven patients underwent surgery with the assistance of the ADPJ system (ADPJ group). In the other 31 patients, surgery was performed without the ADPJ system (Control group). The ADPJ system was used for clot removal and aneurysmal dissection. The clinical impact of the ADPJ system was judged by comparing the rate of premature rupture, degree of clot removal, and clinical outcomes. Intraoperatively, a premature rupture was encountered in 18.2 and 25.8% of cases in the ADPJ and control groups, respectively. Although the differences were not statistically significant, intraoperative observation suggested that the ADPJ system was effective in clot removal and dissection of aneurysms in a safe manner. Computed tomography scans indicated the achievement of higher degrees of clot removal, especially when the ADPJ system was used for cases with preoperative clot volumes of more than 25 ml (p = 0.047, Mann-Whitney U test). Clinical outcomes, including incidence of postoperative brain injury or symptomatic vasospasm, were similar in both groups. We described our preliminary surgical results using the ADPJ system for aSAH. Although further study is needed, the ADPJ system was considered a safe and effective tool for clot removal and dissection of aneurysms.

Concentration-dependent effect of hypocalcaemia on in vitro clot strength in patients at risk of bleeding: a retrospective cohort study.

PubMed

Ho, K M; Yip, C B

2016-02-01

It is uncertain whether hypocalcaemia is associated with an increased risk of bleeding. This study assessed the dose-related relationship between ionised calcium concentrations and in vitro clot strength measured by maximum amplitude (MA) on the thromboelastograph (TEG). A total of 610 patients who were at risk of bleeding or had active bleeding between 2010 and 2014 were considered in this retrospective cohort study. A scatter plot with Pearson correlation coefficient (r) and multiple linear regression was used to assess the dose-related relationship between ionised calcium concentrations and MA on the TEG. The mean ionised calcium of the patients was 1·10 mmol L(-1) (interquartile range: 1·04-1·17) and 235 (38·5%) of them had hypocalcaemia (<1·1 mmol L(-1) ). Hypocalcaemia was more common in patients with significant coexisting coagulopathy. Ionised calcium concentrations (r = 0·285, 95% confidence interval (CI) 0·211-0·356, P = 0·001), as well as fibrinogen concentrations, platelet counts, international normalised ratio (INR) and activated Partial Thromboplastin Time (aPTT), had a significant linear correlation with the MA on the TEG. Ionised calcium concentrations and its interaction term with platelet count were both significantly associated with the MA on the TEG (slope of the regression line 1·1 per 0·1 mmol L(-1) increment, 95%CI 0·3 to 1·9, P = 0·011), after adjusting for fibrinogen concentrations, platelet counts, INR and aPTT. Ionised calcium concentrations had a concentration-dependent association with in vitro clot strength after adjusting for other coagulation abnormalities in patients with coexisting coagulopathy. Maintaining a normal ionised calcium concentration, >1 mmol L(-1) , during critical bleeding is recommended. © 2016 British Blood Transfusion Society.

Optical aggregometry of red blood cells associated with the blood-clotting reaction in extracorporeal circulation support.

PubMed

Sakota, Daisuke; Kosaka, Ryo; Nishida, Masahiro; Maruyama, Osamu

2016-09-01

The aggregability of red blood cell (RBCs) is associated with the contribution of plasma proteins, such as fibrinogen and lipoproteids, to blood-clotting. Hence, we hypothesized that RBC aggregability reflects the blood-clotting reaction. A noninvasive optical monitoring method to measure RBC aggregability for the assessment of blood-clotting stage during mechanical circulatory support was developed. An in vitro thrombogenic test was conducted with a rotary blood pump using heparinized fresh porcine blood. Near-infrared laser light at a wavelength of 785 nm was guided by an optical fiber. The fibers for detecting incident, forward-, and backward-scattered light were fixed on the circuit tubing with an inner diameter of 1/4 inch. Because there is substantial RBC aggregation at low shear flow rates, a pulsatile flow was generated by controlling the pump rotational speed. The flow rate was changed from 0 to 8.5 L/min at a period of 40 s. The intensities of forward- and backward-scattered light changed dramatically when the flow stopped. The aggregability was evaluated by the increase ratio of the transmitted light intensity from the flow stopping in the low-flow condition. The experiment started when the anticoagulation was stopped by the addition of protamine into the circulating blood. Reduction in RBC aggregability was associated with a decrease in the amount of fibrinogen and the number of platelets. Continuous, noninvasive monitoring of thrombosis risk is possible using optical measurements combining pulsatile flow control of a rotary blood pump. RBC aggregometry is a potential label-free method for evaluating blood-clotting risk.

Evaluation of Optical Coherence Tomography for the Measurement of the Effects of Activators and Anticoagulants on the Blood Coagulation In Vitro

PubMed Central

Geng, Jinhai; Liu, Gangjun; Chen, Zhongping

2013-01-01

Optical properties of human blood during coagulation were studied using optical coherence tomography (OCT) and the parameter of clotting time derived from the 1/e light penetration depth (d1/e) versus time was developed in our previous work. In this study, in order to know if a new OCT test can characterize the blood-coagulation process under different treatments in vitro, the effects of two different activators (calcium ions and thrombin) and anticoagulants, i.e., acetylsalicylic acid (ASA, a well-known drug aspirin) and melagatran (a direct thrombin inhibitor), at various concentrations are evaluated. A swept-source OCT system with a 1300 nm center wavelength is used for detecting the blood-coagulation process in vitro under a static condition. A dynamic study of d1/e reveals a typical behavior due to coagulation induced by both calcium ions and thrombin, and the clotting time is concentration-dependent. Dose-dependent ASA and melagatran prolong the clotting times. ASA and melagatran have different effects on blood coagulation. As expected, melagatran is much more effective than ASA in anticoagulation by the OCT measurements. The OCT assay appears to be a simple method for the measurement of blood coagulation to assess the effects of activators and anticoagulants, which can be used for activator and anticoagulant screening. PMID:23392340

Evaluation of optical coherence tomography for the measurement of the effects of activators and anticoagulants on the blood coagulation in vitro.

PubMed

Xu, Xiangqun; Geng, Jinhai; Liu, Gangjun; Chen, Zhongping

2013-08-01

Optical properties of human blood during coagulation were studied using optical coherence tomography (OCT) and the parameter of clotting time derived from the 1/e light penetration depth (d(1/e)) versus time was developed in our previous work. In this study, in order to know if a new OCT test can characterize the blood-coagulation process under different treatments in vitro, the effects of two different activators (calcium ions and thrombin) and anticoagulants, i.e., acetylsalicylic acid (ASA, a well-known drug aspirin) and melagatran (a direct thrombin inhibitor), at various concentrations are evaluated. A swept-source OCT system with a 1300 nm center wavelength is used for detecting the blood-coagulation process in vitro under a static condition. A dynamic study of d1/e reveals a typical behavior due to coagulation induced by both calcium ions and thrombin, and the clotting time is concentration-dependent. Dose-dependent ASA and melagatran prolong the clotting times. ASA and melagatran have different effects on blood coagulation. As expected, melagatran is much more effective than ASA in anticoagulation by the OCT measurements. The OCT assay appears to be a simple method for the measurement of blood coagulation to assess the effects of activators and anticoagulants, which can be used for activator and anticoagulant screening.

Genome scan of clot lysis time and its association with thrombosis in a protein C-deficient kindred.

PubMed

Meltzer, M E; Hasstedt, S J; Vossen, C Y; Callas, P W; DE Groot, Ph G; Rosendaal, F R; Lisman, T; Bovill, E G

2011-07-01

 Previously, we found increased clot-lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. The genes influencing CLT are as yet unknown.  We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis.  Protein C-deficient family members had shorter CLTs than non-deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. The heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin-activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13. © 2011 International Society on Thrombosis and Haemostasis.

The microscale evolution of the erosion front of blood clots exposed to ultrasound stimulated microbubbles.

PubMed

Acconcia, Christopher N; Leung, Ben Y C; Goertz, David E

2016-05-01

Serial two-photon microscopy of blood clots with fluorescently tagged fibrin networks was conducted during microbubble-mediated sonothrombolysis to examine the microscale evolution of the resulting erosion front. The development of a complex zonal erosion pattern was observed, comprised of a cell depleted layer of fibrin network overlying intact clot which then underwent progressive recession. The fibrin zone architecture was dependent on exposure conditions with 0.1 MPa causing no erosion, 0.39 MPa resulting in homogenous structure, and combination 0.39/0.96 MPa pulses forming large-scale tunnels. High speed imaging and Coulter counter data indicated the fibrin zone formation process involves the ejection of intact erythrocytes.

Comparison of various solutions to dissolve critical care diet clots.

PubMed

Parker, Valerie J; Freeman, Lisa M

2013-01-01

Enteral feeding tubes are frequently placed in animals to provide assisted nutritional support; however, one major reported complication is clogging of the tubes. The goal of this study was to determine which solution is most effective at dissolving in vitro clots made using a veterinary canned critical care diet. Various solutions were tested for their ability to dissolve enteral feed clots, including water, meat tenderizers in water, predetermined amounts of pancreatic enzymes (with and without sodium bicarbonate) in water, carbonated beverages, and cranberry juice. The solution that resulted in the greatest dissolution was ¼ teaspoon pancreatic enzymes and 325 mg sodium bicarbonate in 5 mL water, which was significantly better than all other solutions (water: P = 0.03; ¼ teaspoon pancreatic enzymes in water: P = 0.002; all others: P < 0.001). Water was significantly better than all carbonated beverages and cranberry juice (P < 0.001). The least successful solution was ½ teaspoon pancreatic enzymes and sodium bicarbonate in water. Despite anecdotal reports of using carbonated beverages, cranberry juice, and ½ teaspoon pancreatic enzymes to unclog feeding tubes, all were significantly less effective than water. In vivo studies to evaluate the effectiveness of methods to unclog feeding tubes are warranted to further investigate these findings. © Veterinary Emergency and Critical Care Society 2013.

Thrombin generation potential and clot-forming capacity of thawed fresh-frozen plasma, plasma frozen within 24 h and solvent/detergent-treated plasma (octaplasLG® ), during 5-day storage at 1-6°C.

PubMed

Heger, A; Neisser-Svae, A; Trawnicek, L; Triulzi, D

2018-04-23

To enable rapid availability of plasma in emergency situations, the shelf-life of thawed fresh-frozen plasma (FFP) has been extended from 24 h to 5 days. The aim of this study was to evaluate the thrombin generation (TG) potential and clot-forming ability during 5 days of refrigerated storage of thawed FFP, plasma frozen within 24 h and solvent/detergent-treated plasma octaplasLG ® . During storage for 5 days, TG capacity decreased significantly over time, and rotational thromboelastometry showed significantly prolonged clotting times. However, the stability studies confirmed comparable in vitro haemostatic potentials of all three thawed plasma products at day 5. © 2018 International Society of Blood Transfusion.

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

PubMed Central

Stubblefield, William B.; Alves, Nathan J.; Rondina, Matthew T.; Kline, Jeffrey A.

2016-01-01

Background We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Methods Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Results Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. Conclusion The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT. PMID:26866684

Treatment protocol based on assessment of clot quality during endovascular thrombectomy for acute ischemic stroke using the Trevo stent retriever

PubMed Central

Ishikawa, Kojiro; Ohshima, Tomotaka; Nishihori, Masahiro; Imai, Tasuku; Goto, Shunsaku; Yamamoto, Taiki; Nishizawa, Toshihisa; Shimato, Shinji; Kato, Kyozo

2016-01-01

ABSTRACT The optional endovascular approach for acute ischemic stroke is unclear. The Trevo stent retriever can be used as first-line treatment for fast mechanical recanalization. The authors developed a treatment protocol for acute ischemic stroke based on the assessment of clot quality during clot removal with the Trevo. This prospective single-center study included all patients admitted for acute ischemic stroke between July 2014 and February 2015, who underwent emergency endovascular treatment. According to the protocol, the Trevo was used for first-line treatment. Immediately after the Trevo was deployed, the stent delivery wire was pushed to open the stent by force (ACAPT technique). Clot quality was assessed on the basis of the perfusion status after deployment of the Trevo; continued occlusion or immediate reopening either reoccluded or maintained after the stent retriever had been in place for 5 min. If there was no obvious clot removal after the first pass with the Trevo, according to the quality of the clot, either a second pass was performed or another endovascular device was selected. Twelve consecutive patients with acute major cerebral artery occlusion were analyzed. Thrombolysis in cerebral infarction score 2b and 3 was achieved in 11 patients (91.7%) and 9 (75%) had a good clinical outcome after 90 days based on a modified Rankin scale score ≤ 2. Symptomatic intracranial hemorrhage occurred in 1 patient (8.3%). The overall mortality rate was 8.3%. Endovascular thrombectomy using the Trevo stent retriever for first-line treatment is feasible and effective. PMID:27578909

Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma.

PubMed

Dos Santos, Danielle Cabral; Lange, Carla Christine; Avellar-Costa, Pedro; Dos Santos, Katia Regina Netto; Brito, Maria Aparecida Vasconcelos Paiva; Giambiagi-deMarval, Marcia

2016-05-01

Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A Model Incorporating Some of the Mechanical and Biochemical Factors Underlying Clot Formation and Dissolution in Flowing Blood

DOE PAGES

Anand, M.; Rajagopal, K.; Rajagopal, K. R.

2003-01-01

Multiple interacting mechanisms control the formation and dissolution of clots to maintain blood in a state of delicate balance. In addition to a myriad of biochemical reactions, rheological factors also play a crucial role in modulating the response of blood to external stimuli. To date, a comprehensive model for clot formation and dissolution, that takes into account the biochemical, medical and rheological factors, has not been put into place, the existing models emphasizing either one or the other of the factors. In this paper, after discussing the various biochemical, physiologic and rheological factors at some length, we develop a modelmore » for clot formation and dissolution that incorporates many of the relevant crucial factors that have a bearing on the problem. The model, though just a first step towards understanding a complex phenomenon, goes further than previous models in integrating the biochemical, physiologic and rheological factors that come into play.« less

Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition.

PubMed

Cao, Lan; Chang, Mark; Lee, Chi-Ying; Castner, David G; Sukavaneshvar, Sivaprasad; Ratner, Buddy D; Horbett, Thomas A

2007-06-15

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions. (c) 2007 Wiley Periodicals, Inc.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow.

PubMed

Zhu, Shu; Travers, Richard J; Morrissey, James H; Diamond, Scott L

2015-09-17

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) -bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm(2). Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm(2) and sensitive to O1A6 at 0 to 0.2 molecules per µm(2). However, neither antibody reduced fibrin generation at ∼2 molecules per µm(2) when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm(2)) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. © 2015 by The American Society of Hematology.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow

PubMed Central

Zhu, Shu; Travers, Richard J.; Morrissey, James H.

2015-01-01

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) –bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm2. Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm2 and sensitive to O1A6 at 0 to 0.2 molecules per µm2. However, neither antibody reduced fibrin generation at ∼2 molecules per µm2 when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm2) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall. PMID:26136249

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking

PubMed Central

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A.; Moncman, Carole L.

2016-01-01

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate–ribosylation factor 6 (Arf6) is a small guanosine triphosphate–binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)–labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. PMID:26738539

Arf6 controls platelet spreading and clot retraction via integrin αIIbβ3 trafficking.

PubMed

Huang, Yunjie; Joshi, Smita; Xiang, Binggang; Kanaho, Yasunori; Li, Zhenyu; Bouchard, Beth A; Moncman, Carole L; Whiteheart, Sidney W

2016-03-17

Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbβ3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbβ3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbβ3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function. © 2016 by The American Society of Hematology.

Confinement regulates complex biochemical networks: initiation of blood clotting by "diffusion acting".

PubMed

Shen, Feng; Pompano, Rebecca R; Kastrup, Christian J; Ismagilov, Rustem F

2009-10-21

This study shows that environmental confinement strongly affects the activation of nonlinear reaction networks, such as blood coagulation (clotting), by small quantities of activators. Blood coagulation is sensitive to the local concentration of soluble activators, initiating only when the activators surpass a threshold concentration, and therefore is regulated by mass transport phenomena such as flow and diffusion. Here, diffusion was limited by decreasing the size of microfluidic chambers, and it was found that microparticles carrying either the classical stimulus, tissue factor, or a bacterial stimulus, Bacillus cereus, initiated coagulation of human platelet-poor plasma only when confined. A simple analytical argument and numerical model were used to describe the mechanism for this phenomenon: confinement causes diffusible activators to accumulate locally and surpass the threshold concentration. To interpret the results, a dimensionless confinement number, Cn, was used to describe whether a stimulus was confined, and a Damköhler number, Da(2), was used to describe whether a subthreshold stimulus could initiate coagulation. In the context of initiation of coagulation by bacteria, this mechanism can be thought of as "diffusion acting", which is distinct from "diffusion sensing". The ability of confinement and diffusion acting to change the outcome of coagulation suggests that confinement should also regulate other biological "on" and "off" processes that are controlled by thresholds.

High-throughput proteomic characterization of plasma rich in growth factors (PRGF-Endoret)-derived fibrin clot interactome.

PubMed

Anitua, Eduardo; Prado, Roberto; Azkargorta, Mikel; Rodriguez-Suárez, Eva; Iloro, Ibon; Casado-Vela, Juan; Elortza, Felix; Orive, Gorka

2015-11-01

Plasma rich in growth factors (PRGF®-Endoret®) is an autologous technology that contains a set of proteins specifically addressed to wound healing and tissue regeneration. The scaffold formed by using this technology is a clot mainly composed of fibrin protein, forming a three-dimensional (3D) macroscopic network. This biomaterial is easily obtained by biotechnological means from blood and can be used in a range of situations to help wound healing and tissue regeneration. Although the main constituent of this clot is the fibrin scaffold, little is known about other proteins interacting in this clot that may act as adjuvants in the healing process. The aim of this study was to characterize the proteins enclosed by PRGF-Endoret scaffold, using a double-proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS, and 2-DE followed by MALDI-TOF/TOF. The results presented here provide a description of the catalogue of key proteins in close contact with the fibrin scaffold. The obtained lists of proteins were grouped into families and networks according to gene ontology. Taken together, an enrichment of both proteins and protein families specifically involved in tissue regeneration and wound healing has been found. Copyright © 2013 John Wiley & Sons, Ltd.

How pH, Temperature, and Time of Incubation Affect False-Positive Responses and Uncertainty of the LAL Gel-Clot Test.

PubMed

Lourenço, Felipe Rebello; Botelho, Túlia De Souza; Pinto, Terezinha De Jesus Andreoli

2012-01-01

The limulus amebocyte lysate (LAL) test is the simplest and most widely used procedure for detection of endotoxin in parenteral drugs. The LAL test demands optimal pH, ionic strength, temperature, and time of incubation. Slight changes in these parameters may increase the frequency of false-positive responses and the estimated uncertainty of the LAL test. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. LAL tests were performed in nominal conditions (37 °C, 60 min, and pH 7) and in different conditions of temperature (36 °C and 38 °C), time of incubation (58 and 62 min), and pH (6 and 8). Slight differences in pH increase the frequency of false-positive responses 5-fold (relative risk 5.0), resulting in an estimated of uncertainty 7.6%. Temperature and time of incubation affect the LAL test less, showing relative risks of 1.5 and 1.0, respectively. Estimated uncertainties in 36 °C or 38 °C temperatures and 58 or 62 min of incubation were found to be 2.0% and 1.0%, respectively. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses. The limulus amebocyte lysate (LAL) gel-clot test is a simple test for detection of endotoxin from Gram-negative bacteria. The test is based on a gel formation when a certain amount of endotoxin is present; it is a pass/fail test. The LAL test requires optimal pH, ionic strength, temperature, and time of incubation. Slight difference in these parameters may increase the frequency of false-positive responses. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. We find that slight differences in pH increase the frequency of false-positive responses 5-fold. Temperature and time of incubation affect the LAL test less. Simultaneous differences in these parameters

Clustering of Cyclodextrin-Functionalized Microbeads by an Amphiphilic Biopolymer: Real-Time Observation of Structures Resembling Blood Clots.

PubMed

Arya, Chandamany; Saez Cabesas, Camila A; Huang, Hubert; Raghavan, Srinivasa R

2017-10-25

Colloidal particles can be induced to cluster by adding polymers in a process called bridging flocculation. For bridging to occur, the polymer must bind strongly to the surfaces of adjacent particles, such as via electrostatic interactions. Here, we introduce a new system where bridging occurs due to specific interactions between the side chains of an amphiphilic polymer and supramolecules on the particle surface. The polymer is a hydrophobically modified chitosan (hmC) while the particles are uniform polymeric microbeads (∼160 μm in diameter) made by a microfluidic technique and functionalized on their surface by α-cyclodextrins (CDs). The CDs have hydrophobic binding pockets that can capture the n-alkyl hydrophobes present along the hmC chains. Clustering of CD-coated microbeads in water by hmC is visualized in real time using optical microscopy. Interestingly, the clustering follows two distinct stages: first, the microbeads are bridged into clusters by hmC chains, which occurs by the interaction of individual chains with the CDs on adjacent particles. Thereafter, additional hmC from the solution adsorbs onto the surfaces of the microbeads and an hmC "mesh" grows around the clusters. This growing nanostructured mesh can trap surrounding microsized objects and sequester them within the overall cluster. Such clustering is reminiscent of blood clotting where blood platelets initially cluster at a wound site, whereupon they induce growth of a protein (fibrin) mesh around the clusters, which entraps other passive cells. Clustering does not occur with the native chitosan (lacking hydrophobes) or with the bare particles (lacking CDs); these results confirm that the clustering is indeed due to hydrophobic interactions between the hmC and the CDs. Microbead clustering via amphiphilic biopolymers could be applicable in embolization, which is a surgical technique used to block blood flow to a particular area of the body, or in agglutination assays.

Correlation between classical rheometry and supersonic shear wave imaging in blood clots.

PubMed

Bernal, Miguel; Gennisson, Jean-Luc; Flaud, Patrice; Tanter, Mickael

2013-11-01

The assessment of coagulating blood elasticity has gained importance as a result of several studies that have correlated it to cardiovascular pathologic conditions. In this study we use supersonic shear wave imaging (SSI) to measure viscoelastic properties of blood clots. At the same time, classical rheometry experiments were carried out on the same blood samples taken within the first few seconds of coagulation. Using SSI, phase velocities of the shear wave indicated increasing dispersion with time. In all cases, the frequency bandwidth of propagating shear waves changed from 20-50 Hz at the first few min of coagulation to around 300 Hz toward the end of experiments. Using the values of G' and G″ from the rheometry studies, the theoretical shear wave velocities were calculated and correlated with SSI measurements. Results of the two techniques were in very good agreement, confirming that SSI provides accurate measurements of viscoelastic properties as corroborated by conventional rheometric measurements. Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Thrombolytic along with anti-platelet activity of crinumin, a protein constituent of Crinum asiaticum.

PubMed

Singh, Kunwar Awaneesh; Nayak, Manasa K; Jagannadham, Medicherla V; Dash, Debabrata

2011-08-15

Several anticoagulants, anti-platelet and thrombolytic medications are used for the treatment of thrombotic disorders. Anti-coagulants and anti-platelet agents prevent the formation of blood clots but do not dissolve existing clots, whereas thrombolytic agents are able to dissolve a clot but emboli can form even after successful treatment. Thus, none of them provide a permanent and complete solution. In this regard a single molecule that could both dissolve the clot and prevent the formation of new clots would be useful in the treatment of thrombotic diseases. Crinumin, a stable and active (in many adverse conditions) serine protease, shows plasmin-like fibrinolytic activity and inhibits platelet aggregation and P-selectin exposure, as established by photography, phase contrast microscopy, whole blood optical Lumi-aggregometry and flow cytometry. Crinumin could be an efficient and inexpensive therapeutic agent for the treatment and prevention of thromboembolic diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

Magnetically Driven Flows of Suspensions of Rods to Deliver Clot-Busting Drugs to Dead-End Arteries

NASA Astrophysics Data System (ADS)

Bonnecaze, Roger; Clements, Michael

2014-11-01

Suspensions of iron particles in the presence of a magnetic field create flows that could significantly increase the delivery of drugs to dissolve clots in stroke victims. An explanation of this flow rests on the foundation of the seminal works by Prof. Acrivos and his students on effective magnetic permittivity of suspensions of rods, hydrodynamic diffusion of particles, and the flow of suspensions. Intravenous administration of the clot dissolving tissue plasminogen activator (tPA) is the most used therapy for stroke. This therapy is often unsuccessful because the tPA delivery is diffusion-limited and too slow to be effective. Observations show that added iron particles in a rotating magnetic field form rotating rods along the wall of the occluded vessel, creating a convective flow that can carry tPA much faster than diffusion. We present a proposed mechanism for this magnetically driven flow in the form of coupled particle-scale and vessel-scale flow models. At the particle-scale, particles chain up to form rods that rotate, diffuse and translate in the presence of the flow and magnetic fields. Localized vorticity created by the rotating particles drives a macroscopic convective flow in the vessel. Suspension transport equations describe the flow at the vessel-scale. The flow affects the convection and diffusion of the suspension of particles, linking the two scales. The model equations are solved asymptotically and numerically to understand how to create convective flows in dead-end or blocked vessels.

Computational Study of Thrombus Formation and Clotting Factor Effects under Venous Flow Conditions

PubMed Central

Govindarajan, Vijay; Rakesh, Vineet; Reifman, Jaques; Mitrophanov, Alexander Y.

2016-01-01

A comprehensive understanding of thrombus formation as a physicochemical process that has evolved to protect the integrity of the human vasculature is critical to our ability to predict and control pathological states caused by a malfunctioning blood coagulation system. Despite numerous investigations, the spatial and temporal details of thrombus growth as a multicomponent process are not fully understood. Here, we used computational modeling to investigate the temporal changes in the spatial distributions of the key enzymatic (i.e., thrombin) and structural (i.e., platelets and fibrin) components within a growing thrombus. Moreover, we investigated the interplay between clot structure and its mechanical properties, such as hydraulic resistance to flow. Our model relied on the coupling of computational fluid dynamics and biochemical kinetics, and was validated using flow-chamber data from a previous experimental study. The model allowed us to identify the distinct patterns characterizing the spatial distributions of thrombin, platelets, and fibrin accumulating within a thrombus. Our modeling results suggested that under the simulated conditions, thrombin kinetics was determined predominantly by prothrombinase. Furthermore, our simulations showed that thrombus resistance imparted by fibrin was ∼30-fold higher than that imparted by platelets. Yet, thrombus-mediated bloodflow occlusion was driven primarily by the platelet deposition process, because the height of the platelet accumulation domain was approximately twice that of the fibrin accumulation domain. Fibrinogen supplementation in normal blood resulted in a nonlinear increase in thrombus resistance, and for a supplemented fibrinogen level of 48%, the thrombus resistance increased by ∼2.7-fold. Finally, our model predicted that restoring the normal levels of clotting factors II, IX, and X while simultaneously restoring fibrinogen (to 88% of its normal level) in diluted blood can restore fibrin generation to

Treatment of Knee Osteochondral Lesions Using a Novel Clot of Autologous Plasma Rich in Growth Factors Mixed with Healthy Hyaline Cartilage Chips and Intra-Articular Injection of PRGF

PubMed Central

Alentorn-Geli, Eduard; Steinbacher, Gilbert; Álvarez-Díaz, Pedro; Cuscó, Xavier; Seijas, Roberto; Barastegui, David; Navarro, Jordi; Laiz, Patricia; García-Balletbó, Montserrat

2017-01-01

Knee cartilage or osteochondral lesions are common and challenging injuries. To date, most symptomatic lesions warrant surgical treatment. We present two cases of patients with knee osteochondral defects treated with a one-step surgical procedure consisting of an autologous-based matrix composed of healthy hyaline cartilage chips, mixed plasma poor-rich in platelets clot, and plasma rich in growth factors (PRGF). Both patients returned to playing soccer at the preinjury activity level and demonstrated excellent defect filling in both magnetic resonance imaging and second-look arthroscopy (in one of them). The use of a clot of autologous plasma poor in platelets with healthy hyaline cartilage chips and intra-articular injection of plasma rich in platelets is an effective, easy, and cheap option to treat knee cartilage injuries in young and athletic patients. PMID:28798878

Treatment of Knee Osteochondral Lesions Using a Novel Clot of Autologous Plasma Rich in Growth Factors Mixed with Healthy Hyaline Cartilage Chips and Intra-Articular Injection of PRGF.

PubMed

Cugat, Ramón; Alentorn-Geli, Eduard; Steinbacher, Gilbert; Álvarez-Díaz, Pedro; Cuscó, Xavier; Seijas, Roberto; Barastegui, David; Navarro, Jordi; Laiz, Patricia; García-Balletbó, Montserrat

2017-01-01

Knee cartilage or osteochondral lesions are common and challenging injuries. To date, most symptomatic lesions warrant surgical treatment. We present two cases of patients with knee osteochondral defects treated with a one-step surgical procedure consisting of an autologous-based matrix composed of healthy hyaline cartilage chips, mixed plasma poor-rich in platelets clot, and plasma rich in growth factors (PRGF). Both patients returned to playing soccer at the preinjury activity level and demonstrated excellent defect filling in both magnetic resonance imaging and second-look arthroscopy (in one of them). The use of a clot of autologous plasma poor in platelets with healthy hyaline cartilage chips and intra-articular injection of plasma rich in platelets is an effective, easy, and cheap option to treat knee cartilage injuries in young and athletic patients.

Antithrombin, an Important Inhibitor in Blood Clots.

PubMed

Zhu, Ying; Cong, Qing-Wei; Liu, Yue; Wan, Chun-Ling; Yu, Tao; He, Guang; He, Lin; Cai, Lei; Chou, Kuo-Chen

2016-01-01

Blood coagulation is healthy and lifesaving because it can stop bleeding. It can, however, be a troublemaker as well, causing serious medical problems including heart attack and stroke. Body has complex blood coagulation cascade to modulate the blood clots. In the environment of plasma, the blood coagulation cascade is regulated by antithrombin, which is deemed one of the most important serine protease inhibitors. It inhibits thrombin; it can inhibit factors IXa and Xa as well. Interestingly, its inhibitory ability will be significantly increased with the existence of heparin. In this minireview paper, we are to summarize the structural features of antithrombin, as well as its heparin binding modes and anti-coagulation mechanisms, in hopes that the discussion and analysis presented in this paper can stimulate new strategies to find more effective approaches or compounds to modulate the antithrombin.

Clotting of mammalian fibrinogens by papain: a re-examination.

PubMed

Doolittle, Russell F

2014-10-28

Papain has long been known to cause the gelation of mammalian fibrinogens. It has also been reported that papain-fibrin is insoluble in dispersing solvents like strong urea or sodium bromide solutions, similar to what is observed with thrombin-generated clots in the presence of factor XIIIa and calcium. In those old studies, both the gelation and subsequent clot stabilization were attributed to papain, although the possibility that the second step might be due to contaminating factor XIII in fibrinogen preparations was considered. I have revisited this problem in light of knowledge acquired over the past half-century about thiol proteases like papain, which mostly cleave peptide bonds, and transglutaminases like factor XIIIa that catalyze the formation of ε-lysyl-γ-glutamyl cross-links. Recombinant fibrinogen, inherently free of factor XIII and other plasma proteins, formed a stable gel when treated with papain alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intermolecular cross-linking in papain-fibrin leads to γ-chain dimers, trimers, and tetramers, just as is the case with thrombin-factor XIIIa-stabilized fibrin. Mass spectrometry of bands excised from gels showed that the cross-linked material is quite different from what occurs with factor XIIIa, however. With papain, the cross-linking occurs between γ chains in neighboring protofibrils becoming covalently linked in a "head-to-tail" fashion by a transpeptidation reaction involving the α-amino group of γ-Tyr1 and a papain cleavage site at γ-Gly403 near the carboxy terminus, rather than by the (reciprocal) "tail-to-tail" manner that occurs with factor XIIIa and that depends on cross-links between γ-Lys406 and γ-Gln398.

Confinement Regulates Complex Biochemical Networks: Initiation of Blood Clotting by “Diffusion Acting”

PubMed Central

Shen, Feng; Pompano, Rebecca R.; Kastrup, Christian J.; Ismagilov, Rustem F.

2009-01-01

Abstract This study shows that environmental confinement strongly affects the activation of nonlinear reaction networks, such as blood coagulation (clotting), by small quantities of activators. Blood coagulation is sensitive to the local concentration of soluble activators, initiating only when the activators surpass a threshold concentration, and therefore is regulated by mass transport phenomena such as flow and diffusion. Here, diffusion was limited by decreasing the size of microfluidic chambers, and it was found that microparticles carrying either the classical stimulus, tissue factor, or a bacterial stimulus, Bacillus cereus, initiated coagulation of human platelet-poor plasma only when confined. A simple analytical argument and numerical model were used to describe the mechanism for this phenomenon: confinement causes diffusible activators to accumulate locally and surpass the threshold concentration. To interpret the results, a dimensionless confinement number, Cn, was used to describe whether a stimulus was confined, and a Damköhler number, Da2, was used to describe whether a subthreshold stimulus could initiate coagulation. In the context of initiation of coagulation by bacteria, this mechanism can be thought of as “diffusion acting”, which is distinct from “diffusion sensing”. The ability of confinement and diffusion acting to change the outcome of coagulation suggests that confinement should also regulate other biological “on” and “off” processes that are controlled by thresholds. PMID:19843446

New photoacoustic platform for early detection of circulating clots to prevent stroke and other fatal thromboembolic complications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Carey, Kai A.

2017-03-01

Nearly 800,000 people in the U.S. experience an incident of stroke each year; 80% of these are first time occurrences and 87% are ischemic in nature. Someone dies of a stroke every few minutes in the U.S. but despite its prevalence there have been minimal advances in the early detection and screening of thromboembolic events, especially during patient post-operative periods or in genetically predisposed individuals. Environmental or genetic factors may disrupt the balance between coagulation and lysis of micro-thrombi in circulation and increase the risk of stroke. We introduced here a novel in vivo multicolor negative-contrast photoacoustic (PA) flow cytometry (PAFC ) platform with many innovations including customized high pulse repetition rate 1064 laser from IPG Photonics Corporation, powerful laser diode array, multichannel optical schematic, and time-resolved recording system. Using animal models, we verified the potential of this technology to detect small clots in relatively large vessels in vivo. If future clinical trials using a cost-effective, easy-to-use, safe, watch-like, wearable PA probe are successful, PAFC could provide breakthroughs in early monitoring of the growth in size and number of small clots that may predict and potentially prevent fatal thromboembolic complications. We also believe that this technology could be utilized to assess therapeutic benefits of anticoagulants and develop more efficient dosage in treatments by analyzing changes in the composition and frequency of micro-thrombi

Effects of fibrinogen concentrate after shock/resuscitation – A comparison between in vivo microvascular clot formation and thromboelastometry

PubMed Central

Martini, Judith; Cabrales, Pedro; Fries, Dietmar; Intaglietta, Marcos; Tsai, Amy G.

2014-01-01

Objective Dilutional coagulopathy after resuscitation with crystalloids/colloids clinically often appears as diffuse microvascular bleeding. Administration of fibrinogen reduces bleeding and increases maximum clot firmness (MCF), measured by thromboelastometry. Study objective was to implement a model where microvascular bleeding can be directly assessed by visualizing clot formation in microvessels, and correlations can be made to thromboelastometry. Design Randomized animal study. Setting University research laboratory. Subjects Male Syrian Golden hamsters. Interventions Microvessels of Syrian Golden hamsters fitted with a dorsal window chamber were studied using videomicroscopy. After 50% hemorrhage followed by 1 hr of hypovolemia resuscitation with 35% of blood volume using a high molecular weight (MW) HES solution (Hextend®, Hospira, MW 670 kD) occurred. Animals were then treated with 250 mg/kg fibrinogen iv (Laboratoire français du Fractionnement et des Biotechnologies (LFB), Paris, France) or an equal volume of saline before venular vessel wall injuries were made by directed laser irradiation and the ability of microthrombus formation was assessed. Measurements and main results Thromboelastometric measurements of MCF were performed at the beginning and at the end of the experiment. Resuscitation with HES and sham treatment significantly decreased FIBTEM MCF from 32 ± 9 at baseline vs. 13 ± 5 mm after sham treatment (p < 0.001). Infusion of fibrinogen concentrate significantly increased MCF, restoring baseline levels (baseline 32 ± 9 mm; after fibrinogen administration 29 ± 2 mm). In vivo microthrombus formation in laser injured vessels significantly increased in fibrinogen treated animals compared with sham (77% vs. 18%). Conclusions Fibrinogen treatment leads to increased clot firmness in dilutional coagulopathy as measured with thromboelastometry. At the microvascular level this increased clot strength, corresponds to an increased incidence of

The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles.

PubMed

Johnson, Lacey; Coorey, Craig P; Marks, Denese C

2014-08-01

Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend the shelf life from 5 days to 2 years. Cryopreserved PLTs are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. As such, the aim of this study was to understand the mechanisms responsible for the hemostatic potential of cryopreserved PLTs and the contribution of the reconstitution solution to this activity. DMSO (5% final concentration) was added to buffy coat-derived PLTs, followed by prefreeze removal of DMSO and storage at -80°C. Cryopreserved PLTs (n=8 per group) were thawed at 37°C, reconstituted with either 1 unit of thawed frozen plasma or PLT additive solution (PAS-G). In vitro assays were performed before freezing and after thawing to assess the hemostatic activity of PLTs. Cryopreserved PLTs expressed high levels of phosphatidylserine and contained significantly more phosphatidylserine-positive PLT microparticles than liquid-stored PLTs. This was accompanied by a significant decrease in the time to clot formation and clot strength, as measured by thromboelastography. The supernatant from cryopreserved PLTs was sufficient to reduce the phosphatidylserine-dependent clotting time and increase the thrombin generation potential. Overall, plasma-reconstituted cryopreserved PLTs were more procoagulant than those reconstituted in PAS-G. PLT cryopreservation results in the generation of phosphatidylserine-expressing PLT microparticles which contribute to the hemostatic activity. Understanding the hemostatic activity of these components may assist in extending the use of these specialized components beyond military applications. © 2014 Australian Red Cross Blood Service. Transfusion © 2014 AABB.

Percutaneous transvenous mitral commissurotomy in mitral stenosis and left atrial appendage clot patients in special conditions: Hospital-based study.

PubMed

Rajbhandari, Rajib; Malla, Rabi; Maskey, Arun; Bhatta, Yadav; Limbu, Yubraj; Sharma, Ranjit; Singh, Satish; Adhikari, Chandramani; Mishra, Sundeep

The percutaneous transvenous mitral commissurotomy is an important procedure for the treatment of mitral stenosis. A lot of mitral stenosis cases have left atrial appendage clot which precludes the patient from the benefit of this procedure. The aim of the study was to study the feasibility and safety of the procedure in a patient with appendage clot in the setup of certain urgent conditions. All cases of mitral stenosis with significant dyspnea and mitral valve area <1.5cm 2 with left atrial appendage clot and a condition which would preclude the patient from continuing on anticoagulation and needed urgent intervention were included in the study. From January 2011 to December 2013, twenty patients coming to Shahid Gangalal National Heart Centre, Kathmandu were selected for the procedure with conventional sampling technique. Informed written consent was obtained from the patients explaining all possible complications. The approval of the study was taken from the ethical committee of the hospital. Mean mitral valve area increased from 0.90cm 2 (SD±0.14) to 1.5cm 2 (SD±0.21) (p=0.02). Left atrial mean pressure decreased from mean of 20 to 10mmHg. Subjective improvement was reported in all. All of the patients had fulfilled criteria for successful PTMC. There was no mortality during hospital stay or in one-week follow-up period. There were no neurological complications or any need for emergency surgery. The immediate result of percutaneous transvenous mitral commissurotomy in selected cases of mitral stenosis with left atrial appendage clot is safe and acceptable in certain urgent situations in experienced hands. Copyright © 2016. Published by Elsevier B.V.

A commercial soy-based phospholipid emulsion accelerates clot formation in normal canine whole blood and induces hemolysis in whole blood from normal and dogs with inflammatory leukograms.

PubMed

Behling-Kelly, Erica L; Wakshlag, Joseph

2018-05-01

To compare lipid emulsion-induced hemolysis in blood from dogs with inflammatory leukograms to blood from healthy dogs, and determine the impact of a prototypical soy-based phospholipid emulsion on coagulation in whole blood from healthy dogs. Ex vivo study using EDTA and citrated whole blood from healthy dogs and EDTA anticoagulated whole blood from dogs with inflammatory leukograms. University research laboratory. Healthy dogs (total of 16, 9 for hemolysis assays and 6 for thromboelastography) included student- and staff-owned animals. Blood samples from dogs with inflammatory leukograms (8) were obtained from the clinical pathology laboratory after the complete blood count was performed as part of patient care. For the purposes of this study, an inflammatory leukogram was defined as a neutrophilia with a left-shift (minimum of 3% band neutrophils) and evidence of toxic change. None. Hemolysis was measured via spectrophotometric quantification of released hemoglobin and expressed as a percent of a water-lysed control. The soy emulsion caused hemolysis in blood from healthy dogs, ranging from 3.6% to 16.4% as the dose increased, and 4.1% to 25.0% in blood from dogs with inflammatory leukograms. Hemolysis between these patient groups was significantly different at the highest dose. Coagulation was assessed by native thromboelastography. Treatment of whole blood with the lipid emulsion caused a significant decrease in the time to clot formation (R) and a shorter time to reach a clot amplitude of 20 mm (K). Soy-based lipid emulsions cause hemolysis that is more severe in blood from dogs with inflammatory leukograms and accelerate clot formation in canine blood. The in vivo significance of these findings is not clear at this time, but warrants additional investigation given the use of these emulsions in clinical practice. © Veterinary Emergency and Critical Care Society 2018.

Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit.

PubMed

Salehi, Mahmoud; Aghamaali, Mahmoud Reza; Sajedi, Reza H; Asghari, S Mohsen; Jorjani, Eisa

2017-05-01

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The K m and V max values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl 2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses. Copyright © 2017 Elsevier B.V. All rights reserved.

Cationic PAMAM Dendrimers Aggressively Initiate Blood Clot Formation

PubMed Central

Jones, Clinton F.; Campbell, Robert A.; Brooks, Amanda E.; Assemi, Shoeleh; Tadjiki, Soheyl; Thiagarajan, Giridhar; Mulcock, Cheyanne; Weyrich, Andrew S.; Brooks, Benjamin D.; Ghandehari, Hamidreza; Grainger, David W.

2012-01-01

Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated through electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos (ZFE) were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regards to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity. PMID:23062017

Pharmacological Differentiation of Thrombomodulin Alfa and Activated Protein C on Coagulation and Fibrinolysis In Vitro.

PubMed

Tanaka, Kosuke; Tawara, Shunsuke; Tsuruta, Kazuhisa; Hoppensteadt, Debra; Fareed, Jawed

2018-01-01

Although thrombomodulin alfa (TM alfa), recombinant human soluble thrombomodulin, exerts antithrombogenic effects through activated protein C (APC), clinical trials suggested that TM alfa has a lower bleeding risk than does recombinant human APC. To address the mechanism explaining this difference, effects of TM alfa and APC on thrombogenic, coagulation, and fibrinolytic processes were compared in vitro. TM alfa and APC inhibited generation of thrombogenic markers, thrombin, and prothrombin fragment F1+2 and prolonged coagulation parameters, activated clotting time (ACT), and activated partial thromboplastin time (APTT). Concentrations of TM alfa effective for thrombin and F1+2 generation inhibition were comparable to those of APC. However, effects of TM alfa on ACT and APTT were clearly weaker than those of APC. TM alfa significantly prolonged clot lysis time (CLT) and decreased LY30, a parameter of degree of fibrinolysis in thromboelastography, whereas APC significantly shortened CLT and increased LY30. These results suggested that while the antithrombogenic effects of TM alfa were similar to those of APC, its anticoagulant effects were lower. In addition, effects of TM alfa were antifibrinolytic, while those of APC were profibrinolytic.

Prostatic fossa gauze-packing in the prevention of blood clot obstruction of the bladder after transvesical prostatectomy.

PubMed

Umunna, J I

2010-01-01

Clot obstruction often complicates transvesical prostatectomy. Any measure that prevents this will be a great relief to both surgeon and patient. To demonstrate that packing the prostatic fossa with roller gauze bandage after transvesical prostatectomy can prevent post-operative clot blockage of bladder drainage . Charts of all patients who had transvesical prostatectomy at Jasman Hospital Udo by me from 1988 to 1997 were sorted into two groups , Group A, not packed and Group B, packed. Information sought included patient's age, type of prostatectomy performed, whether the prostatic fossa was packed or not , average duration of catheter drainage, and complications. There were 68 patients who had no fossa packing and 72 in Group B with fossa packing. The age range of the two groups A and B were respective 45-85 year-old and 50-83 years. In both groups the highest number of patients was in the 60 t0 79 age bracket (48 in group A , 70%, and 56 in group B (78%). Bladder blockage occurred in 32(47%) patients without packing and none (0%) in group B with packing. Average duration of bladder drainage was 14 days in each group. There was no persisting vesico-cutaneous fistula. Temporary urinary incontinence occurred in three (3%) patients who had no packing and in five (7%) with packing. Gauze-packing of the prostatic fossa during transvesical prostatectomy can prevent bladder obstruction from clot retention without undue complications.