A critical role for Gly25 in the B chain of human thrombin.

We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25(330)S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25(330)S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by Taipan snake or Echis carinatus venoms. We show that the G25(330)S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25(330) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin.

Thrombin-induced platelet PAR4 activation: role of glycoprotein Ib and ADP.

Thrombin activates human platelets via the cleavage of two protease-activated G-protein coupled receptors (PARs), PAR1 and PAR4 that respond to low and high concentrations of thrombin, respectively. The aim of the present study was to examine the relative contributions of GPIbalpha and ADP receptors in response to thrombin-induced PAR1 and PAR4 stimulation. Platelet responses (aggregation, secretion and calcium mobilization) elicited by low thrombin concentrations were impaired when thrombin interaction with GPIbalpha was blocked. In contrast, blockade of thrombin interaction with GPIbalpha had no effect when PAR4-coupled responses were specifically elicited by high thrombin concentrations in the presence of PAR1 antagonists or after PAR1 desensitization. These results confirmed that unlike PAR1, PAR4 does not require GPIbalpha as a cofactor for thrombin-mediated activation. Both apyrase and selective antagonists of P2Y1 and P2Y12 inhibited PAR1-coupled responses but did not modify PAR4-coupled responses, indicating that in contrast to PAR1, PAR4 signals are not reinforced by ADP secretion and binding to the platelets. These results provide the direct evidence that, in human platelets, GPIbalpha and ADP act in synergy to amplify PAR1 coupled responses while PAR4 is activated independently of GPIbalpha and ADP.

Antiphospholipid antibodies and the coagulation cascade.

Hemostasis is a highly controlled system of associated biophysical and biochemical events requiring a number of molecular and cellular interactions, among which molecular assembly at surfaces is an obligatory mechanism. The exposure of flowing blood to subendothelial components results in platelet adhesion, activation, and aggregation with simultaneous exposure of negatively charged phospholipids, which serves as a template for the formation of enzyme-cofactor-substrate complexes. The locally formed proteases activate surface-bound zymogens in a sequence culminating in the formation of thrombin. Fibrinogen is transformed into fibrin by thrombin, which may also activate protein C on phospholipid membranes when bound to TM. Activated protein C is a potent anticoagulant that inactivates coagulation-activated cofactors Va and VIIIa. During this process, proteins bound to the phospholipid surfaces may adopt new configurations and expose neoepitopes, which may elicit an immunologic response giving rise to the generation of antiphospholipid antibodies. These antibodies may then interfere with the procoagulant or anticoagulant activities of the target protein-phospholipid complexes. The apolipoprotein beta 2GPI and prothrombin are the most frequently found cofactors for antiphospholipid antibodies. Components of the protein C pathway have also been identified as cofactors. The pathophysiologic effects of antiphospholipid antibodies on the thrombotic accidents observed in patients with the antiphospholipid syndrome have not been established yet.

Current outcome of portal vein thrombosis in adults: risk and benefit of anticoagulant therapy.

BACKGROUND & AIMS: The outcome of portal vein thrombosis in relation to associated prothrombotic states has not been evaluated. We assessed current outcome and predictors of bleeding and thrombotic events in a cohort of 136 adults with nonmalignant, noncirrhotic portal vein thrombosis, of whom 84 received anticoagulant therapy. METHODS: Multivariate Cox model analysis for event-free survival and analysis taking into account multiple events were used. RESULTS: Median follow-up was 46 months. The incidence rate of gastrointestinal bleeding was 12.5 (95% confidence interval [CI], 10-15) per 100 patient-years. Large varices were an independent predictor for bleeding. Anticoagulant therapy did not increase the risk or the severity of bleeding. The incidence rate of thrombotic events was 5.5 (95% CI, 3.8-7.2) per 100 patient-years. Underlying prothrombotic state and absence of anticoagulant therapy were independent predictors for thrombosis. In patients with underlying prothrombotic state, the incidence rates of splanchnic venous infarction were 0.82 and 5.2 per 100 patient-years in periods with and without anticoagulant therapy, respectively (P = 0.01). Two nonanticoagulated patients died of bleeding and thrombosis, respectively. CONCLUSIONS: In patients with portal vein thrombosis, the risk of thrombosis is currently as clinically significant as the risk of bleeding. The benefit-risk ratio favors anticoagulant therapy.

Factor V Leiden related Budd-Chiari syndrome.

BACKGROUND: The role of factor V Leiden as a cause of Budd-Chiari syndrome has only recently been described. AIMS: To assess the specific features of factor V Leiden related Budd-Chiari syndrome. PATIENTS: Sixty three consecutive patients with hepatic vein or terminal inferior vena cava thrombosis. METHODS: Standardised chart review. RESULTS: Factor V Leiden was found in 20 patients (31% (95% CI 20-43)). In the subgroup of patients with, compared with the subgroup without, factor V Leiden, a combination of prothrombotic states was more common (70% (95% CI 50-90) v 14% (95% CI 3-24)); inferior vena cava thrombosis was more frequent (40% (95% CI 19-61) v 7% (95% CI 0-14)); and distribution of initial alanine aminotransferase values was bimodal (almost normal or extremely increased) versus unimodal (p=0.003). Factor V Leiden accounted for four of five cases of massive ischaemic necrosis (transaminases >50-fold the upper limit of normal values) (p=0.014), and also for all three cases developing during pregnancy. Patients with and without factor V Leiden did not differ with regard to mortality, portosytemic shunting, or listing for liver transplantation. Hepatocellular carcinoma developed in two patients; both had factor V Leiden and indolent obstruction of the inferior vena cava. CONCLUSIONS: In patients with Budd-Chiari syndrome, factor V Leiden (a) is common; (b) precipitates thrombosis mostly when combined with another risk factor; (c) is associated with one of two contrasting clinical pictures: indolent thrombosis-particularly of the inferior vena cava-or massive ischaemic necrosis; and (d) is a major cofactor of Budd-Chiari syndrome developing during pregnancy.

Cause of portal or hepatic venous thrombosis in adults: the role of multiple concurrent factors.

According to a recent hypothesis, venous thrombosis results from the concurrence of several factors. This hypothesis was assessed in patients with portal or hepatic venous thrombosis by simultaneously investigating most of the currently identified prothrombotic disorders, local precipitating factors, and other risk factors such as oral contraceptive use. Patients with a tumorous obstruction and patients with cirrhosis with portal vein thrombosis were excluded. The prothrombotic disorders that were investigated included classical and occult myeloproliferative disorders; antiphospholipid syndrome; protein C; protein S and antithrombin deficiency; factor V Leiden; factor II; and methylene-tetrahydrofolate-reductase gene mutations. We found 1 or several prothrombotic disorders and a local precipitating factor in 26 and 10 of the 36 patients with portal vein thrombosis, respectively; and in 28 and none of the 32 patients with hepatic vein thrombosis, respectively. We found a combination of prothrombotic disorders in 5 and 9 patients with portal and hepatic vein thrombosis, respectively, whereas such a combination is expected in less than 1% of asymptomatic subjects. Of the 10 patients with a local precipitating factor, 8 had a prothrombotic disorder. Of the 13 patients who use oral contraceptives, 10 had a prothrombotic disorder. We conclude that portal or hepatic venous thrombosis should be regarded as an index for 1 or several prothrombotic disorders, whether or not local precipitating factors or oral contraceptive use are found. Concurrence of prothrombotic disorders is more common than expected. Extensive investigation of prothrombotic disorders and anticoagulation should be considered in patients with portal or hepatic venous thrombosis.

A chronic hypercoagulable state in patients with beta-thalassaemia major is already present in childhood.

A higher than normal incidence of thromboembolic events has been observed in adult patients with beta-thalassaemia major (TM) and certain haemostatic anomalies found in these patients suggest the existence of a chronic hypercoagulable state. Thalassaemic red blood cells (RBC) were demonstrated to facilitate thrombin formation due to altered asymmetry of the membrane phospholipids with enhanced exposure of phosphatidylserine. Since RBC anomalies exist in thalassaemia from the first months of life, we studied markers of hypercoagulability and thrombophilia in 36 adult patients (range 19-38 years) and 26 children (range 2-18 years) with beta-TM. All the patients were in steady state and none had experienced clinical signs or symptoms of thrombosis. Highly elevated urinary levels of 11-dehydro-thromboxane B2 and significantly elevated plasma levels of thrombin-antithrombin III (TAT) complexes were observed to the same extent in TM children and adults. The levels of factor II were decreased while factors V, VII + X and plasminogen were within the normal range. The natural coagulation inhibitors, protein C (PC) and protein S (PS) were significantly decreased in all TM patients investigated, regardless of age, but the PS binding protein (C4bBP) and antithrombin III levels were normal. The frequency of other thrombophilic mutations was not increased. Thus, a chronic hypercoagulable state already exists in thalassaemia in childhood and may contribute to the cardiac and pulmonary anomalies and the thrombotic events which occur later.

[Thrombin and its pharmacologic regulation]. / La thrombine et sa régulation pharmacologique.

THROMBUS FORMATION: Thrombin plays a crucial role in thrombus formation. It transforms fibrinogen into fibrin and activates platelets. Thrombin has both stimulating and inhibiting effects controlling the feedback regulation of thrombus formation. Thrombin also stimulates circulating cells other than platelets and vascular cells and thus participates in inflammation and wound repair accompanying hemostasis and thrombosis. Thrombin's interaction with cells is mediated by proteolysis activated receptors (PAR), mainly PAR-1 and probably by the recently cloned PAR-3. MECHANISM OF ACTION: Thrombin is an ellipsoid-shaped serine protease whose active site is located at the bottom of a deep groove. One end of the groove bearing the active site carries exosite 1 which links with the C-terminal end of hirudine, PAR-1 and PAR-3, fibrinogen and fibrin, thrombomoduline and platelet GPIb. Exosite 2 is carried on the top side of the molecule opposite the active site. It binds to certain glycoaminoglycanes such as heparin. Thrombin's high substrate specificity results from multiple interactions which occur between different functional domains and complementary domains on the substrate. ACTION OF HEPARIN: The antithrombotic effect of heparin results from its catalytic effect on antithrombin III (AT) inhibition of thrombin. This effect is however limited because heparin is dependent on the concentration of AT and on the of inactivating proteins such as platelet factor 4. In addition, the inhibitory effect of the AT-heparin complex is limited for thrombin bound to the thrombus. Finally, heparin can produce a rare but severe complication, heparin-induced thrombocytopenia. Because of these different drawbacks, research has been focused on other thrombin inhibitors. DIRECT THROMBIN INHIBITORS: The target of these inhibitors is the active site of thrombin. Many compounds are under study, including several which can be administrated orally. The most advanced clinical trials have been conducted with hirudin. Hirudin binds to thrombin at several sites, blocking all the known functions of thrombin. The problem of choosing an agent to maintain antithombotic therapy in patients with heparin-induced thrombocytopenia remains unresolved. Hirudin would be a likely choice as it has no cross reactivity with heparin or heparinoids. Promising early results have been reported.

Characteristics of the interaction between thrombin exosite 1 and the sequence 269-287 [correction of 269-297] of platelet glycoprotein Ibalpha.

The interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbalpha and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbalpha to interact with thrombin. Three peptides were synthesized, including Ibalpha 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibalpha 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibalpha 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibalpha 269-287 and alpha-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when gamma-thrombin was substituted for alpha-thrombin. Ibalpha 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with alpha-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibalpha 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibalpha 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

Inhibition of thrombin-catalyzed factor V activation by bothrojaracin.

We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives beta- and gamma-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.

Sickle cell disease: relation between procoagulant activity of red blood cells from different phenotypes and in vivo blood coagulation activation.

In the present study we examined if, among other mechanisms, the abnormal exposure of phosphatidylserine at the surface of sickle red blood cells (RBCs) contributes to the hypercoagulability which characterizes homozygous sickle cell disease (SCD). The question was addressed by comparison of the procoagulant properties of RBCs from subjects with various phenotypes (SS, SC and AS) that differ in clinical presentation. As previously reported, SS-RBCs accelerated the prothrombin activation by factor Xa, by decreasing the Km of the reaction compared to normal RBCs. SC-RBCs and AS-RBCs also promoted prothrombin activation although their procoagulant properties were milder compared to SS-RBCs. A significant increase of the thrombin-antithrombin complexes was observed in SS subjects. Prothrombin fragment 1+2 (F1+2) was elevated in half of the SS subjects, but the difference with controls did not reach significance. Elevated levels of thrombin-antithrombin complexes were observed in a number of SC (4/11) and AS (3/12) subjects, but the difference with controls was not significant. A significant correlation was observed between the plasma levels of thrombin-antithrombin complexes in the subjects with SS, AS and AA phenotypes, and the procoagulant properties of RBCs. Our results strongly suggest that the procoagulant properties which characterize SS-RBCs also affect SC-RBCs and AS-RBCs, and that exposure of phosphatidylserine by RBCs contributes to the hypercoagulable state observed in SCD.

Molecular cloning and expression of bothrojaracin, a potent thrombin inhibitor from snake venom.

Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-108021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.

Polymorphism in exon 10 of the human coagulation factor V gene in a population at risk for sickle cell disease.

In Caucasians, the R506Q mutation in exon 10 of the factor V gene (FV Leiden) confers an increased risk of thromboembolism. We have scanned this region of the gene for possible mutations in 450 subjects from populations at risk for sickle cell disease (SCD). The R506Q mutation was absent in subjects from sub-Saharan Africa, whereas its allelic frequency was 2.5% in the West Indies. Only one other substitution with no functional consequences in vitro (R485K) was found (32.4% allelic frequency) in sub-Saharan Africa. Thus, we found no mutations in exon 10 of the FV gene constituting an additional risk factor for thrombosis in SCD in sub-Saharan Africa. This suggests that the putative selective advantage conferred by R506Q does not exist in these populations, unless R485K has functional consequences in vivo. If further suggests that R506Q in American Africans is of Caucasian origin. Our data are the first to document ethnic variations in the frequency of the R485K polymorphism.

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with beta-thalassaemia major.

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with beta-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelerate thrombin generation in vivo which, in turn, triggers platelet activation.

Increased procoagulant activity of red blood cells from patients with homozygous sickle cell disease and beta-thalassemia.

It has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous beta-thalassemia behave as procoagulant cells. The procoagulant activity of beta-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i.e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with beta-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or beta-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 microM) than in the absence of cells (26 microM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 microM) or beta-thalassemia RBCs (mean value: 1.5 microM) was significantly lower compared to normal RBCs (p < 0.001). No significant difference was observed between SS-RBCs and beta-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and beta-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both beta-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

Bothrojaracin: a potent two-site-directed thrombin inhibitor.

The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.

Thrombin interaction with platelet membrane glycoprotein Ib.

Platelet activation by low doses of thrombin allows the amplification thrombin formation and thereby plays an important role in the development of thrombi. Although thrombin-induced platelet activation is elicited via the cleavage of its specific receptor (TR), platelet membrane glycoprotein Ib (GPIb) is required for responses to low concentrations of thrombin, as evidenced from the observation that GPIb-deficient platelets are characterized by a decreased sensitivity to thrombin and a low rate of activation. Glycoprotein Ib is an integral membrane protein composed of two disulfide-linked chains noncovalently associated to glycoproteins IX and V. As the receptor of the von Willebrand factor (vWF), GPIb plays a main role in platelet adhesion to the subendothelium. There are 25,000 copies of GPIb at the platelet surface but only a limited number of them appear to be involved in the high-affinity binding of thrombin. The catalytic site of thrombin is not involved in the interaction with GPIb. In contrast, competitive inhibition of GPIb-thrombin interaction by the C-terminal tail of hirudin, fibrin(ogen), and thrombomodulin indicates that thrombin exosite 1 is essential for GPIb binding. A hydrophylic domain located on the 45-kd N-terminal domain of GPIb alpha is involved in thrombin binding, and in particular, a stretch of negatively charged residues appears to make ionic interactions with thrombin. The same region of GPIb also contributes to the vWF binding site that should be very close to and even overlapping the thrombin-binding site. Despite GPIb and TR both interacting with thrombin exosite 1, the soluble fragment of GPIb does not modify the hydrolysis by thrombin of its target peptidic bond on TR, indicating that these two proteins bind to discrete subsites within exosite 1 and that the promoting effect of GPIb on TR-coupled responses depends on the anchorage of these proteins to the platelet membrane.

Functional consequences of the Ser334-->Pro mutation in a human factor X variant (factor XMarseille).

A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T-->C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334-->Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser-->Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.