Factor XIIa regulates the structure of the fibrin clot independently of thrombin generation through direct interaction with fibrin.

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombin-deficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and α-FXIIa, but not ß-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that FXIIa modulates fibrin clot structure independently of thrombin generation through direct binding of the N-terminus of FXIIa to fibrin(ogen). Modification of fibrin structure by FXIIa represents a novel physiologic role for the contact pathway that may contribute to the pathophysiology of thrombosis.

Notecarin D binds human factor V and factor Va with high affinity in the absence of membranes.

Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ∼55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ∼5,000- and ∼80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ∼85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding.

Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo.

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (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 ProT(S195A) 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 ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), 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-CH(2)Cl ([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.

Combined formative and summative professional behaviour assessment approach in the bachelor phase of medical school: a Dutch perspective.

BACKGROUND: Teaching and assessment of professional behaviour (PB) has been receiving increasing attention in the educational literature and educational practice. Although the focus tends to be summative aspects, it seems perfectly feasible to combine formative and summative approaches in one procedural approach. AIMS AND METHOD: Although, many examples of frameworks of professionalism and PB can be found in the literature, most originate from North America, and only few are designed in other continents. This article presents the framework for PB that is used at Maastricht medical school, the Netherlands. RESULTS: The approach to PB used in the Dutch medical schools is described with special attention to 4 years (2005-2009) of experience with PB education in the first 3 years of the 6-year undergraduate curriculum of Maastricht medical school. Future challenges are identified. CONCLUSIONS: The adages 'Assessment drives learning' and 'They do not respect what you do not inspect' [Cohen JJ. 2006. Professionalism in medical education, an American perspective: From evidence to accountability. Med Educ 40, 607-617] suggest that formative and summative aspects of PB assessment can be combined within an assessment framework. Formative and summative assessments do not represent contrasting but rather complementary approaches. The Maastricht medical school framework combines the two approaches, as two sides of the same coin.

Effect of oral contraceptives on thrombin generation measured via calibrated automated thrombography.

In a study population consisting of healthy men (n = 8), women not using oral contraceptives (OC) (n = 28) and women using different kinds of OC (n = 187) we used calibrated automated thrombography (CAT) in the absence and presence of added activated protein C (APC) to compare parameters that can be obtained from thrombin generation curves, i.e. lag time, time to peak, peak height and endogenous thrombin potential (ETP). Both with and without APC, plasmas of OC users exhibited the shortest lag time and time to peak, and the highest peak height and ETP. In the absence of APC none of these parameters differed between users of OC containing different progestogens. In contrast, in the presence of APC shorter lag times and time to peak, and higher peak height and ETP were observed in plasma of users of gestodene-, desogestrel-, drospirenone- and cyproterone acetate-containing OC than in plasma of users of levonorgestrel- containing OC. The ETP determined in the absence of APC (ETP(-APC)) had no predictive value for the APCsr (r = 0.11; slope 0.9 x 10(-3); 95% CI: -0.1 x 10(-3) to 2.0 x 10(-3)) whereas the ETP measured in the presence of APC (ETP+APC) showed an excellent correlation with the APCsr (r = 0.95; slope 6.6 x 10(-3); 95% CI: 6.3 x 10(-3) to 6.9 x 10(-3)) indicating that the APCsr is entirely determined by the ETP+APC. In conclusion, OC use increases thrombin generation, but differential effects of second and third generation OCs on the protein C system likely determine the differences in the risk of venous thrombosis between these kinds of OC.

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels.

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.

Effect of oral contraceptives on the anticoagulant activity of protein S in plasma.

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.

The Ser460Pro mutation in recombinant protein S Heerlen does not affect its APC-cofactor and APC-independent anticoagulant activities.

Protein S is a vitamin K-dependent plasma protein that functions as an APC-cofactor, but also exhibits anticoagulant activity in the absence of APC. The Heerlen polymorphism of protein S is characterized by a Ser460Pro substitution and lacks glycosylation at Asn458. It is associated with decreased protein S levels due to selective deficiency of free protein S Heerlen. To understand the lack of thrombotic complications associated with the protein S Heerlen mutation, we compared recombinant protein S Heerlen, wild type (wt) protein S and plasma-derived protein S. wt-Protein S and protein S Heerlen each bound 1:1 to C4BP with dissociation constants of 0.27 and 0.33 nM, respectively. Both wt-protein S and protein S Heerlen, either free or in complex with C4BP, were equally active as prothrombinase inhibitors in the absence of APC. All three protein S preparations stimulated APC-catalyzed inactivation of normal FVa, FVa Leiden and FVIIIa to the same extent. If extrapolated to plasma, it is not likely that the decreased free protein S levels in carriers of the protein S Heerlen mutation are compensated by an increased anticoagulant activity of protein S Heerlen-C4BP complexes. It is possible that an unrecognized plasma factor selectively enhances the anticoagulant activity of protein S Heerlen. If not, the reduction of free protein S levels in heterozygous protein S Heerlen-carriers combined with (low) normal total protein S levels apparently minimally affects the total anticoagulant activity of protein S (APC-cofactor and APC-independent activity) and hence is not associated with increased risk of venous thrombosis.

Reduced factor V concentration and altered FV1/FV2 ratio do not fully explain R2-associated APC-resistance.

Carriership of the factor V (FV) R2 haplotype is associated with mild APC-resistance, moderately reduced FV levels and a relative increase of the more thrombogenic FV isoform, FV1. Since low FV levels and increased FV1 can theoretically cause APC-resistance, we investigated whether these alterations can quantitatively account for R2-associated APC-resistance. In order to determine the effect of FV concentration and FV isoform composition on the APC-response, we reconstituted FV-deficient plasma with purified FV1 and FV2 in different molar ratios and to varying FV concentrations. APC sensitivity ratios (APCsr) were determined with the Coatest APC Resistance V, which probes the effect of APC on both FVa- and FVIIIa-inactivation, and with the Immunochrom APC response test, which only quantifies the effect of APC on FVIII(a)-inactivation. In both assays, low FV concentrations and/or high relative amounts of FV1 rendered plasma samples more resistant to APC. APCsr were also determined in FV-deficient plasma reconstituted with purified FV at levels and isoform ratios observed in R2-homozygotes (98% FV, 42% FV1) and age-matched controls (119% FV, 26% FV1). In both tests the APCsr of reconstituted control plasma was the same as that of plasma from controls, whereas reconstituted R2-plasma was less APC-resistant than plasma from homozygous carriers of the R2 haplotype. We conclude that the low FV levels and altered FV isoform ratio cannot fully explain R2-associated APC-resistance.

Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test.

BACKGROUND: Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis. METHOD: We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor V(Leiden) (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60). RESULTS: The thrombin generation-based APC resistance test was sensitive for the factor V(Leiden) and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases. INTERPRETATION: The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.

Effects of (pre-)analytical variables on activated protein C resistance determined via a thrombin generation-based assay.

The normalized activated protein C sensitivity ratio (nAPC-sr) determined with an assay that quantifies the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway identifies hereditary and acquired defects of the protein C system. We investigated the influence of assay conditions (analytical variables) and plasma handling (pre-analytical variables) on nAPC-sr obtained with this APC resistance test. The effect of the analytical variables (CaCl2, phospholipid and APC concentrations and the concentration and source of tissue factor) was determined in pooled normal plasma. Inhibition of thrombin formation by APC was dependent on the APC concentration and was also affected by the tissue factor, Ca2+ and phospholipid concentrations. Thus, strict standardization of reactant concentrations is required to obtain reproducible nAPC-sr. Three different tissue factor preparations were compared by determining nAPCsr in plasma samples obtained from 90 healthy individuals. nAPC-sr were similar for all three tissue factor preparations although, compared with the noncommercially available tissue factor used in earlier studies, values determined with commercial tissue factor preparations showed larger variation. Pre-analytical variables, investigated in plasma of nine volunteers (3 normal individuals and 6 individuals with an APC-resistant phenotype) were: concentration of anticoagulant (3.2% vs. 3.8% trisodiumcitrate), time before processing of blood (0, 4 and 24 h), centrifugation speed, storage temperature of plasma (-20 degrees C vs. -80 degrees C) and sample thawing. Multiple linear regression analysis showed that only the citrate concentration affected the nAPC-sr, which was higher in samples collected in 3.2% trisodiumcitrate than in samples collected in 3.8% trisodiumcitrate.

Endogenous factor V synthesis in megakaryocytes contributes negligibly to the platelet factor V pool.

BACKGROUND AND OBJECTIVES: Coagulation factor V (FV) is distributed between two pools: 80% circulates in plasma and 20% is stored in platelets. The aim of the study was to determine the origin of platelet FV. DESIGN AND METHODS: We investigated a FV Leiden heterozygous patient who had received an allogeneic bone marrow transplant from a normal donor. The patient had been referred to our laboratory for his marked activated protein C (APC) resistance in the apparent absence of FV Leiden. Analysis of the DNA from a buccal swab showed that the patient was indeed a heterozygous carrier of FV Leiden. The difference in FV genotype between the hepatocytes (heterozygous FV Leiden) and the blood cells (homozygous normal) of the patient provided a good model to investigate the origin of platelet FV. Platelets were isolated from the patient and the bone marrow donor and activated with thrombin and ionomycin to release and activate FV. APC was then added and the inactivation of platelet FVa was followed over time with a highly sensitive prothrombinase-based assay. RESULTS: While the donor's platelet FVa showed a normal inactivation time course, the patient's platelet FVa was considerably resistant to APC. The kinetic pattern of APC-catalyzed inactivation of the patient's platelet FVa was indistinguishable from that of plasma FVa from a FV Leiden heterozygote. INTERPRETATION AND CONCLUSIONS: These data indicate that platelet FV is derived from plasma and that endogenous FV synthesis by megakaryocytes contributes negligibly to the platelet FV pool.

Re-evaluation of the role of the protein S-C4b binding protein complex in activated protein C-catalyzed factor Va-inactivation.

Protein S expresses cofactor activity for activated protein C (APC) by enhancing the APC-catalyzed proteolysis at R(306) in factor Va. It is generally accepted that only free protein S is active and that complex formation with C4b-binding protein (C4BP) inhibits the APC-cofactor activity of protein S. However, the present study shows that protein S-C4BP expresses APC-cofactor activity and stimulates APC-catalyzed proteolysis at R(306) more than 10-fold, but instead inhibits proteolysis at R(506) by APC 3- to 4-fold. Free protein S stimulates APC-catalyzed cleavage at R(306) approximately 20-fold and has no effect on cleavage at R(506). The resulting net effect of protein S-C4BP complex formation on APC-catalyzed factor Va inactivation is a 6- to 8-fold reduction in factor Va inactivation when compared with free protein S, which is not explained by inhibition of APC-cofactor activity of protein S at R(306), but by generation of a specific inhibitor for APCcatalyzed proteolysis at R(506) of factor Va. These results are of interest for carriers of the factor V(Leiden) mutation (R(506)Q), as protein S-C4BP effectively enhances APC-catalyzed factor Va (R(306)) inactivation in plasma containing factor V(Leiden).

Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation.

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.

The role of thrombin exosites I and II in the activation of human coagulation factor V.

Human blood coagulation Factor V (FV) is a plasma protein with little procoagulant activity. Limited proteolysis at Arg(709), Arg(1018), and Arg(1545) by thrombin or Factor Xa (FXa) results in the generation of activated FV, which serves as a cofactor of FXa in prothrombin activation. Both thrombin exosites I and II have been reported to be involved in FV activation, but the relative importance of these regions in the individual cleavages remains unclear. To investigate the role of each exosite in FV activation, we have used recombinant FV molecules with only one of the three activation cleavage sites available, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV activating enzymes from the venom of Russell's viper (RVV-V) and of Levant's viper (LVV-V) known to activate FV by specific cleavage at Arg(1545). Our results indicate that thrombin exosite II is not involved in cleavage at Arg(709) and that both thrombin exosites are important for recognition and cleavage at Arg(1545). Efficient thrombin-catalyzed FV activation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg(709), Arg(1018), and Arg(1545) show different cleavage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respective cleavage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleavage site specificity as compared with activation by RVV-V or LVV-V.

Venous thrombosis and oral contraceptives: current status.

The use of oral contraceptives is associated with an increased risk of venous thrombosis. It is now generally accepted that women who use oral contraceptives that contain so-called third-generation progestins (desogestrel or gestodene) are exposed to a twofold higher risk of venous thrombosis than women who use oral contraceptives that contain the second-generation progestin levonorgestrel. Coagulation studies demonstrated that oral contraceptives increase the plasma level of prothrombin, decrease the level of protein S and induce acquired activated protein C resistance. The changes in hemostatic parameters can explain why women who use oral contraceptives are exposed to an increased risk of venous thrombosis and why the risk is further increased in third-generation oral contraceptive users.

Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor.

Tissue factor (TF) plays an important role in hemostasis, inflammation, angiogenesis, and the pathophysiology of atherosclerosis and cancer. In this article we uncover a mechanism in which protein S, which is well known as the cofactor of activated protein C, specifically inhibits TF activity by promoting the interaction between full-length TF pathway inhibitor (TFPI) and factor Xa (FXa). The stimulatory effect of protein S on FXa inhibition by TFPI is caused by a 10-fold reduction of the K(i) of the FXa/TFPI complex, which decreased from 4.4 nM in the absence of protein S to 0.5 nM in the presence of protein S. This decrease in K(i) not only results in an acceleration of the feedback inhibition of the TF-mediated coagulation pathway, but it also brings the TFPI concentration necessary for effective FXa inhibition well within range of the concentration of TFPI in plasma. This mechanism changes the concept of regulation of TF-induced thrombin formation in plasma and demonstrates that protein S and TFPI act in concert in the inhibition of TF activity. Our data suggest that protein S deficiency not only increases the risk of thrombosis by impairing the protein C system but also by reducing the ability of TFPI to down-regulate the extrinsic coagulation pathway.

Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions.

Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.

Changes of hemostatic variables during oral contraceptive use.

The use of oral contraceptives (OCs) has been known for many years to affect significantly almost all hemostatic parameters, but the challenge to relate these changes in a meaningful way to OC-induced increased venous thrombotic risk has not been met. New insights indicate that at least part of the answer can be found in the net effect of OC use on the efficacy with which the protein C pathway down-regulates thrombin formation. During OC use the (blood) plasma of a woman becomes resistant to the anticoagulant action of activated protein C (APC). The extent of this so-called acquired APC resistance as determined in a thrombin generation-based assay correlates remarkably well with the risk increases observed in clinical studies. Recent evidence indicates that the prothrombotic effect of the estrogen component ethinylestradiol in combined OC is counteracted by the progestagen component present in these preparations and that third-generation progestagens such as desogestrel or gestodene are less efficient with respect to this than the second-generation progestagen levonorgestrel.