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Sriram Krishnaswamy - One of the best experts on this subject based on the ideXlab platform.
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occlusion of anion binding exosite 2 in Meizothrombin explains its impaired ability to activate factor v
Journal of Biological Chemistry, 2019Co-Authors: Harlan N Bradford, Sriram KrishnaswamyAbstract:The proteolytic conversion of factor V to factor Va is central for amplified flux through the blood coagulation cascade. Heterodimeric factor Va is produced by cleavage at three sites in the middle of factor V by thrombin, yielding an N terminus-derived heavy chain and a C terminus-derived light chain. Here, we show that light chain formation resulting from the C-terminal cleavage is the rate-limiting step in the formation of fully cleaved Va. This rate-limiting step also corresponded to and was sufficient for the ability of cleaved factor V to bind Xa and assemble into the prothrombinase complex. Meizothrombin, the proteinase intermediate in thrombin formation, cleaves factor V more slowly than does thrombin, resulting in a pronounced defect in the formation of the light chain. A ∼100-fold reduced rate of Meizothrombin-mediated light chain formation by Meizothrombin corresponded to equally slow production of active cofactor and an impaired ability to amplify flux through the coagulation cascade initiated in plasma. We show that this defect arises from the occlusion of anion-binding exosite 2 in the catalytic domain by the covalently retained propiece in Meizothrombin. Our findings provide structural insights into the prominent role played by exosite 2 in the rate-limiting step of factor V activation. They also bear on how factor V is converted into a cofactor capable of assembling into prothrombinase.
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a single cleavage reaction in the proteolytic activation of factor v to the cofactor is rate limiting in a step that is selectively dependent on anion binding exosite 2 of thrombin
Blood, 2017Co-Authors: Harlan N Bradford, Sriram KrishnaswamyAbstract:Abstract Human factor V (hFV) is a procofactor with a domain organization of A1-A2-B-A3-C1-C2. It requires proteolytic activation before it can assemble within the prothrombinase complex and function as a cofactor. Activation by thrombin results from cleavages following Arg709, Arg1018 and Arg1545 to release the B domain in two fragments and form heterodimeric factor Va (FVa, A1-A2/A3-C1-C2). While A1-A2/A3-C1-C2 is the prototypic active cofactor, variable cofactor function is attributed to partially cleaved species. Several studies have proposed that Meizothrombin (mIIa), an intermediate produced during prothrombin activation, is also an efficient activator of membrane-bound hFV and possibly responsible for FVa formation early in the clotting process. However, this is inconsistent with the proposed importance of anion binding exosite 2 (ABE2) for hFV activation by thrombin which is occluded by the covalently attached propiece in mIIa. We have studied the kinetics of hFV cleavage using a quantitative two-color immunoblotting approach to simultaneously and unambiguously identify intermediates and products containing A1-A2 and/or A3-C1-C2. We have also compared hFV cleavage by thrombin by cleavage with a stable mIIa variant and by mIIa lacking both the 4-carboxyglutamic acid and kringle 1 domains (mIIa-desF1) in the presence of membranes. Addition of 0.1 nM thrombin yielded quantitative cleavage of hFV (50 nM) in ~6 minutes, producing products consistent with cleavage at Arg709 and/or Arg1018. However, the C-terminal intermediate beginning at 1019 but uncleaved at Arg1545 persisted for more than 90 minutes. A3-C1-C2, arising from cleavage at Arg1545, appeared at a ~20-fold slower rate than the A1-A2 species. Thus, for the reaction with thrombin, cleavage at Arg1545 represents the rate limiting step in the conversion of hFV to heterodimeric FVa. Similar rates of hFV consumption and appearance of species cleaved at Arg709 and/or Arg1018 were seen with mIIa. However, cleavage at Arg1545 to produce A3-C1-C2 was substantially slower, by a factor of ~100, than A1-A2 formation. Equivalently large differences in the rates of A1-A2 and A3-C1-C2 formation were evident in the absence of membranes except the rate of all cleavages by mIIa was further reduced. Because the results obtained with mIIa were replicated with mIIa-desF1, the data imply that it is covalent occlusion of ABE2 in these mIIa variants that underlies their selectively impaired ability to cleave at Arg1545 and produce heterodimeric Va but does not affect cleavage at Arg709 or Arg1018. The binding of Xa to activated FV, essential for membrane-dependent prothrombinase assembly, is a prerequisite for cofactor function. We correlated hFV cleavage with the development of cofactor function by binding measurements using Xa inactivated with a fluorescent probe covalently tethered to the active site with a peptidyl chloromethylketone. For hFV cleavage by thrombin, Xa binding and the assembly of prothrombinase proceeded unexpectedly slowly and correlated with cleavage at Arg1545, widely separated in time from rapid cleavage at Arg709 and/or Arg1018. Therefore, slow cleavage at Arg1545 dictates both the formation of heterodimeric FVa and the ability of the product to bind Xa with high affinity to form prothrombinase. Only a small fraction of Xa binding developed within an hour in hFV cleavage reactions initiated with mIIa in comparison to the same concentration of thrombin. Our findings indicate that ABE2 in thrombin variants plays an essential role in the rate-limiting cleavage at Arg1545 required for the formation of heterodimeric FVa and for the assembly of prothrombinase. Occlusion of ABE2 by the covalently linked propiece in mIIa variants greatly impairs their ability to activate hFV to the active cofactor by selectively decreasing the rate of cleavage at Arg1545. The surprising and near absolute need for cleavage at Arg1545 to produce cofactor is consistent with new biochemical and structural evidence implicating an essential role played by an acidic sequence immediately N-terminal to Arg1545 in concert with a basic sequence C-terminal to Arg1018 within the B domain in restricting the binding of Xa to the incompletely cleaved procofactor and preventing cofactor function. Disclosures Krishnaswamy: Novo Nordisk: Consultancy; Janssen: Consultancy, Research Funding; Portola: Research Funding.
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Anticoagulation by a thrombin precursor
2016Co-Authors: Vascular Biology, Sriram Krishnaswamy, Of Philadelphia UniversityAbstract:A prothrombin mutant that is activated to yield stable meizo-thrombin produces dominant inhibition of clot formation in a mouse model of carotid artery injury. S tudies of clot formation in vivo that drawfrom established details of coagulation biochemistry have the potential for yielding unexpected insights into the regulation of co-agulation or suggesting novel strategies for its inhibition. In this vein, Shim and colleagues (page 415) report studies directed at evaluating the potential contribution of Meizothrombin to thrombus formation in a mouse carotid ar-tery injury model. Thrombin catalyzes a spectrum of cleavage reactions relevant to both the procoagulant and anticoagulant pathways of blood coagula
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crystal structure of the prothrombinase complex from the venom of pseudonaja textilis
Blood, 2013Co-Authors: Bernhard C Lechtenberg, Thomas A Murrayrust, Daniel J D Johnson, Ty E Adams, Rodney M Camire, Sriram Krishnaswamy, James A. HuntingtonAbstract:The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate Meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex—the molecular engine of blood coagulation.
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active site labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo
Journal of Biological Chemistry, 2011Co-Authors: Heather K Kroh, Guido Tans, Peter Panizzi, Jan Rosing, Sriram Krishnaswamy, Svetlana N Tchaikovski, Regan T Baird, Bruce Furie, Paul E. BockAbstract:Abstract Mouse and human prothrombin (ProT) active site specifically labeled with d-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProTS195A mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProTS195A as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProTS195A, the pathway of FPR-ProT cleavage by prothrombinase was redirected from Meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH2Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.
Marcia M B Da Silva - One of the best experts on this subject based on the ideXlab platform.
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a prothrombin activator from bothrops erythromelas jararaca da seca snake venom characterization and molecular cloning
Biochemical Journal, 2003Co-Authors: Claudio A. M. Sampaio, Marcia M B Da Silva, Mirta Schattner, Celso Raul Romero Ramos, Inacio L M Junqueiradeazevedo, M C Guarnieri, Maria A Lazzari, Roberto Gabriel Pozner, Janaina De Souza VenturaAbstract:A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into Meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
Elke Bucha - One of the best experts on this subject based on the ideXlab platform.
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drug monitoring of argatroban using the ecarin chromogenic assay
Seminars in Thrombosis and Hemostasis, 2008Co-Authors: Götz Nowak, Ute Lange, Elke BuchaAbstract:The ecarin chromogenic assay (HaemoSys ECA; JenAffin GmbH, Jena, Germany) was developed for quantitative determination of direct thrombin inhibitors. As a further development of the ecarin clotting time (ECT), the ECA is based on the same principle of measurement: the activation of prothrombin by ecarin, a snake venom from Echis carinatus. In the ECA, the prothrombin activation products Meizothrombin and Meizothrombin-des-F1 cleave a chromogenic substrate. The activity of Meizothrombin/ Meizothrombin-des-F1 is inhibited in a concentration-dependent manner by direct thrombin inhibitors. The ECA-T is an assay for quantitative determination of active site directed synthetic thrombin inhibitors. For argatroban ECA-T, there is a very precise and sensitive method of quantitative drug monitoring. Only very low interindividual variations were found compared with activated partial thromboplastin time and even ECT. ECA-T is independent of variations in prothrombin levels of the plasma samples. Heparin does not influence the measuring result. ECA-T is performed on manual coagulation analyzers with an option for optical measurement but can also be applied to automated laboratory systems. By the use of ECA-T as a drug-monitoring method, the drug safety of argatroban, most of all in critically ill patients, can be increased.
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ecarin chromogenic assay innovativer test zur quantitativen bestimmung direkter thrombininhibitoren im plasma
Hamostaseologie, 2005Co-Authors: Ute Lange, Goetz Nowak, Andrea Olschewski, Elke BuchaAbstract:Der ECA (ecarin chromogenic assay) wurde zur quantitativen Bestimmung von direkten Thrombininhibitoren entwickelt. Er ist eine Weiterentwicklung der ECT (ecarin clotting time) und basiert wie diese auf der Prothrombinaktivierung durch Ecarin, einem Schlangengiftenzym aus Echis carinatus. Durch die entstehenden Aktivierungsprodukte Meizothrombin und Meizothrombin-Des-Fragment 1 wird im ECA ein chromogenes Substrat gespalten, wahrend im Gerinnungsassay ECT plasmatisches Fibrinogen zu Fibrin umgesetzt wird. Die Aktivitat von Meizothrombin und Meizothrombin-Des- Fragment 1 wird konzentrationsabhangig durch direkte Thrombininhibitoren gehemmt. Der ECA kann als ECA-H zur quantitativen Hirudinbestimmung und als ECA-T zur Bestimmung von synthetischen Thrombinhemmstoffen eingesetzt werden. Am Beispiel von Hirudin, Argatroban und Melagatran erwies sich der ECA als auserst prazise und sensitive Methode, die die Vorteile der ECT mit denen chromogener Tests verbindet. Im Vergleich zu aPTT und ECT weist der ECA sehr geringe interindividuelle Schwankungen auf. Er wird weder von der Prothrombin- noch von der Fibrinogenkonzentration im Plasma beeinflusst.
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ecarin chromogenic assay a new method for quantitative determination of direct thrombin inhibitors like hirudin
Pathophysiology of Haemostasis and Thrombosis, 2003Co-Authors: Ute Lange, Götz Nowak, Elke BuchaAbstract:A new sensitive and precise method for quantitative determination of direct thrombin inhibitors is described, the ecarin chromogenic assay (ECA). Ecarin is used as the specifi c prothrombin-activating principle. The cleavage of a chromogenic substrate by Meizothrombin is inhibited in a concentration-dependent fashion by direct thrombin inhibitors. For the ECA, the linear measuring range is about 0.1–3.0 � g hirudin/ml plasma. Coeffi cients of variations between 2.3 and 4% over the whole concentration range were achieved. The ECA has proved to be more sensitive than the compared tests (ecarin clotting time and a thrombin-based chromogenic assay); a detection limit of 0.011 � g hirudin/ml and a quantitation limit of 0.032 � g hirudin/ml were calculated. The ECA is independent of the variations of the coagulation variables fi brinogen and prothrombin. Neither heparin nor oral anticoagulants interfere with the ECA.
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Quantitative Determination of Hirudin in Blood and Body Fluids
Seminars in Thrombosis and Hemostasis, 1996Co-Authors: Goetz Nowak, Elke BuchaAbstract:Recent clinical studies using hirudin as anticoagulant have demonstrated that an efficient method to determine the current blood level of hirudin is imperative for exact dose finding and adjustment. Only the exact determination of the hirudin content in blood, performed within a few minutes, prevents overdosage involving side effects or, otherwise, a subtherapeutic dose regimen. Therefore, a method for rapid, sensitive, and reproducible measurement of hirudin in blood, plasma, and other body fluids has been developed. The method, which is based on coagulation measurement, is called ecarin clotting time (ECT). In this test, ecarin, a purified enzyme of the Echis carinatus snake venom, acts as a prothrombin activator. In contrast to the "solid phase" prothrombin activation by prothrombinase, the ecarin-induced prothrombin activation proceeds in an alternative way, i.e., without the need of cofactors, resulting in intermediates such as Meizothrombin. Compared to thrombin, Meizothrombin has a lower procoagulant activity, but it still binds hirudin, which leads to the inhibition of Meizothrombin. Depending on the sample's concentration of hirudin, ecarin forms a residual, nonhirudin-bound amount of intermediates of the prothrombin-thrombin conversion that are able to concentration-dependently convert fibrinogen to fibrin. There is an excellent linear correlation between ECT prolongation and the hirudin content of the sample in a range from 50 to 5,000 ng/mL blood or plasma. This allows immediate measurement not only of the therapeutic blood level of hirudin, but also of its concentration in blood following under- or overdosage. The ECT method is nearly independent of variations in the sample's content of fibrinogen (from 60% to 100%) and prothrombin (from 20% to 100%.) Heparin is not able to catalyze the very low antithrombin inhibition of Meizothrombin. Therefore, it is also possible to determine hirudin in blood containing varying amounts of heparin. Another advantage of the method is that it can be applied to different mechanical measuring systems used in coagulation diagnostics.
Janaina De Souza Ventura - One of the best experts on this subject based on the ideXlab platform.
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a prothrombin activator from bothrops erythromelas jararaca da seca snake venom characterization and molecular cloning
Biochemical Journal, 2003Co-Authors: Claudio A. M. Sampaio, Marcia M B Da Silva, Mirta Schattner, Celso Raul Romero Ramos, Inacio L M Junqueiradeazevedo, M C Guarnieri, Maria A Lazzari, Roberto Gabriel Pozner, Janaina De Souza VenturaAbstract:A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into Meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
J. E. Sadler - One of the best experts on this subject based on the ideXlab platform.
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r268a inhibits thrombosis in a model of acute carotid artery injury a recombinant murine Meizothrombin precursor prothrombin r157a
2013Co-Authors: Kyuhwan Shim, Hongfa Zhu, Lisa A. Westfield, J. E. SadlerAbstract:Ł € Hemostasis, Thrombosis, and Vascular Biology (2497 articles)Articles on similar topics can be found in the following Blood collectionshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at:http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at:
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activation induced exposure of the thrombin anion binding exosite interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite specific antibody
Journal of Biological Chemistry, 1994Co-Authors: Virginie Picard, Martine Aiach, J. E. SadlerAbstract:Abstract The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on Meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on Meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of Meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.
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ligand specificity of human thrombomodulin equilibrium binding of human thrombin Meizothrombin and factor xa to recombinant thrombomodulin
Journal of Biological Chemistry, 1992Co-Authors: M Tsiang, Steven R. Lentz, J. E. SadlerAbstract:Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), Meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for Meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled Meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human Meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.