Thrombin Generation Responses to Human Factor XIa in Plasma of Animal Species

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5142-5142
Author(s):  
Yideng Liang ◽  
Evi B Struble ◽  
Li Ma ◽  
Samuel A Woodle ◽  
Naveen Jha ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pig ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Zealand White Rabbit ◽  
Coagulation Factor ◽  
Species Selection ◽  
Plasma Samples ◽  
Factor Xia ◽  
Generation Test

Abstract Abstract 5142 Thromboembolic events (TEE), including deep venous thrombosis and myocardial infarction, have been reported in patients receiving immunoglobulin intravenous (IVIG). Recent research showed that many TEE-induced IVIG samples were contaminated with human coagulation factor XIa (FXIa). At present, there is no systematic investigation of animal models that can be used to assess FXIa dependent thrombogenicity risk in animals and be predictive for human response. To assist in species selection for in vivo animal studies, we compared the magnitude and kinetic parameters of thrombin generation in commercially available human and animal plasma samples in response to human FXIa. A low sample volume, highly reproducible thrombin generation test was developed for this study. In our assay, thrombin generation in plasma (20 microliters) is initiated by physiological activator tissue factor in the presence of procoagulant phospholipids, sample (FXIa), buffer and calcium (combined volume of all additions to plasma is 20 microliters). The thrombin generation response to human FXIa was evident from increased thrombin peak height and/or shortened time to thrombin peak. The minimal effective FXIa concentration was species-dependent and ranged from below 0. 1 pM in monkey plasma to over 100 nM in BalbC mouse. The FXIa sensitivity of commercial plasma samples was aligned in the following order: Rhesus and Cynomolgus Monkeys > Human Donors > New Zealand White Rabbit and Sprague Dawley Rat > Hartley Guinea Pig and CD1 Mouse > Lewis Rat > Wistar and Fisher 344 Rats > C57BL6 and Blab C Mice. However, further experiments on freshly collected guinea pig plasma demonstrated that preanalytical variables, notably, blood collection and plasma freezing and thawing techniques, are critical to achieve optimal sensitivity of the thrombin generation test to FXIa. Therefore, variable quality of commercial plasma may have affected observed interspecies differences in thrombin generation. Collectively, these findings will help to clarify differences in minimal thrombogenic doses of FXIa observed in various animal models and levels of FXIa associated with thrombotic events in humans. This research was supported by the FDA Office of Women's Health grant (2012). Disclosures: No relevant conflicts of interest to declare.

Comparison of Branded Enoxaparin (Lovenox) and a Biosimilar Version of Enoxaparin (Fibrinox)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4385-4385
Author(s):  
Walter Jeske ◽  
Elizabeth McGeehan ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Average Molecular Weight ◽  
Molecular Profile ◽  
Distribution Analysis ◽  
Thrombin Time ◽  
Plasma Samples ◽  
Branded Product ◽  
Inhibition Of Thrombin

Abstract Abstract 4385 Several biosimilar versions of branded enoxaparin (Lovenox, Sanofi-Aventis, Paris, France) have recently become available throughout the world. These biosimilar enoxaparin preparations are distributed by multiple suppliers in Asia and in North and South America. Enoxaparin represents a complex mixture of oligosaccharides obtained by alkaline depolymerization of porcine mucosal heparin. It is the most widely used low molecular weight heparin which has been validated for clinical use in multiple indications. While the molecular profile and anti-Xa potencies of some of the biosimilar versions of enoxaparin are comparable, product based differences have been reported amongst some of the biosimilar versions of enoxaparin. The purpose of this study was to compare the biochemical and pharmacologic profile of one biosimilar version of enoxaparin, namely Fibrinox (Sandoz SA, Buenos Aires, Argentina) with the branded product Lovenox. The products were compared in equigravimetric amounts, assuming equivalent potency (100 AXa U/mg). Both products exhibited comparable molecular weight profiles in terms of average molecular weight and oligosachharide distribution. Analysis of the antithrombin binding hexasaccharide fractions of Fibrinox and Lovenox indicated the presence of eight distinct hexasaccharides. The relative proportions these hexasaccharides differed between Fibrinox and Lovenox. The anti-Xa and anti-IIa activities were comparable. In the whole blood clot-based assays such as TEG and ACT, both agents produced similar anticoagulant effects. In the plasma based assays such as the APTT, Heptest and thrombin time, both products showed comparable anticoagulant effects in the normal human pooled plasma samples. However, in plasma samples collected from patients with liver disease who were apparently anticoagulant free, the two products showed differences in their anticoagulant effects in the APTT assay (p<0.05). In the TF mediated thrombin generation assay, Fibrinox produced a stronger inhibition of thrombin generation compared to Lovenox (IC50; Fibrinox, 1.6 μ g/ml, Lovenox 2.2 μ g/ml). No differences were observed between the two products in the agonist induced platelet aggregation assays. However in the 14C serotonin release study, Fibrinox produced a stronger HIT serum mediated 14C release (p<0.05). Differences in the fibrinokinetic profile and the inhibition of thrombin activatable fibrinolytic inhibitor activation were observed with these LMWHs. These studies suggest while both the molecular profile and the pharmacopoeial potency of Fibrinox is similar to the branded product, these drugs can be differentiated in some of the other assays and should be evaluated in terms of additional pharmacologic mechanisims to demonstrate bioequivalence. Disclosures: No relevant conflicts of interest to declare.

Reversion of the Experimental Hemodilutional Coagulopathy Induced by Crystalloids and Colloids Using Different Coagulation Factor Concentrates

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4349-4349
Author(s):  
Carolina Caballo ◽  
Ana M Galan ◽  
Maribel Diaz-Ricart ◽  
Irene Lopez-Vilchez ◽  
Miguel Lozano ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Human Albumin ◽  
Massive Bleeding ◽  
Trauma Patients ◽  
Beneficial Effects ◽  
Ringer’S Lactate ◽  
The Impact

Abstract Abstract 4349 BACKGROUND: Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding. STUDY DESIGN AND METHODS: Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed. RESULTS: Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect. CONCLUSION: Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation. Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009 Disclosures: No relevant conflicts of interest to declare.

Persistant Inhibition of Thrombin Generation After Intravenous Administration of Enoxaparin in Primates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2095-2095
Author(s):  
Evangelos Litinas ◽  
Angel Gray ◽  
Nasir Sadeghi ◽  
Josephine Cunanan ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Clinical Effects ◽  
Plasma Samples ◽  
Thrombin Inhibition ◽  
Biologic Effects ◽  
Antithrombotic Effects ◽  
Sustained Inhibition ◽  
Inhibition Of Thrombin

Abstract Abstract 2095 Poster Board II-72 The biologic half life (T12) of low molecular weight heparin (LMWH) is usually measured in terms of the circulating anti-Xa levels. Enoxaparin represents an unique LMWH whose biologic T12 is relatively longer than most LMWHs. Moreover, it is known that the antithrombotic effects of this agent last longer in comparison to the measurable circulating anti-Xa levels. Therefore besides the anti-Xa activity, additional non-measurable biologic effects are contributory to the clinical effects of this agent. Plasma based thrombin generation assays have recently become available to assess the effects of LMWHs such as enoxaparin. In these assays blood plasma samples are activated using different activators and the generated thrombin inhibition is measured. To measure the time course of thrombin generation inhibitory activity after an IV bolus dose of 0.5 mg/kg of enoxaparin into groups of primates (n=6-8), a commercially available thrombin generation method was employed (Technoclone, Vienna, Austria/DiaPharma, West Chester,OH). Blood samples were drawn from each of the primates injected at varying time points for up to 28 hours. A thromboplastin/phospholipids based reagent was used to generate thrombin and the results were recorded in terms of nm of thrombin formed. The baseline values ranged from 500-900 nm (710±60 nm), although a complete inhibition of thrombin generation was noted at 1 hour (24±8 nm), a slow and gradual reduction in the thrombin generation inhibition was noted with a T12 of 9 hours. Even at 28 hours after the administration of enoxaparin, sustained inhibition of thrombin generation was noted (30-50%). Interestingly, the circulating anti-Xa and anti-IIa activity gradually diminished to an almost non-detectable level at 6 hours. These studies suggest that enoxaparin produces antithrombotic actions by multiple mechanisms. Furthermore thrombin generation methods in plasma samples may provide a more sensitive assay for the monitoring of the effect of LMWH. Disclosures: No relevant conflicts of interest to declare.

Thrombin generation in plasma of healthy adults and children: Chromogenic versus fluorogenic thrombogram analysis

2007 ◽  
Vol 98 (09) ◽  
pp. 600-613 ◽  
Author(s):  
Katrien Devreese ◽  
Walter Wijns ◽  
Isabelle Combes ◽  
Soetkin Vankerckhoven ◽  
Marc Hoylaerts
Keyword(s):  
Thrombin Generation ◽  
Healthy Adult ◽  
Coagulation Factor ◽  
Experimental Tests ◽  
Curve Analysis ◽  
Coagulation System ◽  
Plasma Samples ◽  
Coagulation Tests ◽  
Adults And Children ◽  
Coagulation Pathway

SummaryCoagulation tests and coagulation factor assays have been complemented recently with experimental tests to measure the total amount of thrombin formed.We have presently analyzed thrombin generation of healthy adult and paediatric plasma samples via a fluorogenic and a chromogenic method.The chromogenic method was performed on the fully automated Behring Coagulation System® (BCS®) and fluorogenic assays via Calibrated Automated Thrombography ® (CAT),after coagulation induction by various tissue factor (TF) concentrations.Sample distribution and variability were analyzed for the four main coagulation parameters, derived via computerized curve analysis in each method. Results for both methods were correlated.At the recommended TF concentration (300 pM), thrombin generation via BCS was less variable than via CAT (1–6 pM), but at comparable TF concentrations (1–6 pM), the CAT sensitivity was higher than that of BCS. Inhibition of intrinsic coagulation with the anti-factor VIII antibody BO2C11 revealed that the BCS detected extrinsic coagulation exclusively, at all TF concentrations tested. In contrast, at low TF concentrations (1 and 2.5 pM), via CAT, intrinsic coagulation pathway amplification was measured. At standardizedTF concentrations (300 pM in BCS vs. 2.5 pM in CAT), different reference values between adults and children were found, for all parameters, except Tmax. In adult samples, the best correlation between both methods was observed for ETPCAT versus ETPBCS and for Peak heightCAT versus CmaxBCS, when thrombin generation was exclusively extrinsic (300 pM in BCS vs. 6 pM in CAT). In conclusion, differential thrombin generation characteristics in BCS and CAT are relevant for their clinical applicability.

Microparticles Modulate Formation, Structure, and Properties of Fibrin Clots

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2807-2807
Author(s):  
Rosa M. Nabiullina ◽  
Dzhigangir A. Faizullin ◽  
Chandrasekaran Nagaswami ◽  
Yuriy F. Zuev ◽  
Ilshat G. Mustafin ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Clot Lysis ◽  
Outer Cell ◽  
Plasma Samples ◽  
Fibrin Polymerization ◽  
Fibrin Formation ◽  
Fibrin Clots

Abstract Microparticles (MPs) are phospholipid vesicles about 0.1-1 μm in size released into blood from vascular and blood cells upon activation and apoptosis. The mechanism of MP formation by the exocytic budding of the outer cell membrane provides them with procoagulant activity, mainly due to phosphatidylserine exposure and tissue factor expression. MPs are present in the peripheral blood of healthy subjects but their level is elevated in a variety of vascular, infectious, and immune-mediated pathologies. While there are extensive studies on multiple roles of MPs in various diseases, including thrombotic disorders, the question remains open as to whether MPs are involved in the formation and properties of fibrin, the scaffold of hemostatic clots and obstructive thrombi. To investigate this problem, we studied in vitro the effects of MPs on the kinetics of fibrin polymerization, fibrin clot structure and susceptibility to fibrinolysis. Blood was drawn from healthy donors according to an IRB protocol. MPs were removed from citrated platelet-free plasma (PFP) by filtration through 0.1-μm pores, yielding microparticle-depleted plasma (MDP). To restore the phospholipids eliminated, MDP was replenished with cephalin (MDP-C). Fresh samples of PFP, MDP, and MDP-C from the same donor were analyzed in parallel. Concentrations of MPs and expression of platelet-specific CD61 were studied by flow cytometry and confocal microscopy. A thrombin generation test in recalcified defibrinated plasma was performed using a chromogenic substrate. Fibrin polymerization induced by recalcification of PFP, MDP, and MDP-C was studied by dynamic turbidimetry. tPA-induced fibrinolysis was also followed optically as a decrease of a plasma clot turbidity. Fibrin ultrastructure was studied with scanning electron microscopy (SEM) in a FEI Quanta 250 microscope. Each test was performed with plasma samples from at least 3 donors and the results were averaged. Enumeration of MPs in PFP vs. MDP showed that about 90% of total MPs were removed by filtration, including 99.6% of CD61-positive platelet-derived MPs. The rate of thrombin generation was significantly reduced in MDP vs. PFP and was fully restored in MDP-C, confirming an essential role of MPs in the intrinsic coagulation pathway. Based on the lag times and slopes of the turbidimetric curves, fibrin formation in recalcified plasma samples was significantly delayed in the absence of MPs (MDP) and was much faster in their presence (PFP and MDP-C). The maximum optical density was consistently and significantly higher in MDP than in PFP and MDP-C, suggesting that fibrin clots formed in the absence and presence of MPs have fibers of different thickness. This presumption was confirmed by SEM of fibrin clots formed upon recalcification of the plasma samples. Clots formed in PFP were less porous and had thinner fibers (170 ± 38 nm), while clots from MDP had larger pores and were built of thicker fibers (214 ± 53 nm, p<0.01). Addition of phospholipids to MDP (MDP-C) resulted in the formation of densely packed thin fibrin fibers (141 ± 34 nm), similar to the initial clots from PFP. Consistent with the dissimilarity in ultrastructure, the clots displayed a distinct susceptibility to fibrinolysis: the rate of clot lysis was significantly higher in MDP than in PFP and MDP-PL. A novel and important finding from the SEM of fibrin clots is that fibers formed in MDP were quite smooth, while fibers formed in the presence of MPs (PFP and MDP-C) had relatively rough surfaces with multiple sub-micron size particles attached, suggesting that MPs might directly bind fibrin. Consistent with the SEM of the appearance of MPs on the fibers, confocal microscopy of PFP-clots stained for CD61 with FITC-labeled antibodies, unlike MDP-clots, revealed fluorescent spots associated with fibrin, indicating that MPs were adsorbed on fibrin fibers. In conclusion, MPs have profound effects on fibrin formation and on the final structure and characteristics of fibrin clots via at least two mechanisms. One is an indirect kinetic effect based on the MP-dependent rate of thrombin generation. The other is direct binding of MPs to fibrin fibers during and after fibrin assembly. Both mechanisms underlie a previously underappreciated potential role of MPs in hemostasis and thrombosis as modulators of fibrin formation and properties. (Research supported by NIH grant HL090774 and the Program of Competitive Growth of Kazan Federal University) Disclosures No relevant conflicts of interest to declare.

Thrombin Generation Mediators and Markers in Sepsis Associated Coagulapathy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3341-3341
Author(s):  
Debra Hoppensteadt ◽  
Josephine Cunanan ◽  
Hussein Khan ◽  
Evangelos Litinas ◽  
Gautam Sharma ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Inflammatory Process ◽  
Conflicts Of Interest ◽  
Functional Method ◽  
Control Group ◽  
Normal Male ◽  
Fibrinopeptide A ◽  
Plasma Samples ◽  
Male And Female ◽  
D Dimer

Abstract Abstract 3341 Thrombin plays a crucial role in the pathogenesis of sepsis associated disseminated intravascular coagulation (DIC). The purpose of this study was to profile prothrombin fragment (F1.2), fibrinopeptide A (FPA) and thrombin antithrombin complex (TAT), along with tissue factor (TF), microparticles (MP), D-dimer (DD) and antithrombin (AT). Baseline plasma samples from one hundred patients diagnosed with sepsis associated DIC were analyzed for various parameters by using ELISA methods. The MP levels were measured by using a functional method utilizing annexing trapping whereas the antithrombin was measured using both the functional and immunologic methods. Plasma samples from 50 normal male and female individuals comprised the control group. The results are tabulated in the following table. Both TF and MP levels were markedly elevated in comparison to the control group. All of the other markers of thrombin generation showed elevated levels F1.2 (3.4 fold), FPA (4.3 fold) and TAT (4.0 fold). Marked elevation of D-dimer was also noted (5.0 fold). Both the immunologic and functional levels of AT were also decreased. While the data was widely scattered, these results show that DIC represents a hypercoagulable state along with other hemostatic abnormalities and the activation of the inflammatory process. Modulation of these activation processes such as the antithrombin, thrombomodulin and TFPI may play an important regulatory role in the pathogenesis of this syndrome.MarkerNormal ControlsDIC BaselineF 1.2 (pM)108.6+12.3473.7+320FPA (ng/ml)1.6+0.86.8+3.1MP (nM)8.47+1.124.56+2.1AT (% NHP)94.2+2.780.4+3.1DD (ng/ml)415.4+65.23685+112TAT (ng/ml)3.4+0.317.0+3.1 Disclosures: No relevant conflicts of interest to declare.

Novel Plasma Based Microfluidic Assay for Measuring Fibrin Formation,Thrombin Generation and Fibrinolysis Under Flow: Monitoring Patients with Hemophilia on Inhibitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2579-2579
Author(s):  
Abimbola Jarvis ◽  
Katherine Ruegg ◽  
Linda Jacobson ◽  
Brian Branchford ◽  
Elizabeth Villalobos-Menuey
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Substrate Surface ◽  
Lag Time ◽  
Clot Formation ◽  
Plasma Samples ◽  
Flow Monitoring ◽  
Fibrin Formation

Abstract INTRODUCTION The unpredictable clinical response of patients to bypassing therapy and the lack of a proper laboratory tools to measure clot formation and stability renders prophylaxis and surgery on these patients a huge challenge. These patients are at a risk for bleeding or thromboembolic complications. AIMS In this study we introduce a novel plasma based microfluidic assay that can qualitatively and quantitatively measure fibrin deposition, thrombin and plasmin generation, and fibrinolysis under flow. We then examined the dynamics of thrombus formation in patients with hemophilia and their response to replacement and bypass therapies under flow conditions. METHODS Coagulation in the plasma based assay was initiated by spherical 1µm lipidated- Tissue Factor biomimetic silica beads which were patterned into 200µm circles on a substrate surface. Plasma samples were obtained from Hemophilia patients and inhibitor patients, before and after replacement or bypassing treatment and perfused over the tissue factor rich surfaces at a sheer rate of 100 s-1 Fibrinolysis was initiated with the addition of tPAto the plasma samples before perfusion. RESULTS The microfluidic assay was sensitive enough to measure the activation of coagulation triggered by the bypassing agents. Fibrin generation and thrombin generation were measurable both qualitatively and quantitatively using three metrics; lag time, rate of production and maximum quantity produced. Individuals on replacement therapy showed normalized fibrin formation with a 69% increase in fibrin formation, a decrease in lag time and an overall increase in maximum fibrin and thrombin production (See attached Figure). The microfluidic assay was also able to show an increase in overall fibrin generation in certain Individuals that were given more bypassing treatment than needed. Compared to healthy controls the rate of fibrin generation and maximum fibrin was greater, thereby identifying a risk for prothrombotic state. (See attached Figure). Finally, using the microfluidic assay we were able to observe both clot formation and lysis and asses the the stability of the fibrin clot produced when these inhibitor patients were on and off treatments, which reflects a more complete picture of the coagulation process. CONCLUSION We are able to show that individuals, treated by replacement therapies showed normalized clot formation. Individuals with hemophilia treated by bypassing therapies also showed normalized clot formation. Sometimes however, the fibrin production is more than a normal control, which could lead to a risk of prothrombosis. By using microfluidic assay, the treatment can be given in doses and fibrin production observed, to decrease the overall fibrin formation from a hypocoagulable to a hemostatic state, avoiding hypercoagulability. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma

2008 ◽  
Vol 99 (04) ◽  
pp. 767-773 ◽  
Author(s):  
Brigitte Pan-Petesch ◽  
Bertrand Arnaud ◽  
Marie-Thérèse Blouch ◽  
Jean-François Abgrall ◽  
Jérôme Duchemin
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Lag Time ◽  
Factor Vii ◽  
Fibrinogen Concentration ◽  
Thrombotic Risk ◽  
Generation Test ◽  
Factor Concentration

SummaryThe thrombin generation test is used to study coagulation in patients with haemorrhagic diseases or with high thrombotic risk. To our knowledge, this is the first study investigating the relative influence of coagulation factors on thrombin generation in plasma. The aim was to investigate the influence of coagulant factors, anticoagulant factors, and tissue factor (TF) on three parameters: endogenous thrombin potential (ETP), peak thrombin concentration, and lag time for the appearance of thrombin. At a low TF concentration, all factors except factor XI influenced thrombin generation. At a high TF concentration, only the factors of the extrinsic pathway exerted an influence. ETP and peak thrombin were linearly correlated to factor II concentration. Factor V and factor VII effects increased hyperbolically with factor concentration. The influence of factor X on thrombin generation depended onTF concentration. In the absence of factorVIII and factor IX, ETP fell to 60–70% of the normal when peak thrombin fell to 25–30% of the normal. Fibrinogen concentration influenced ETP and peak thrombin and decreasing fibrinogen levels shortened the lag time. As expected, decreasing antithrombin concentration caused dramatic increases in thrombin generation. Protein S prolonged the lag time, especially at a low TF concentration. No effect of protein C was observed, likely due to the absence of thrombomodulin. The thrombin generation test was more sensitive to factor deficiencies at low than at high TF concentration. ETP was not the most critical parameter for studying coagulation factor deficiencies. Instead, peak thrombin was the most sensitive parameter.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Experimental Studies on a Recombinant Hirudin, CGP 39393

1991 ◽  
Vol 65 (04) ◽  
pp. 355-359 ◽  
Author(s):  
E Gray ◽  
J Watton ◽  
S Cesmeli ◽  
T W Barrowcliffe ◽  
D P Thomas
Keyword(s):  
Thrombin Generation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Experimental Studies ◽  
Venous Stasis ◽  
Antithrombotic Agent ◽  
Recombinant Hirudin ◽  
Excessive Bleeding ◽  
Generation Test

SummaryThe in vitro anticoagulant activities of recombinant desulphatohirudin (r-hirudin) were studied in the activated partial thromboplastin time (APTT) and the thrombin generation test : systems. In the APTT at concentrations below 5 μg/ml, r-hirudin showed a dose-response curye. At concentrations above 5 μg/ml, the plasma became unclottable, but in the thrombin generation test , at least 10 μg/ml of r-hirudin was required for full inhibition of thrombin generation. The antithrombotic effect was assessed using a rabbit venous stasis model; 150 μg/ml r-hirudin completely prevented thrombus formation at 10 and 20 min stasis. At antithrombotic dose, the mean bleeding time ratio measured in a rabbit ear template model, was not prolonged over control values. At higher doses, the bleeding time ratios were higher than those observed for the same dosage of heparin. These data indicate that while r-hirudin is an effective antithrombotic agent, antithrombotic doses have to be carefully titrated to avoid excessive bleeding.

Sign in / Sign up

Export Citation Format

Share Document