scholarly journals Venom-Induced Blood Disturbances by Palearctic Viperid Snakes, and Their Relative Neutralization by Antivenoms and Enzyme-Inhibitors

2021 ◽  
Vol 12 ◽  
Author(s):  
Abhinandan Chowdhury ◽  
Christina N. Zdenek ◽  
Matthew R. Lewin ◽  
Rebecca Carter ◽  
Tomaž Jagar ◽  
...  
Keyword(s):  
Enzyme Inhibitors ◽  
Enzyme Inhibitor ◽  
Factor X ◽  
Additional Species ◽  
Metalloprotease Inhibitors ◽  
Body Sizes ◽  
Factor X Activation ◽  
Species Being ◽  
Vipera Ammodytes

Palearctic vipers are medically significant snakes in the genera Daboia, Macrovipera, Montivipera, and Vipera which occur throughout Europe, Central Asia, Near and Middle East. While the ancestral condition is that of a small-bodied, lowland species, extensive diversification has occurred in body size, and niche specialization. Using 27 venom samples and a panel of in vitro coagulation assays, we evaluated the relative coagulotoxic potency of Palearctic viper venoms and compared their neutralization by three antivenoms (Insoserp Europe, VIPERFAV and ViperaTAb) and two metalloprotease inhibitors (prinomastat and DMPS). We show that variation in morphology parallels variation in the Factor X activating procoagulant toxicity, with the three convergent evolutions of larger body sizes (Daboia genus, Macrovipera genus, and Vipera ammodytes uniquely within the Vipera genus) were each accompanied by a significant increase in procoagulant potency. In contrast, the two convergent evolutions of high altitude specialization (the Montivipera genus and Vipera latastei uniquely within the Vipera genus) were each accompanied by a shift away from procoagulant action, with the Montivipera species being particularly potently anticoagulant. Inoserp Europe and VIPERFAV antivenoms were both effective against a broad range of Vipera species, with Inoserp able to neutralize additional species relative to VIPERFAV, reflective of its more complex antivenom immunization mixture. In contrast, ViperaTAb was extremely potent in neutralizing V. berus but, reflective of this being a monovalent antivenom, it was not effective against other Vipera species. The enzyme inhibitor prinomastat efficiently neutralized the metalloprotease-driven Factor X activation of the procoagulant venoms. In contrast, DMPS (2,3-dimercapto-1-propanesulfonic acid), which as been suggested as another potential treatment option in the absence of antivenom, DMPS failed against all venoms tested. Overall, our results highlight the evolutionary variations within Palearctic vipers and help to inform clinical management of viper envenomation.

scholarly journals A Clot Twist: Extreme Variation in Coagulotoxicity Mechanisms in Mexican Neotropical Rattlesnake Venoms

2021 ◽  
Vol 12 ◽  
Author(s):  
Lorenzo Seneci ◽  
Christina N. Zdenek ◽  
Abhinandan Chowdhury ◽  
Caroline F. B. Rodrigues ◽  
Edgar Neri-Castro ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Enzyme Inhibitors ◽  
Treatment Strategies ◽  
Factor X ◽  
Clotting Factors ◽  
Adjunct Treatment ◽  
Rattlesnake Venom ◽  
Factor X Activation

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus, and C. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

scholarly journals Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1069-1073 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Activate Factor ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Hemophilic Patient ◽  
Factor X Activation ◽  
Rate Of Activation

Abstract Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

scholarly journals Ixolaris: a Factor Xa heparin-binding exosite inhibitor

2005 ◽  
Vol 387 (3) ◽  
pp. 871-877 ◽  
Author(s):  
Robson Q. MONTEIRO ◽  
Alireza R. REZAIE ◽  
José M. C. RIBEIRO ◽  
Ivo M. B. FRANCISCHETTI
Keyword(s):  
Tissue Factor Pathway Inhibitor ◽  
Enzyme Inhibitor ◽  
Factor Xa ◽  
Factor X ◽  
Heparin Binding ◽  
Chloromethyl Ketone ◽  
Prothrombinase Complex ◽  
Tissue Factor Pathway ◽  
Inhibitor Interaction

Ixolaris is a two-Kunitz TFPI (tissue factor pathway inhibitor) from the tick salivary gland. In contrast with human TFPI, Ixolaris binds tightly to the zymogen FX (Factor X) and to dansyl-Glu-Gly-Arg-chloromethyl ketone-treated FXa (DEGR-FXa; active-site-blocked FXa), indicating that exosites are involved in the FX(a)–Ixolaris interaction. Here we provide evidence that Ixolaris binds specifically to the FXa HBE (heparin-binding exosite), since (i) it markedly decreases the inhibition of FXa by the antithrombin–heparin but not the antithrombin–pentasaccharide complex, (ii) it impairs FXa binding to Sepharose-immobilized heparin, and (iii) it allosterically modulates the catalytic activity of FXa for small chromogenic substrates (S-2765). By using a series of recombinant FXa mutants in which the HBE is mutated, we have identified the importance of amino acids involved in the enzyme–inhibitor interaction as being in the following order: Arg-93≫Arg-165≥Lys-169>Lys-236>Lys-96>Arg-240>Arg-125. Ixolaris at appropriate concentrations also inhibits thrombin formation in vitro by the assembled prothrombinase complex, a process that is critically dependent on the FXa HBE. Ixolaris is the first inhibitor characterized to date that binds specifically to the FXa HBE.

scholarly journals Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1069-1073 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Activate Factor ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Hemophilic Patient ◽  
Factor X Activation ◽  
Rate Of Activation

Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

Depolymerized Holothurian Glycosaminoglycan and Heparin Inhibit the Intrinsic Tenase Complex by a Common Antithrombin-Independent Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1011-1011 ◽  
Author(s):  
John P. Sheehan ◽  
Erik N. Walke
Keyword(s):  
Low Molecular Weight ◽  
Factor X ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Relative Affinity ◽  
Antithrombotic Efficacy ◽  
Factor X Activation

Abstract Depolymerized holothurian glycosaminoglycan (DHG) is a low molecular weight form (M.W. 12,500) of fucosylated chondroitin sulfate isolated from the sea cucumber Stichopus japonicus . DHG demonstrates antithrombotic efficacy in models of thrombin-induced pulmonary thromboembolism in the mouse, venous thrombosis in the rat, and dialysis during renal failure in the dog. The in vitro anticoagulant activities and antithrombotic efficacy of DHG are antithrombin-independent, and associated with lower bleeding tendency compared to unfractionated or low molecular weight heparin (LMWH). DHG has several potential mechanisms of action including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). DHG demonstrates significant affinity for both factor VIIIa and factor IXa, but the specific mechanism for inhibition of the intrinsic tenase complex (ITC) is undefined. We recently established the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of the ITC by LMWH (Yuan et al. Biochemistry44:3615–3625, 2005). The mechanism and molecular target for ITC inhibition by DHG was likewise determined, and compared to inhibition by LMWH. DHG completely inhibited factor X activation with a 50-fold higher apparent affinity (KI ~2 nM) than observed for partial inhibition by LMWH (KI ~111 nM). DHG reduced the Vmax(app) for factor X activation, without a significant effect on the KM(app), consistent with non-competitive inhibition. DHG did not affect the in vitro half-life of factor VIIIa activity, or inhibit chromogenic substrate cleavage by factor IXa-phospholipid. However, DHG reduced the affinity (KD(app)) of factor IXa for factor VIIIa in a dose dependent fashion, suggesting that the decreased Vmax(app) for factor X resulted from reduced complex assembly. DHG competed the binding of factor IXa to immobilized LMWH with an EC50 ~ 35-fold lower than soluble LWMH, suggesting that the binding sites for DHG and LMWH overlap on the protease. Likewise, the relative affinity of DHG for factor IXa compared to LMWH correlated with inhibitor potency. Kinetic analysis of ITC inhibition employing factor IXa with mutations in the heparin-binding exosite demonstrated that relative affinity for DHG (KI) was: wild type>K241A>H92A>R170A>>R233A; with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity, and the potency of LMWH for the ITC. Submaximal inhibitory concentrations of DHG also accelerated decay of the ITC, under condition where the half-life is primarily dependent on dissociation of the factor VIIIa A2 domain. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interaction(s). These structurally diverse glycosaminoglycans share a common mechanism for inhibition of factor X activation by the ITC. This inhibition occurs at DHG concentrations that are significantly lower (KI ~ 2 nM) than required for optimal acceleration of thrombin inhibition by HCII (~2.4 μM), or inhibition of factor VIII activation by thrombin (> 80 nM). Accordingly, DHG represents a lead compound for analysis of this novel antithrombotic mechanism in the absence of confounding antithrombin-dependent activities.

A Variant of Recombinant Factor VIIa with Enhanced Procoagulant and Antifibrinolytic Activities in a Model of Hemophilia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 767-767 ◽  
Author(s):  
Alisa S. Wolberg ◽  
Egon Persson ◽  
Ulla Hedner ◽  
Mirella Ezban ◽  
Geoffrey A. Allen
Keyword(s):  
Thrombin Generation ◽  
Factor Viia ◽  
Factor Xa ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Factor X ◽  
High Concentrations ◽  
Vitro Model ◽  
Factor X Activation

Abstract Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. We have shown that high concentrations of rFVIIa improve parameters of thrombin generation and fibrin clot formation and stability in an in vitro model of hemophilia (Wolberg et al. 2005 Brit J Haem 131:645–55). Recently, new rFVIIa analogs with increased procoagulant activity have been described. One of these analogs, rFVIIa with mutations V158D/E296V/M298Q (NN1731), exhibits increased activity in in vitro and in vivo models. In the present study, we compared the abilities of rFVIIa and NN1731 to specifically modulate factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability in an in vitro model of hemophilia. RFVIIa and NN1731 similarly increased factor X activation on tissue factor−bearing monocytes; however, NN1731 exhibited 30−fold higher factor Xa generation rates on activated platelets than similar concentrations of rFVIIa. We employed a complete, cell−based, reconstituted model of coagulation to compare the activities of rFVIIa and NN1731 in hemophilic conditions. The model system consists of tissue−factor bearing monocytes, purified pro− and anti−coagulant proteins (XI, X, IX, VIII, VIIa, V, II, AT, TFPI), and freshly−isolated, unactivated platelets. Fibrinogen is included in certain wells to examine clot formation. “Hemophilia” is simulated by omitting factors VIII and IX. In this assay, NN1731 produced 4 – 10−fold higher maximal thrombin generation rates than equal concentrations of rFVIIa. In contrast to even very high concentrations of rFVIIa (up to 500 nM), NN1731 was able to normalize the rate of thrombin generation. NN1731 did not directly activate platelets and thrombin generation was not detected prior to platelet activation, suggesting that the presence of NN1731 would not lead to thrombin generation in the absence of injury. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at lower concentrations than were required of rFVIIa. In a plasmin challenge assay, in which clot formation is initiated in the presence of plasmin, both rFVIIa and NN1731 increased fibrin formation and the length of time that fibrin was present in the absence of factors IX and VIII; however, NN1731 was effective at lower concentrations than were required of rFVIIa. Our results indicate that NN1731 increases factor Xa and thrombin generation and promotes clot formation and stability to a greater degree than equal concentrations of rFVIIa. For these reasons, NN1731 may be especially efficacious in situations in which rapid formation of a firm fibrin clot with increased resistance to fibrinolysis is necessary to achieve hemostasis.

Embryonic cell invasiveness: an in vitro study of chick gastrulation

1991 ◽  
Vol 98 (3) ◽  
pp. 403-407
Author(s):  
E.J. Sanders
Keyword(s):  
Basement Membrane ◽  
Enzyme Inhibitors ◽  
In Vitro Study ◽  
Primitive Streak ◽  
Enzymic Activity ◽  
Embryonic Cell ◽  
Cell Surface Carbohydrate ◽  
Metalloprotease Inhibitors ◽  
Primary Determinant

An investigation has been made into some of the possible mechanisms underlying the invasionary activity of gastrulating cells at the primitive streak of the early chick embryo. At gastrulation, epithelial cells in the upper epiblast layer of the embryo undergo a transformation into fibroblastic mesenchyme cells by passage through the primitive streak and penetration of a basement membrane. The resulting cells constitute the first embryonic mesoderm, which then invades the underlying tissue space. This phenomenon has been studied in vitro using the invasion of Matrigel, a reconstituted basement membrane, as a model. Mesoderm cells explanted into this matrix were subjected to treatments aimed at perturbing a number of putative mechanisms for cellular invasion. Application of inhibitors of glycosylation (tunicamycin) and oligosaccharide processing (castanospermine, deoxymannojirimycin, swainsonine) resulted in various degrees of inhibition of invasion. By contrast, cell binding fragments from fibronectin and laminin did not impede invasion, and neither did a panel of enzyme inhibitors, including serine protease and metalloprotease inhibitors. It is concluded that the primary determinant of the invasionary behaviour of these cells at gastrulation is a change in cell surface carbohydrate determinants, and that there is no evidence for the participation of localized enzymic activity. The medial disruption of the basement membrane seen at the primitive streak is therefore most likely to be due to local failure of synthesis, rather than local degradation.

scholarly journals Osteoblast-osteoclast co-culture amplifies inhibitory effects of FG-4592 on osteoclast formation and reduces bone resorption activity

2019 ◽  
Author(s):  
Philippa A Hulley ◽  
Ioanna Papadimitriou-Olivgeri ◽  
Helen J Knowles
Keyword(s):  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
Enzyme Inhibitors ◽  
Enzyme Inhibitor ◽  
Osteoclast Formation ◽  
Potential Contribution ◽  
Non Union

AbstractThe link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilise HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis and skeletal fracture and non-union. However, bone-resorbing osteoclasts also play a central role in bone remodelling and pathological osteolysis and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the final outcome of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function, and consider the potential contribution of inhibitory interactions with other musculoskeletal cells.The PHD enzyme inhibitor FG-4592 stabilised HIF protein and stimulated osteoclast-mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG-4592, but increased sensitivity to reduced concentrations of RANKL. Co-culture with osteoblasts amplified inhibition of osteoclastogenesis by FG-4592, whether the osteoblasts were proliferating, differentiating or in the presence of exogenous M-CSF and RANKL. Osteoblast co-culture dampened the ability of high concentrations of FG-4592 to increase bone resorption.This data provides support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for treatment of osteolytic pathologies, and indicates that FG-4592 might also act to inhibit the formation and activity of the osteoclasts that drive osteolysis.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Factor X Activation by washed human platelets

1979 ◽  
Author(s):  
E van Wijk ◽  
L Kahlé ◽  
J ten Cate
Keyword(s):  
Human Factors ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Factor Xa ◽  
Human Platelets ◽  
Soybean Trypsin Inhibitor ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

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