Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Summary.  Background:  Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. Objectives:  To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. Methods:  Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. Results:  We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 p m FVIIIa (i.e . 16 mU mL⁻¹ or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. Conclusion:  Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.     

Factor IX mutants with enhanced catalytic activity

Summary.  Background : Activated coagulation factor IX (FIXa) has low catalytic activity towards its physiologic substrate FX when activated FVIII (FVIIIa) is absent. One reason for this is that the FIX surface loop 99 stabilizes FIXa in a conformation that limits access of FX to the active site. Objectives : To investigate the effect of mutations in loop 99 and in the active site on FIXa activity with and without FVIIIa. Methods : Five full-length FIX mutants with amino acid exchanges in the catalytic domain of FIX were constructed and characterized by measuring their activity in FX activation in model systems and in plasma. Results and Conclusions : The mutants showed no or marginally improved catalytic properties in FX activation by the intrinsic tenase complex (FIXa–FVIIIa–Ca²⁺–phospholipid). The combination of mutations Y94F and K98T hardly affected FX activation in the presence of FVIIIa, but yielded a FIX molecule that, in FIX-depleted plasma, had ∼ 2.5-fold higher clotting activity and ∼ 3.5-fold higher activity in a thrombin generation assay than plasma-derived FIX (pdFIX). Two FIXa mutants had considerably increased activities towards FX in the absence of FVIIIa. FIXa-Y94F/K98T/Y177F/I213V/E219G (FIXa-L) and FIXa-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIXa-M) activated FX with catalytic efficiencies ( k _cat/ K _m) that, as compared with activated pdFIX, were increased 17-fold and six-fold, respectively. However, in plasma, their zymogen forms performed similarly to pdFIX. This indicates that the introduced mutations not only affected the activity of FIXa but may have also influenced the lifetime of the activated mutant molecules in plasma by modifying their activation and/or inhibition rates.     

Recombinant human factor VIIa (rFVIIa) can activate factor FIX on activated platelets

The studies reported here show that factor (F)VIIa can activate factor (F)IX on activated platelets in the absence of tissue factor. Both FIX and FIXa bind to the activated platelet surface with a K(d) of 8 nM and 2 nM, respectively. With factor (F)VIIIa, FIXa binds more tightly to platelets (K(d) 0.6 nM). At rFVIIa concentrations < 100 nm, no direct binding to the activated platelet surface can be detected with electrophoretic light scattering. However, in the presence of FIX, rFVIIa binding to platelets at concentrations as low as 10 nm rFVIIa can be detected. This is reflected by a decrease in the FIX K(d) from 8 to 1.6 nM. When rFVIIa is added to activated platelets in the presence of both FIX and FVIIIa, the K(d) for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on the surface of activated platelets in the absence of tissue factor. The activation of FIX by FVIIa on activated platelets can also be demonstrated by a functional assay for FIXa. These data show that pharmacological doses of rFVIIa result in the direct activation of FIX by rFVIIa to form additional tenase complexes ultimately resulting in improved thrombin generation. These results may explain, at least in part, the mechanism of action of rFVIIa in hemorrhagic conditions seen in otherwise normal patients who develop an acquired coagulopathy due to trauma, surgery or a variety of other events in which rFVIIa has been found to be effective.     

Role of the N-terminal EGF module of coagulation factor IX in activation of factors IX and X

Absence or reduced activity of coagulation factor IX (FIX) causes the severe bleeding disorder haemophilia B. FIX contains a Gla module, two epidermal growth factor-like (EGF) modules, and a serine protease region. I characterized a monoclonal antibody and found that it recognizes an epitope around residues 72 and 80 in the C-terminal part of EGF1 in human FIX. The antibody exhibited 10-fold greater affinity for activated FIX (FIXa) than for the zymogen FIX, indicating the existence of intra-molecular communication between the serine protease region and EGF1. Binding of the antibody did not affect the amidolytic activity of FIXa, hence I could use the antibody during activation of FIX to show that the C-terminal part of EGF1 is of importance for the interaction with FXIa but not with FVIIa/TF. Considering activation of FX, it is a matter of debate whether EGF1 or FIXa interacts directly with FVIIIa. I activated FX in the presence and absence of the antibody and/or FVIIIa. The addition of antibody caused only a minor decrease in k cat,app , and the major increase in k cat,app caused by the addition of FVIIIa occurred even in the presence of the antibody. This implies that EGF1 of FIXa is not directly involved in interaction with FVIIIa in the Xase complex. A model of the FIXa-FVIIIa complex, based on my findings and results from the literature, was constructed and indicated that EGF1 of FIXa does not interact directly with FVIIIa.     

Structural and functional features of factor XI

Summary.  Factor XI (FXI) has structural and mechanistic features that distinguish it from other coagulation proteases. A relatively recent addition to vertebrate plasma coagulation, FXI is a homodimer, with each subunit containing four apple domains and a protease domain. The apple domains form a disk structure with binding sites for platelets, high molecular weight kininogen, and the substrate factor IX (FIX). FXI is converted to the active protease FXIa by cleavage of the Arg³⁶⁹−Ile³⁷⁰ bond on each subunit. This converts the catalytic domains to the active forms, and unmasks exosites on the apple domains required for FIX binding. FXI activation by factor XIIa or thrombin proceeds through an intermediate with only one activated submit (1/2-FXIa). 1/2-FXIa activates FIX in a similar manner to FXIa. While the importance of the homodimeric structure of FXI is not certain, it may represent a strategy for binding to FIX and a platelet surface simultaneously.     

Functional role of O-linked and N-linked glycosylation sites present on the activation peptide of factor X

Summary.  Background : There are two O-linked and two N-linked glycosylation sites on the activation peptide of factor X (FX) involving residues Thr-17, Thr-29, Asn-39 and Asn-49. Objectives : The purpose of this study was to explore the contribution of carbohydrates of the FX activation peptide to zymogen recognition by physiological activators. Methods : The putative glycosylation sites were substituted individually or in combinations with Ala and mutants were expressed in mammalian cells. The entire activation peptide up to the P3 residue was deleted in another construct. Results : It was discovered that activation of zymogen mutants by both FVIIa and FIXa on negatively charged phospholipid vesicles has been improved 2–40-fold independent of a cofactor. These mutants were activated with slightly lower catalytic efficiency ( k _cat/ K _m) by FVIIa in the extrinsic Xase complex, though both K _m and k _cat constants for mutants were elevated. With the exception of ∼3-fold improvement in the activation of N49A, the catalytic efficiency of FIXa toward mutants was decreased 2–5-fold in the intrinsic Xase complex. Conclusions : The carbohydrate chains of the FX activation peptide play an important role in restricting the specificity of zymogen recognition by both FVIIa and FIXa, thereby preventing the cofactor-independent activation of FX by these proteases. On the other hand, the carbohydrates contribute to the cofactor-dependent recognition of the zymogen by both extrinsic and intrinsic Xase complexes.     

Functional analysis of the EGF-like domain mutations Pro55Ser and Pro55Leu, which cause mild hemophilia B

Summary.  We studied the functional role of two mutations, Pro55Ser and Pro55Leu, located in the N-terminal Epidermal Growth Factor-like domain (EGF1) of coagulation factor (F) IX. Both mutations cause mild hemophilia B with habitual FIX coagulant activities of 10–12% and FIX antigen levels of 50%. We found that activation by FVIIa/TF and FXIa was normal for FIXPro55Ser, but resulted in proteolysis of FIXPro55Leu at Arg318-Ser319 with a concomitant loss of amidolytic activity, suggesting intramolecular communication between EGF1 and the serine protease domain in FIX. This was further supported by experiments using an anti-EGF1 monoclonal antibody. Activation of FX by FIXaPro55Ser was impaired in both the presence and the absence of phospholipid or FVIIIa, indicating that Pro55 is not directly involved in binding to FVIIIa. We also studied the effect of the two Pro55 mutations on Ca²⁺ affinity and found only small changes. Thus, the Pro55Ser mutation causes hemophilia primarily through to an impaired ability to activate FX whereas at least in vitro the Pro55Leu defect interferes with the activation of FIX.     

Specific detection of human coagulation factor IX in cynomolgus macaques

Summary.  After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg⁻¹; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 ± 37.6 ng mL⁻¹ at 1 h after the injection and gradually decreased to 1.79 ± 1.1 ng mL⁻¹ by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.     

Clustered basic residues within segment 484–510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation

Summary.  Residues 484–510 of factor (F)VIIIa A2 subunit comprise a prominent epitope for inhibitor antibodies, suggesting that this region is critical for cofactor function. To address the role of this region in catalysis, FVIIIa forms were evaluated following conversion of conserved charged residues to Ala, either in clusters or individually. The two cluster mutants, Lys496Ala/Lys499Ala/Asp500Ala and Glu507Ala/Lys510Ala, were indistinguishable from wild type. The mutation Arg489Ala/Arg490Ala/Lys493Ala (489-3A) possessed near-normal affinity for FIXa and showed no effect on the Km for FX, but exhibited ∼ 3-fold and ∼ 30-fold reduced kcat values for FXase in the presence and absence of surface, respectively. However, the single-site mutants Arg489Ala, Arg490Ala and Lys493Ala exhibited affinity and kcat values similar to wild type. Furthermore, the 489-3A mutant showed a marked reduction in the positive electrostatic potential within this region of A2, consistent with the hypothesis that the cumulative basic charge in this region of A2 subunit modulates cofactor function.     

Coagulation procofactor activation by factor XIa

Summary. Background: In the extrinsic pathway, the essential procofactors factor (F) V and FVIII are activated to FVa and FVIIIa by thrombin. In the contact pathway and its clinical diagnostic test, the activated partial thromboplastin time (APTT) assay, the sources of procofactor activation are unknown. In the APTT assay, FXII is activated on a negatively charged surface and proceeds to activate FXI, which activates FIX upon the addition of Ca²⁺. FIXa feeds thrombin generation through activation of FX. FIXa is an extremely poor catalyst in the absence of its FVIIIa cofactor, which, in the intrinsic FXase complex, increases FXa generation by ～ 10⁷. One potential APTT procofactor activator in this setting is FXIa. Objective: To test the hypothesis that FXIa can activate FVIII and FV. Methods: Recombinant FVIII and plasma FV were treated with FXIa, and the activities and integrities of each procofactor were measured using commercial clotting assays and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Results: Kinetic analyses of FXIa‐catalyzed activation and inactivation of FV and FVIII are reported, and the the timing and sites of cleavage are defined. Conclusions: FXIa activates both procofactors at plasma protein concentrations, and computational modeling suggests that procofactor activation during the preincubation phase of the APTT assay is critical to the performance of the assay. As the APTT assay is the primary tool for the diagnosis and management of hemophilias A and B, as well as in the determination of FVIII inhibitors, these findings have potential implications in the clinical setting.     

The action of high-dose factor VIIa (FVIIa) in a cell-based model of hemostasis

We have developed a cell-based model of hemostasis. This model suggests that the defect in hemophilia is specifically a failure of platelet-surface factor Xa (FXa) generation, leading to a failure of platelet surface thrombin generation. Activation of FX by FVIIa/tissue factor (TF) does not compensate for a lack of FXa activation on the platelet surface by the FVIIIa/FIXa complex. This is because plasma protease inhibitors prevent FXa from moving through the fluid phase from the TF-bearing cell to the platelet surface. We have previously proposed a platelet-dependent mechanism of action for high-dose factor VIIa (FVIIa; Novoseven, Novo Nordisk, Copenhagen, Denmark). Our data suggest that, when present at high levels, FVIIa binds to activated platelets and activates small amounts of FX independent of TF. This platelet-surface FXa can partially restore platelet-surface thrombin generation in hemophilia. Recently, van't Veer and colleagues reported results from an in vitro model in which coagulation reactions were initiated by relipidated TF. The authors concluded that high-dose FVIIa may exert a hemostatic effect in hemophilia by overcoming inhibition of FVIIa/TF activity by zymogen FVII. By contrast, we found that plasma levels of FVII did not slow thrombin generation in a model system initiated with cell-associated TF. This discrepancy highlights the potential differences between the studies of the coagulation reactions assembled on living cells compared to phospholipid vesicles. Our data suggest that in a cellular system high-dose FVIIa acts primarily by enhancing the rate of thrombin generation on platelet surfaces and not by overcoming inhibition by zymogen FVII of TF-dependent activation of FX.     

The first epidermal growth factor-like domains of factor Xa and factor IXa are important for the activation of the factor VII--tissue factor complex.

During tissue factor (TF)-induced coagulation, the factor (F)VIIa-TF complex activates factor (F)X and factor (F)IX. Through positive feedback, the generated FXa and FIXa activate FVII-TF. The first epidermal growth factor-like (EGF1) domains of FX and FIX serve as important TF-recognition motifs when FVIIa-TF activates FX or FIX. Here, we investigated the role of EGF1 domains of FXa and FIXa during the activation of FVII-TF and inhibition by tissue factor pathway inhibitor (TFPI). FXaPCEGF1 (EGF1 domain of FXa replaced with that of protein C), and FXaQ49P (EGF1 domain mutant with impaired calcium-binding), and the corresponding FIXa mutants were generated, and their abilities to activate FVII-TF were compared with the wild-type (WT) enzymes. In the absence of TF, the rates of FVII activation were similar between WT enzymes and mutant FXa and FIXa proteases. In the presence of either soluble TF (sTF) or relipidated TF, each mutant of FXa or FIXa activated FVII-TF at a slower rate than the corresponding WT enzyme. Kinetics of inhibition of the amidolytic activity of WT and the mutant FXa proteases by either two-domain or full-length TFPI were similar. However, compared with the complex of TFPI-FXaWT, the abilities of the complexes of TFPI-FXa mutants to inhibit FVIIa-TF were impaired. We conclude that the EGF1 domains of FXa and FIXa are important for the activation of FVII-TF and for the formation of FVIIa-TF-FXa-TFPI complex.     

A highly-sensitive plasma von Willebrand factor ristocetin cofactor (VWF:RCo) activity assay by flow cytometry

Summary.  Background:  Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD) and for monitoring therapy. However, current manual or automated VWF:RCo assay methods have relatively poor operating characteristics. Our goal was to develop and validate a simple, accurate, specific and sensitive platelet-based VWF:RCo assay. Methods:  Using green or red fluorochrome-labeled, fixed normal platelets and normal or patient plasma, ristocetin-dependent and VWF-mediated platelet aggregation was detected by flow cytometry. VWF:RCo activity was assayed as the number of double-positive events (green and red) among all green or red events, relative to the calibrator plasma signal (6–150% or IU dL⁻¹), and reported as percent or IU dL⁻¹. We tested plasma samples from normal donors ( n  = 51) and known VWD patients (type 1, n  = 16; type 2, n  = 17) based on clinical history, levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregometry/agglutination assay), factor (F) VIII activity and VWF multimer analysis. Results:  For normal donors and type 1 VWD patients, VWF:RCo activity by flow cytometry vs. manual platelet aggregation correlated closely ( R ² = 0.74), and VWF:RCo/VWF:Ag ratios did not differ significantly. In contrast, VWF:RCo/VWF:Ag ratios for type 2 VWD subtypes were significantly lower using VWF:RCo by flow cytometry vs. manual platelet aggregation assay ( P  < 0.01), especially for type 2A VWD patients. Conclusions:  This new flow cytometry-based VWF:RCo assay is simple, accurate, specific and sensitive, particularly for type 2 VWD.     

Fibrin stimulates platelets to increase factor VIIIa binding site expression.

Factor (F)VIII functions as an enzymatic cofactor on the membranes of stimulated platelets. However, thrombin stimulates platelets to express only a small number of binding sites for FVIII. We wished to determine whether molecules that are likely to be present in a developing thrombus stimulate platelets to up-regulate FVIII binding site expression. Flow cytometry was utilized to measure binding of fluorescein-labeled FVIIIa to activated platelets and a FXase assay was utilized to measure platelet-dependent function. Various agonists as well as normal and mutant fibrinogens and fibrin were evaluated as co-stimuli. Thrombin-stimulated platelets expressed 214 +/- 67 binding sites for thrombin-activated FVIII (FVIIIa) and none of the established soluble agonists enhanced binding site exposure. However, the presence of 5 micro g mL(-1) fibrin increased the number of FVIIIa binding sites/platelet three- to eight-fold (1470 +/- 130, range 600-1800) with a parallel increase in platelet-based FXase assay. Binding site up-regulation was not stimulated by fibrinogen and was blocked by inhibitors of GPIIbIIIa. Mutant fibrin lacking the gamma-chain C-terminal four residues was ineffective while fibrin with altered RGD sequences did stimulate expression of FVIIIa binding sites indicating that co-stimulation is mediated by the fibrin gamma-chain termini. Fibrin-enhanced expression of FVIIIa binding sites was not supported by D364H fibrin, which does not aggregate normally, and was blocked by the GPRP peptide, which inhibits fibrin polymerization. Polymerized fibrin can function as a platelet co-stimulus, up-regulating expression of binding sites for FVIIIa.     

How factor VIIa works in hemophilia

Summary.  The influence of elevated platelet concentration and recombinant factor VIIa (rFVIIa) on thrombin generation at 5 pM tissue factor (TF) in a synthetic mixture corresponding to hemophilia B (SHB) and 'acquired' hemophilia B blood (AHBB) produced in vitro by an antifactor IX antibody was evaluated. (a) Thrombin generation in SHB and AHBB was delayed and reduced; (b) with 10 nM rFVIIa or 5× normal platelets (10 × 10⁸/mL) SHB and AHBB showed a slight increase in thrombin generation; (c) in the absence of TF, almost no thrombin generation was detected in SHB and AHBB in the presence of 10 nM rFVIIa and 10 × 10⁸/mL activated platelets (5× normal); (d) with TF, 10 nM rFVIIa and 3–5× normal nonactivated platelets (6–10 × 10⁸/mL), thrombin levels approaching normal values were attained. FVIIa appears to function effectively and locally by the combined effect of TF expression and platelet accumulation at the site of a vascular lesion.     

Molecular models of the procoagulant Factor VIIIa–Factor IXa complex

BACKGROUND: Formation of the intrinsic tenase complex is an essential event in the procoagulant reactions that lead to clot formation. The tenase complex is formed when the activated serine protease, Factor IXa (FIXa), and its cofactor Factor VIIIa (FVIIIa) assemble on a phospholipid surface to proteolytically convert the zymogen Factor X (FX) into its active form FXa. The physiological relevance of the tenase complex is evident in hemophilia A or B patients who present with bleeding disorders. OBJECTIVES: The purpose of this study was to establish three-dimensional (3D) models of the FVIIIa-FIXa complex. METHODS: First, we built two new theoretical models of FVIIIa via homology modeling, inter-domain docking and loop simulation algorithms as well as a model for FIXa. This was followed by pseudo-Brownian protein-protein docking in internal coordinates with the ICM (Internal Coordinates Mechanics) program between the two FVIIIa and the FIXa structures. RESULTS: Ten representative models of this complex are presented based on agreements with known experimental data and according to structural criteria. CONCLUSIONS: These novel 3D models will help guide future site directed mutagenesis aimed at improving the functionality of FVIIIa and/or FIXa and will contribute to a better understanding of the role of this macromolecular complex in the blood coagulation cascade.     

Protein C inhibitor directly and potently inhibits activated hepatocyte growth factor activator

Background:  Hepatocyte growth factor (HGF) plays an important role in tissue repair and regeneration. HGF activator (HGFA), a factor XIIa-like serine protease, activates HGF precursor to HGF. The precursor of HGFA, proHGFA, is activated by thrombin generated at sites of tissue injury. It is known that protein C inhibitor (PCI), an inhibitor of activated protein C (APC), also inhibits thrombin–thrombomodulin (TM) complex. Objectives:  In the present study we evaluated the effect of PCI on thrombin-catalyzed proHGFA activation in the presence of TM, and on HGFA activity. Results:  PCI did not inhibit thrombin-TM-mediated proHGFA activation, but it directly inhibited activated HGFA by forming an enzyme inhibitor complex. The second-order rate constants ( m ⁻¹ min⁻¹) of the reaction between HGFA and PCI in the presence or absence of heparin (10 U mL⁻¹) were 4.3 × 10⁶ and 4.0 × 10⁶, respectively. The inhibition of HGFA by PCI resulted in a significant decrease of HGFA-catalyzed activation of HGF precursor. Exogenous HGFA added to normal human plasma formed a complex with plasma PCI, and this complex formation was competitively inhibited by APC in the presence of heparin, but very weakly in the absence of heparin. We also demonstrated using recombinant R362A-PCI that Arg362 residue of PCI is important for HGFA inhibition by PCI as judged from the three-dimensional structures constructed using docking models of PCI and HGFA or APC. Conclusion:  These observations indicate that PCI is a potent inhibitor of activated HGFA, suggesting a novel function for PCI in the regulation of tissue repair and regeneration.     

A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa

BACKGROUND: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. OBJECTIVE: Based on our finding of low-specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. RESULTS: The rate of activity decay of E1829A was approximately fourteen fold faster than wild-type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one-stage and two-stage assays were approximately 14% and approximately 11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A-A3C1C2 was comparable to wt A3C1C2 (K(d) = 20.1 +/- 3.4 nM for E1829A, 15.3 +/- 3.7 nM for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by approximately 3.6-fold as a result of the mutation (K(d) = 526 +/- 107 nM for E1829A, 144 +/- 21 nM for wt). CONCLUSION: As modeling data suggest that Glu1829 is located at the A2-A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa.     

Novel insights into the regulation of coagulation by factor V isoforms,tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro‐ and anticoagulant pathways. It circulates as a single‐chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane‐bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV‐Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV‐Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C‐terminus of the FV‐Short B domain, which binds the positively charged C‐terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV‐Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.     

The function of factor XI in tissue factor-initiated thrombin generation

Summary.  The influence of plasma and platelet factor (F)XI on thrombin generation initiated with 10 p m tissue factor (TF) in a synthetic coagulation model was evaluated in the presence of either 2 × 10⁸ mL⁻¹ platelets or the equivalent (2 µ m ) phospholipids. In either system, with all proteins present at physiological concentrations, FXI (30 n m ) had no effect on thrombin generation. With phospholipids in the absence of FXI, an increase in vitamin K-dependent proteins (VKDP) (up to 500%) significantly prolonged the initiation phase of thrombin generation and decreased maximum thrombin levels. The inhibition was principally caused by the elevated prothrombin and FIX concentrations. When 30 n m FXI was added with elevated VKDP and phospholipids, the initiation phase was decreased and the maximum thrombin levels generated substantially increased. In experiments with platelets (with and without plasma FXI), an increase in VKDP had little effect on the initiation phase of thrombin generation. These data indicate that (i) FXI has no effect on thrombin generation at 10 p m TF and physiological concentrations of VKDP; (ii) platelets and plasma FXI are able to compensate for the inhibitory effects of elevated VKDP.