A Plasma-Based Assay to Measure the Susceptibility of Factor V(a) to Inhibition By TFPIα

Abstract Background: Coagulation factor V (FV) is the precursor of activated FV (FVa), which assembles with factor Xa (FXa) on phospholipid surfaces to form the prothrombinase complex, accelerating prothrombin activation >1000-fold. FV is activated by FXa or thrombin via limited proteolysis at Arg709, Arg1018 and Arg1545. These cleavages progressively expose the FXa-binding site, generating activation intermediates with increasing affinities for FXa. Recently, it has been shown that tissue factor pathway inhibitor α (TFPIα) inhibits both the activation of FV (by interfering with cleavage at Arg1545) and the ability of partially activated FV(a) species to enhance prothrombin activation. These effects are mediated by interactions of the C-terminus of TFPIα with an acidic region in the B-domain of FV as well as with the FV(a) heavy chain. The pathophysiological relevance of these novel anticoagulant activities of TFPIα is still unexplored, but evidence has been provided that prothrombinase complexes assembled with FV(a) Leiden are less susceptible to TFPIα inhibition. Moreover, FV splicing variants (FV-short) with increased affinity for TFPIα have recently been discovered in two unrelated families (from East Texas and Amsterdam, respectively) with bleeding tendencies. Rationale and Aim: The FV present in plasma from different individuals may differ in its sensitivity to inhibition by TFPIα, with potential implications for the risk of venous thrombosis or bleeding. Therefore, the aim of this study was to develop a plasma-based assay that measures the susceptibility of FV(a) to TFPIα inhibition. Methods: FV in 1/1000 diluted plasma was activated for 3 minutes with a suboptimal FXa concentration on 20/60/20 DOPS/DOPC/DOPE lipids in the presence or absence of a peptide mimicking the C-terminus of TFPIα (TFPIα C-term). Purified prothrombin and a chromogenic substrate for thrombin were then added, and the activity of the prothrombinase complex was monitored continuously up to 30 minutes. The parabolic absorbance curves were fitted to second-order polynomial equations and the rate of prothrombin activation was calculated from the coefficient of the x2-term. The assay outcome was expressed as residual prothrombinase ratio (RP-ratio), defined as the ratio between the rates of prothrombin activation obtained in the presence and absence of TFPIα C-term. The assay was validated using plasma from 4 FV Leiden homozygotes and 4 normal controls. In addition, we tested plasma from 3 members of the FV Amsterdam family (2 carriers of the mutation up-regulating FV-short Amsterdam and 1 non-carrier). Results: The rate of prothrombin activation in the absence of peptide was a function of plasma FV level and pre-incubation time, and was inhibited by TFPIα C-term in a dose-dependent manner. A pre-incubation time of 3 minutes and a peptide concentration of 100 nM, yielding an RP-ratio of 0.30 in normal pooled plasma, were chosen. The RP-ratio was independent of the plasma FV level in the 75-150% range. Moreover, control experiments indicated that, at this high dilution, the plasma background did not influence the assay outcome. The intra- and inter-assay coefficients of variation of the RP-ratio were 5.4% and 12%, respectively. FV Leiden homozygotes had higher RP-ratios than normal controls (0.45 ± 0.04 vs. 0.30 ± 0.03, p=0.002), indicating resistance to inhibition by TFPIα C-term. Differently, the 2 carriers of the FV Asterdam mutation, who express high levels of FV-short Amsterdam, had markedly reduced RP-ratios (0.18 and 0.16 vs. 0.29 in the non-carrier), as expected from the high affinity of FV-short Amsterdam for TFPIα. Conclusions: We have developed and validated an assay that measures the susceptibility of plasma FV(a) to inhibition by TFPIα. This assay can be used to test whether TFPIα-mediated inhibition of FV activation and prothrombinase activity differs for (genetically) different FV variants and whether it correlates with the risk of thrombosis or bleeding. Supported by grant 2014-1 from the Dutch Thrombosis Foundation. Disclosures No relevant conflicts of interest to declare.

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Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα

Thrombosis and Haemostasis ◽

10.1055/s-0039-1700516 ◽

2019 ◽

Vol 120 (01) ◽

pp. 055-064

Author(s):

Peter van Doorn ◽

Jan Rosing ◽

Elena Campello ◽

Saskia Middeldorp ◽

Paolo Simioni ◽

...

Keyword(s):

Tissue Factor Pathway Inhibitor ◽

Bleeding Risk ◽

Factor V ◽

Prothrombinase Complex ◽

C Terminus ◽

Normal Individuals ◽

Splicing Variants ◽

Tissue Factor Pathway ◽

Fv Leiden ◽

Prothrombinase Activity

Abstract Background Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. Materials and Methods FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). Results The TFPIr (0.25–0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28–0.31) to FVL heterozygotes (0.35, 95% CI 0.34–0.37) and homozygotes (0.39, 95% CI 0.37–0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. Conclusion We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

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Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia

Blood ◽

10.1182/blood.v120.21.3391.3391 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3391-3391

Author(s):

Georgios Pongas ◽

Swapan Dasgupta ◽

Perumal Thiagarajan

Keyword(s):

Conflicts Of Interest ◽

Hospital Setting ◽

Fluorescence Emission ◽

Cross Reactivity ◽

Platelet Factor 4 ◽

Fluorescence Emission Spectrum ◽

Dependent Manner ◽

Platelet Factor ◽

Gram Negative ◽

Normal Controls

Abstract Abstract 3391 Introduction The anti-platelet factor 4(PF4)/heparin antibodies, arising as a result of previous heparin exposure, are causally related to the procoagulant state due to platelet and monocyte activation. Formation of these antibodies with subsequent thrombocytopenia or thrombosis has also been described in patients, who have not been previously exposed to heparin. The presence of anti-PF4/heparin antibodies in individuals correlates with the severity of periodontal disease, implying that their occurrence may be triggered by periodontal pathogens. In this study, we determined the presence of anti-PF4/heparin antibodies in gram-negative bacteremic patients in a hospital setting and propose a pathophysiologic mechanism of their presence. Method We developed an in house ELISA for quantifying anti-PF4/heparin antibodies using therapeutic heparin and PF4 isolated from platelets. We used serum from a patient with high optical density as a standard and assigned an unit of 100 arbitrarily to construct a standard curve. We tested the sera from gram negative bacteremic patients (n= 34) in the quantitative ELISA along with normal controls (n=10). We also developed an in house ELISA for studying cross reactivity between anti-PF4/heparin antibodies and lipopolysaccharide (LPS)/PF4. We tested the sera from patients (n=5) with heparin induced thrombocytopenia in this cross reactivity ELISA. To test the interaction of LPS with PF4, we labeled PF4 with Alexa488 and measured its binding to LPS by monitoring the changes in fluorescence emission spectrum following excitation at λ480. Results Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls (26.4 ± SD 33 units, N=34 versus 6.3 ± SD 2.38 units, N=10, P=0.032). Bacterial LPS interacted with alexa488-labeled PF4 in a concentration-dependent manner, as measured by the quenching of the excitation spectrum. Patients with ant-PF4/heparin antibodies also reacted with LPS/PF4 complex in ELISA. Prior absorption of serum with PF4/heparin complex coated on ELISA plates decreased the reactivity of the serum towards PF4/LPS complex (19–46%) in two out of the five patients tested suggesting some were cross-reaction between PF4/Heparin and PF4/LPS complex. Conclusions PF4 forms a complex with lipopolysaccharide and this complex is immunogenic. Antibodies to PF4/LPS complex can cross-react with PF4/heparin complex raising the possibility that these antibodies may be responsible for the detection of PF4/heparin in individuals never been exposed to heparin previously. These antibodies may also be at least partly responsible for increased thrombosis associated with infection. Disclosures: No relevant conflicts of interest to declare.

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Specific Leucine, Isoleucine and Threonine Residues In The TFPIα C-Terminal Region Are Necessary For Inhibition Of Prothrombinase

Blood ◽

10.1182/blood.v122.21.3562.3562 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3562-3562

Author(s):

Jeremy P Wood ◽

Alan E. Mast

Keyword(s):

Inhibitory Activity ◽

Thrombin Generation ◽

Factor Viia ◽

Specific Binding ◽

Mammalian Species ◽

Limited Proteolysis ◽

Dependent Manner ◽

Factor Va ◽

C Terminus ◽

Acidic Region

Abstract Background In addition to regulating the initiation of coagulation through inhibition of the tissue factor/factor VIIa complex, we have recently demonstrated a previously unrecognized anticoagulant function of TFPIα: inhibition of the prothrombinase complex (factor Xa (FXa), factor Va (FVa), Ca++, and phospholipids). No endogenous protein has previously been identified to inhibit prothrombinase under physiologically relevant conditions. The inhibition of prothrombinase is mediated by two specific binding events: (1) binding of TFPI’s second Kunitz domain to the FXa active site; and (2) an essential high–affinity exosite interaction between the TFPIα C-terminus and an acidic region within the factor V B-domain, retained in forms of FVa present in platelet alpha granules or generated through limited proteolysis with FXa. The TFPIα C-terminus contains a basic region (LIKTKRKRK) nearly identical to one found in the FV B-domain (LIKTRKKKK). Both of these sequences are highly conserved across mammalian species, suggesting they have an important physiological function. The basic residues of these sequences are necessary for the charge-based interaction with the FVa B-domain acidic region. We sought to determine the function of the absolutely conserved L, I, and T residues of this sequence. Methods Seven peptides containing different changes in the LIKT portion of the sequence, as shown below, were synthesized and tested in thrombin generation assays using forms of Factor Va containing (FVaAR) or lacking (FVaIIa) the acidic region of the B-domain. Results The wild type peptide (LIKTKRKRKK) inhibited FVaAR prothrombinase (IC50 = 1.03 µM) but did not inhibit FVaIIa prothrombinase at concentrations up to 225 µM, confirming that inhibition requires the presence of the B-domain acidic region. Substitution of LIKT with AAAA (AAAAKRKRKK) essentially abolished inhibitory activity with only ∼20% inhibition observed at 350 µM peptide, as did substitution of the L, I, and T (AAKAKRKRKK), demonstrating that the positively charged K and R residues alone do not mediate the inhibitory activity. Individual Ala substitutions of the L, I, and T residues resulted in decreased, but measurable, inhibitory function (IC50= 70.2 µM, 16.7 µM, and 107 µM, respectively). Finally, the peptide LIETKRKRKK was made to assess the effect of a K254E mutation, which has been identified in the NHLBI Exome Sequencing Project. This also essentially abolished inhibitory activity with only 20% inhibition observed in the presence of 340 µM peptide. Conclusions TFPIα inhibits thrombin generation by prothrombinase assembled with forms of FVa that retain the acidic region of the B-domain, which serves as a key exosite, binding the TFPIα basic C-terminus in a charge-dependent manner. The peptide studies presented here demonstrate that the hydrophobic residues L, I, and T are also absolutely essential for exosite binding and inhibition of thrombin generation. In addition, a natural mutation in the LIKT sequence (LIET) results in complete loss of prothrombinase inhibition, and therefore may represent a previously unrecognized prothrombotic risk factor. Thus, the regulation of coagulation occurring through TFPIα-mediated inhibition of prothrombinase appears to be relevant during normal hemostasis, as well as under pathologic conditions. Disclosures: Mast: Novo Nordisk: Honoraria, Research Funding.

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The C-Terminus of Tfpiα Inhibits Factor V Activation By Protecting the Arg1545 Cleavage Site

Blood ◽

10.1182/blood.v128.22.257.257 ◽

2016 ◽

Vol 128 (22) ◽

pp. 257-257

Author(s):

Peter van Doorn ◽

Jan Rosing ◽

Simone Wielders ◽

Tilman M. Hackeng ◽

Elisabetta Castoldi

Keyword(s):

Thrombin Generation ◽

Full Length ◽

Model Systems ◽

Factor V ◽

Dependent Manner ◽

High Affinity ◽

C Terminus ◽

Dose Dependent ◽

Acidic Region ◽

Basic Region

Abstract Coagulation factor V (FV) is the inactive precursor of FVa, which acts as an essential cofactor of factor Xa (FXa) in the prothrombinase complex. FV is maintained in the inactive state by the interaction between a basic and acidic region in the B-domain. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) is highly homologous to the FV basic region and also binds to the acidic region of FV. In fact, a large fraction of plasma TFPIα circulates in complex with FV. Thanks to this interaction, FV acts as a cofactor of TFPIα in the inhibition of FXa and TFPIα inhibits prothrombinase complexes containing forms of FVa that retain the acidic region. However, when FV is activated through cleavage at Arg709, Arg1018 and Arg1545 by FXa or thrombin, it loses its anticoagulant properties and becomes a strong procoagulant. Recently, a FV splicing variant (FV-short) that lacks the basic region and binds TFPIα with high affinity has been described. FV-short is present in all individuals and represents ~5% of all plasma FV. To gain more insight in the functional implications of the FV-TFPIα interaction, we studied the effects of a peptide identical to the TFPIα C-terminus (TFPIα C-term) on thrombin generation in plasma and on FV activation in model systems. All major findings were confirmed with full-length TFPIα. TFPIα C-term (0-5 µM) prolonged the lag time and decreased the peak height of tissue factor- and FXa-triggered thrombin generation in a dose-dependent manner. These effects were more pronounced at low procoagulant stimuli and in the presence of plasma TFPIα. TFPIα C-term also inhibited thrombin generation in FV-depleted plasma reconstituted with FV, but not in FV-depleted plasma reconstituted with FVa, suggesting an effect on FV activation and/or prothrombinase. In model systems, TFPIα C-term inhibited the activation of purified FV by FXa and thrombin in a dose-dependent manner. This could be due to inhibition of FV proteolysis and/or to inhibition of prothrombinase in the assay used to quantify FVa activity. Therefore, FV activation was also followed by SDS-PAGE and Western blotting. This showed that TFPIα C-term (1 µM) interferes with FV proteolysis by both FXa and thrombin by selectively impairing cleavage of FV at Arg1545, which is located close to the FV acidic region (residues 1493-1537). The effect of TFPIα C-term on FV activation by thrombin was 3-fold stronger for FV-short than for full-length FV, in line with their respective affinities for the TFPIα C-terminus. Full-length TFPIα (10 nM) also inhibited FV cleavage at Arg1545 and delayed FV activation by thrombin. Its effect was also more pronounced on FV-short than on FV. In summary, binding of the TFPIα C-terminus to the acidic region of FV inhibits FV activation by FXa or thrombin by blocking access to the Arg1545 cleavage site. Since cleavage at this site marks the transition of FV from an anticoagulant form (TFPIα-cofactor) to a procoagulant form (FXa-cofactor), this may represent an important new anticoagulant function of TFPIα. The main target of this anticoagulant mechanism is presently unclear, but it is unlikely to be intact FV, whose plasma concentration is 100-fold higher than the TFPIα concentration. More likely candidates are low-abundance FV species that lack the basic region but retain the acidic region, and therefore bind TFPIα with high affinity, such as FV-short, early FV activation intermediates and/or platelet FV. Supported by grant nr. 2014-1 from the Dutch Thrombosis Foundation. Disclosures No relevant conflicts of interest to declare.

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Amino Acids Asp695 and Tyr696 of Factor Va Are Essential for Optimal Rate of Prothrombin Activation by Prothrombinase.

Blood ◽

10.1182/blood.v114.22.3163.3163 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3163-3163

Author(s):

Jamila Hirbawi ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Catalytic Efficiency ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Prothrombinase Complex ◽

Factor Va ◽

Sds Page ◽

Prothrombin Activation

Abstract Abstract 3163 Poster Board III-101 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271 to yield human alpha-thrombin. This pathway is responsible for the generation of a transient intermediate, meizothrombin, that is enzymatically active with increased chromogenic substrate activity, but yields poor clotting activity. Factor Va is composed of heavy and light chains that play a crucial role during thrombin formation. Portions of the fVa heavy chain have been found to interact with proexosite 1 (pro1) of prothrombin and influence prothrombinase activity. It has been recently demonstrated that deletion of the COOH-terminal region of the factor Va heavy chain causes accumulation of meizothrombin due to delayed cleavage of prothrombin at Arg271. Site-directed mutagenesis was performed to generate recombinant mutant molecules in order to identify the specific amino acids of this COOH-terminal region that regulate cleavage. Mutants with the 695DYDY698→DFDY(fVaDFDY), KFDY(fVaKFDY),DEDE(fVaDEDE),DFDF(fVaDFDF) substitutions were constructed. These recombinant molecules along with wild type factor V (fVWT) were transiently expressed in COS7 cells purified to homogeneity and assessed for their capability to promote prothrombin activation. Prothrombin activation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVaKFDY and fVaDFDY was delayed, while prothrombinase assembled with fVaDEDE and fVaDFDF had no significant effects when compared to fVaWT. Two- stage clotting assays (PT times) revealed that fVaKFDY and fVaDFDY both had reduced clotting activity when compared to fVaWT, while fVaDEDE and fVaDFDF gave similar clotting results as fVaWT. Determination of kcat values for prothrombinase assembled with the various recombinant molecules revealed that prothrombinase assembled fVaKFDY and fVaDFDY had a 20% increase catalytic efficiency as compared with prothrombinase assembled with fVaWT, while fVaDEDE and fVaDFDF gave values that were comparable to prothrombinase assembled with fVaWT. Comparison of the rate of cleavage of two recombinant prothrombin mutant molecules, rMZ that can't be cleaved at Arg271 and rPII that can't be cleaved at Arg320, by prothrombinase assembled with the mutant fVa molecules resulted in normal cleavage of rMZ by all the mutants. Cleavage of rPII, however, was impaired when fVaKFDY and fVaDFDY were incorporated into prothrombinase. The data presented suggests that the 695DY696 portion of the acidic cluster found in the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate interaction during thrombus formation. Disclosures No relevant conflicts of interest to declare.

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The Factor V Activation Paradox

Journal of Biological Chemistry ◽

10.1074/jbc.m400727200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19580-19591 ◽

Cited By ~ 34

Author(s):

Thomas Orfeo ◽

Nicole Brufatto ◽

Michael E. Nesheim ◽

Hung Xu ◽

Saulius Butenas ◽

...

Keyword(s):

Rate Constants ◽

Factor Xa ◽

Catalytic Efficiency ◽

Reaction Pathways ◽

Factor V ◽

Prothrombinase Complex ◽

Factor Va ◽

Activation Products ◽

Initial Activation ◽

Prothrombin Activation

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. α-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.

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Studies On The Interaction Between Factor Va And Factor Xa

10.1055/s-0038-1652218 ◽

1981 ◽

Author(s):

M Lindhout ◽

J Govers-Riemslag ◽

J Rosing ◽

H Hemker

Keyword(s):

Factor Xa ◽

Factor V ◽

Molecular Weights ◽

Prothrombinase Complex ◽

Factor Va ◽

A Value ◽

Wide Range ◽

Carboxyglutamic Acid ◽

Polypeptide Chains ◽

Prothrombin Activation

Thrombin activated bovine factor V is composed of two polypeptide chains with molecular weights 94,000 and 80,000. The two polypeptide chains are complexed via Ca2+ions.Factor Va enhances the rate of thrombin formation by drastically increasing the Vmax of the prothrombin activation. We have undertaken a study of the interactions of factor Va with the different components of the prothrombinase complex (e.g. factor Xa and prothrombin), in order to get more insight in the mode of action of factor Va.Our kinetic experiments in solution show that the functional enzyme in the prothrombinase complex is a equimolar complex of factor Va and factor Xa. The dissociationconstant, as determined over a wide range of prothrombin concentrations, has a value of 3×10-9M.For the stimulating effect of factor Va on the prothrombin activation by factor Xa in solution, the presence of Ca2+ions is required. The dissociationconstant of the Va-Xa complex was found to be independent of the Ca2+ concentration. In order to reveal whether an interaction between Ca2+ and γ- carboxyglutamic acid residues is responsible for the observed Ca2+ requirement, identical experiments were carried out with decarboxyfactor Xa and decarboxyprothrombin. The isolated polypeptide chains of factor Va have, in the presence or absence of factor Va, no effect on the kinetic parameters of the prothrombin activation. This let us conclude that there is no interaction between factor Xa and the separate polypeptide chains of factor Va.The affinity of factor Xa for negatively charged phospholipid or stimulated bloodplatelets is greatly enhanced by the presence of factor Va. Our Kd value measured for the Xa-Va complex in combination with reported dissociationconstants of factor Xa-phospholipid and Factor Va-phospholipid complexes give a quantitative explanation for the above mentioned effect of factor Va on the binding of factor Xa to phospholipid membranes.

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Role of Inherited Bleeding Disorders in Women with Pregnancy Loss

Blood ◽

10.1182/blood.v118.21.4657.4657 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4657-4657

Author(s):

Manuela Krause ◽

Daniele Pillitteri ◽

Ann-Kathrin Pilgrimm ◽

Thomas Scholz ◽

Rainer Schwerdtfeger ◽

...

Keyword(s):

Pregnancy Loss ◽

Conflicts Of Interest ◽

Fetal Loss ◽

Factor V Leiden ◽

Mthfr C677t ◽

Von Willebrand Disease ◽

Factor V ◽

Bleeding Disorders ◽

Increased Risk ◽

Fv Leiden

Abstract Abstract 4657 Introduction: Pregnancy is a hypercoagulable state, and thromboembolism is the leading cause of antepartum and postpartum maternal mortality. Women with thrombophilic mutations (factor V leiden, prothrombin, and MTHFR) and inherited bleeding disorders, such as deficiency of factor XIII and fibrinogen, have been shown to be at increased risk of pregnancy loss. However, the risk of miscarriage in women with other inherited bleeding disorders has been discussed controversially. Due to the lack of data, it cannot be determined if the risk of miscarriage is increased in women with von Willebrand disease (vWD). The aim of our study was to clarify the association between inherited bleeding disorders and pregnancy loss. Patients and Methods: Subjects Concerning this investigation we included 91 female patients with two [n=46] or more [n=45] miscarriages occurring prior to 28 weeks of gestation and/or stillbirth without apparent reason. The median age of the examined group at the time of first fetal loss was 29 years, ranging from 17 to 41 years. Methods At first we compiled a detailed clinical history of bleedings of all patients. Subsequently, we performed various tests to gather information regarding coagulation abnormalities and thrombophilic defects. Therefore a molecular and functional assessment of the following data was performed: Coagulation factors, vWF:Ag, vWF:RCo, phospholipid antibodies, hyperhomocysteinaemia (HHCY), protein S (PS), protein C (PC), antithrombin (AT) and FV-Leiden mutation (G1691A), FII mutation (G20210A) and MTHFR C677T. Results: In our investigated population consisting of 91 women we registered 299 pregnancies of which 240 resulted in fetal loss, 232 prior to week 28 of pregnancy and 8 stillbirths. Seven out of 91 patients (8%) were carriers of inherited coagulation disorders; vWD: n=2 (2%), FVII deficiency: n=3 (3%), thrombocytopathy: n=2 (2%). In our study collective there was no increased rate of patients with vWD. None of the patients showed a FXIII- or fibrinogen deficiency. However, 17 patients (19%) have a bleeding diathesis. In 55 patients (60%) we could detect the following thrombophilic defects: FV-Leiden (G1691A): n=10, MTHFR C677T: n=42, PS: n=1, PC: n=1, APS: n=1. Conclusion: The incidence of vWD patients in our miscarriage collective is the same as the overall incidence of vWD patients in the general population. Therefore vWD is not associated with an increased risk of fetal loss. Disclosures: No relevant conflicts of interest to declare.

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Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016341 ◽

2020 ◽

pp. jbc.RA120.016341

Author(s):

Teodolinda Petrillo ◽

Francis Ayombil ◽

Cornelis van't Veer ◽

Rodney M. Camire

Keyword(s):

Bond Cleavage ◽

Coagulation Factor ◽

Factor V ◽

Functional Connection ◽

Prothrombinase Complex ◽

C Terminus ◽

Coagulation Factor V ◽

Tissue Factor Pathway ◽

Opposing Effects ◽

Necessary And Sufficient

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which form(s) of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ~1 nM) to TFPIα-BR which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (μM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

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Prothrombin Activation in the Presence of Platelets

10.1055/s-0039-1684694 ◽

1979 ◽

Author(s):

B.U. Dahlbäck ◽

J. Stenflo

Keyword(s):

Factor Xa ◽

Factor V ◽

Factor X ◽

Platelet Factor ◽

Platelet Surface ◽

Release Reaction ◽

Prothrombinase Complex ◽

Dependent Part ◽

The One ◽

Prothrombin Activation

The role of fragment 1, the vitamin K-dependent part of prothrombin containing γ-carboxyglutamic acid residues, and of fragment 2, the factor V-binding part of prothrombin for rapid prothrombin activation was studied. Activation rates of bovine prothrombin, acarboxyprothrombin and prethrombin 1 by factor Xa in the presence of platelets on the one hand and by the prothrombinase complex (factor Xa, factor V, phospholipid and Ca2+) on the other were compared. The conversion products of prothrombin in the presence of factor Xa and platelets were found to be the same as those seen when prothrombin was activated by the prothrombinase complex. The complete prothrombinase complex was more efficient even for activation of acarboxyprothrombin and prethrombin 1, which do not bind to phospholipid, than an abortive complex lacking the phospholipid. This was probably due to more effectively bound factor X . For rapid prothrombin activation by factor Xa in the presence of platelets both fragment 1 and fragment were found to be required. Acarboxyprothrombin and prethrombin 1 were slowly activated to thrombin by factor Xa in the presence of platelets but only after the platelet release reaction. The apparent KH of 0.6 uM prothrombin was 6 times lower than that of acarboxyprothrombin and the coefficient for proteolytic efficiency was approximately 50 times higher. The platelet surface ex posed upon the release reaction gradually lost its catalytic property during the prothrombin activation, probably due to destruction of the platelet factor Xa receptor by thrombin.

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