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

Abstract

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 forms 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. 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 forms 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. The proteolytic conversion of factor V (FV) to activated FV (FVa) is central to the amplification of coagulation and necessary for blood clot formation (1Mann K.G. Kalafatis M. Factor V: A combination of Dr Jekyll and Mr Hyde.Blood. 2003; 101: 20-30Crossref PubMed Scopus (176) Google Scholar). FV circulates in the blood as a single-chain, inactive procofactor in which the N-terminal heavy chain (HC; domains A1-A2) and C-terminal light chain (LC; domains A3-C1-C2) are situated between a central B-domain (Fig. 1A) (1Mann K.G. Kalafatis M. Factor V: A combination of Dr Jekyll and Mr Hyde.Blood. 2003; 101: 20-30Crossref PubMed Scopus (176) Google Scholar, 2Camire R.M. Bos M.H. The molecular basis of factor V and VIII procofactor activation.J. Thromb. Haemost. 2009; 7: 1951-1961Crossref PubMed Scopus (66) Google Scholar). Proteolysis of FV at Arg709, Arg1018, and Arg1545 by thrombin or factor Xa (FXa) releases the B-domain and activates it to FVa (3Suzuki K. Dahlback B. Stenflo J. of coagulation factor PubMed Google Scholar, The of factor of activated factor of and of PubMed Google Scholar, R.M. K.G. and of factor PubMed Scopus Google Scholar, K.G. of chain factor PubMed Google Scholar, K.G. of factor V PubMed Google Scholar, Dahlback B. for of factor V by factor J. PubMed Scopus Google Scholar). a FVa binds with high affinity the prothrombinase which to thrombin K.G. in the and of blood PubMed Scopus Google Scholar). FVa is for thrombin as it the at which by of K.G. of the PubMed Google Scholar). the by which FV is as a procofactor and how in the B-domain FV been R.M. Bos M.H. The molecular basis of factor V and VIII procofactor activation.J. Thromb. Haemost. 2009; 7: 1951-1961Crossref PubMed Scopus (66) Google Scholar, R.M. A at blood coagulation factor PubMed Scopus Google Scholar, and of factor V and a a of the PubMed Scopus Google Scholar, of factor PubMed Scopus Google Scholar, R.M. of B-domain from factor V the by which cofactor function is PubMed Scopus Google Scholar). conserved in the B-domain the region and acidic region 1493–1537) by the binding the (Fig. 1A) R.M. the B-domain factor V in an inactive PubMed Scopus Google Scholar, M.H. R.M. A region the B-domain function in factor PubMed Scopus Google Scholar). The the necessary to FV of of the B-domain, the a FV that is as a procofactor M.H. R.M. A region the B-domain function in factor PubMed Scopus Google Scholar). However, removal of the BR or AR2 by a constitutively active which functions like FVa R.M. the B-domain factor V in an inactive PubMed Scopus Google Scholar, M.H. R.M. A region the B-domain function in factor PubMed Scopus Google Scholar). with an binds with high affinity to forms of FV that but the BR The binding of to binding and blocks procoagulant Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar). The of these functional landmarks in the B-domain FV as a factor pathway inhibitor (TFPIα) binds to an AR2 forms of FV and procoagulant function by in prothrombinase R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, of factor pathway inhibitor do not PubMed Google Scholar). the positively charged C-terminal of TFPIα (TFPIα-BR) is to and binding to AR2 R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, Tissue factor pathway PubMed Scopus Google Scholar). FV, which is in the B-domain and from and FV, cleaved by or physiologic forms that by TFPIα R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, of factor pathway PubMed Scopus Google Scholar). forms of FV the BR AR2 to as and are the of in to and factor factor pathway PubMed Google TFPIα to thrombin at the of prothrombinase R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, R.M. in the of factor V and PubMed Scopus Google Scholar). between FV and TFPIα been with J. of factor pathway inhibitor in with factor V PubMed Scopus Google that the and associated with FV is to of TFPIα in that of FV and TFPIα to and of FV from TFPIα a with FV and TFPIα and that FV TFPIα with an affinity of J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar, M. The C-terminus of factor pathway inhibitor is for its with V and Thromb. Haemost. PubMed Scopus Google Scholar). it has been that of of factor pathway inhibitor do not PubMed Google Scholar, M. J. of binding in the of by Haemost. PubMed Google not with FV M. R.M. J. Factor V has an anticoagulant cofactor that the of PubMed Scopus Google Scholar). TFPIα via its BR FV by thrombin or by cleavage at Arg1545 J. The C-terminus of factor pathway factor V by the cleavage Thromb. Haemost. PubMed Scopus Google Scholar). is that FVa with TFPIα as it TFPIα of of factor pathway inhibitor do not PubMed Google Scholar, of factor Xa by factor pathway PubMed Google and the to bind M. The C-terminus of factor pathway inhibitor is for its with V and Thromb. Haemost. PubMed Scopus Google Scholar, B. with FVa and to prothrombinase the of PubMed Scopus Google Scholar). A in the from the of a isoform of FV B. the regulation of coagulation by factor V factor pathway and Thromb. Haemost. PubMed Scopus Google Scholar). in a with a the in the activates a in in an of a in a from PubMed Scopus Google Scholar, Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). for a FV isoform FV-short, which in the B-domain including the BR but AR2 (Fig. with the high of FV-short nM) and TFPIα Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). that FV-short and TFPIα a complex in via the AR2 FV-short Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). The in TFPIα is to to the and from a in the TFPIα is from the to FV-short B. and anticoagulant of factor V in of and J. PubMed Scopus Google Scholar). At in from been that and a FV-short with in TFPIα K. J. A in the V associated with of factor V procoagulant PubMed Scopus Google Scholar, R.M. Factor V A in the in FV-short and Scholar). Importantly, in at a as FV-short is in at Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). The of FV-short is not but a it the of with to B. Factor and as factor pathway inhibitor Thromb. Haemost. PubMed Scopus Google Scholar). these are it unclear which forms of FV are physiologic ligands for TFPIα. is an to as it has a key in coagulation by molecular for its in the coagulation FV-short and its binding to TFPIα to Here, we the binding and regulation of FV and FV-short by TFPIα and examine how cleavage in the B-domain influences these interactions. Our findings show that FV and FVa are not physiologic ligands for TFPIα. TFPIα binds and forms of FV that the BR as FV cleaved at and The mechanistic the regulation of coagulation and the necessary that FV to with TFPIα. A of FV is in FV from the FV and in to its with a FV with a shortened B-domain, and a to FV-short and with at or thrombin cleavage and The cleavage at the (Fig. cleavage of and FV-short the and to FVa. and at Arg709 and Arg1545, not cleaved by thrombin (Fig. a we that FV-short and FV-short to FVa of at Arg709 with thrombin or at Arg1545 with to the not FV-short, and FVa thrombin in a thrombin the and in the which to the FV in in is a the for FV-short, and cleaved FV-short to FVa (Fig. and these show that FV-short is constitutively active like FVa and not cleavage at Arg709 or Arg1545 to it from of FV of FV a in as in The of FV in to and the to The of or FV The to FV-short to the the of at and the the in a is constitutively active and is by and the cofactor and the for the of prothrombinase by or TFPIα of cofactor and the for with an to and the effects of TFPIα to its C-terminal The to FV-short to the are the of at and the the in a The of FV a in as in The of FV in to and the to The of or FV The to FV-short to the the of at and the the and the cofactor and the for the of prothrombinase by or TFPIα of cofactor and the for with an to and the effects of TFPIα to its C-terminal The to FV-short to the are the of at and the the FV-short is from FVa as a region of the B-domain between the and which AR2 (Fig. We and to examine the of FV-short function in full-length TFPIα has of in the we a to the of FVa in a the of FV-short and in the of cleavage of FV-short at Arg709 a the of to FV-short cofactor However, cofactor function FV-short at Arg1545 the show that FV-short function but AR2 is to the The to how FV-short with the FVa by (Fig. A and FV-short to (Fig. and a of a (Fig. and We that FV-short is to with the once coagulation is thrombin or in the FV-short removing its B-domain, by of is to cleavage at Arg1545, as FV-short at Arg1545 to at Arg709 (Fig. FV-short to with the a (Fig. We that which thrombin in of FV-short at Arg709 and Arg1545, which binding to the C-terminal of TFPIα. We the of FV-short to full-length TFPIα or in a the of TFPIα with an FV-short or by with a of FVa and not (Fig. a full-length in the the (Fig. and TFPIα-BR, full-length TFPIα not prothrombinase FVa as the We the in for and full-length TFPIα to TFPIα not by the to between the which of thrombin that TFPIα and FV and a complex in J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar, M. The C-terminus of factor pathway inhibitor is for its with V and Thromb. Haemost. PubMed Scopus Google Scholar, Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). to as or in a and these that binds and blocks the function of FV, to with of how BR with AR2. TFPIα the FV or it binds region of is that TFPIα not bind FV, but and the function of the including forms of FV with an AR2 examine we to to in molecular the of FV-short and FVa (Fig. from full-length TFPIα (Fig. of FV-short with TFPIα or not that TFPIα with FV-short the that the a show that are not for FV-short binding to TFPIα. FV-short at Arg709 TFPIα (Fig. at Arg1545 binding (Fig. FVa not bind TFPIα (Fig. to FV-short, TFPIα as a with or the of an between the (Fig. and at physiologic and FV and TFPIα are to a complex in We binding by in a FV-short a of and it a binding with a binding of and (Fig. with binding with FVa (Fig. or full-length TFPIα that these from FV-short (Fig. The for binding between FV-short and (Kd nM) and TFPIα (Kd of nM) to show that domains and of TFPIα do not binding to with functional and to FV-short cleaved at Arg709, with a affinity (Kd nM) (Fig. of FV-short at Arg1545 binding to the of AR2 to the (Fig. we to binding of or to with the that TFPIα not bind binding it is (μM and to of physiologic Our that FV-short is in which thrombin or is its to TFPIα to cleavage the we of and FV-short and of in and to FV-short of the high from FV-short binding to (Fig. of FV-short and by that the in with the of the to cleavage at Arg1545 (Fig. and with which is cleaved at Arg1545 (Fig. thrombin the of cleavage at Arg709 (Fig. is to the affinity for FV-short is cleaved at FV not bind of or with in a (Fig. However, of the cleaved forms of FV bind to (Fig. the the to proteolytic by of the cleavage of FV by thrombin (Fig. and not for The in with the of the to which from FV cleavage at Arg709 and Arg1018 by thrombin (Fig. The in well with cleavage at However, the to a of FV not cleaved at We that to between nM) and the FV B-domain which the and in the FV cleaved at Arg709 and Arg1018, we not the role of these cleavage we an that cleaved at the of not bind as the at (Fig. that cleaved by thrombin at Arg709 and Arg1545, but to binding (Fig. and show that cleavage of FV at Arg709 has binding of FV to We that is to the that the BR the FV B-domain and with AR2. Our show that FV is at Arg1018 FV and with its AR2. the show that TFPIα not bind FV, but binds proteolytic of FV with a BR. to FV-short we that the of FV-short cleavage by at an by J. The C-terminus of factor pathway factor V by the cleavage Thromb. Haemost. PubMed Scopus Google Scholar). an to the affinity of the between FV BR and AR2 with the between and AR2 in A and thrombin FV-short is the of formation between FV and FV-short (Fig. FV-short and FV cleavage FV, the are with that cleavage at Arg1545 is (3Suzuki K. Dahlback B. Stenflo J. of coagulation factor PubMed Google Scholar, of in its to factor PubMed Scopus Google Scholar). However, a of to the the of formation FV-short cleavage by thrombin to FV (Fig. and or as the not the in FV-short cleavage by thrombin or TFPIα (Fig. these that for FV, the BR binds to AR2 at with the binding of to is an the findings are in with in affinity between between and binding of to J. PubMed Scopus Google Scholar). we show which forms of FV bind and are by TFPIα via its BR. FV-short and cleaved FV but do not full-length FV or FVa. findings the role by the B-domain in TFPIα TFPIα via its thrombin by FV-short and which from or from cleaved at Arg709 and Arg1018 once thrombin or is forms of FV that an AR2 bind TFPIα-BR, which as for the FV BR, these forms of FV in a procofactor state. The BR and AR2 of FV functional landmarks that with and are the B-domain to enforce the FV procofactor (Fig. R.M. Bos M.H. The molecular basis of factor V and VIII procofactor activation.J. Thromb. Haemost. 2009; 7: 1951-1961Crossref PubMed Scopus (66) Google Scholar, R.M. in the of factor V and PubMed Scopus Google Scholar). these it is not that we not binding of FV to TFPIα. The with BR for binding to which has a affinity (Fig. TFPIα binding FV cleaved at Arg709 and Arg1018 (Fig. The of AR2 to However, it is that is between and the which the and binding and cofactor to assessing the to which cleaved FV is or not by is We to that binding FV cleaved at Arg709 (Fig. cleavage the that the BR associated with AR2 cleavage at Arg709 and cleavage at Importantly, and with Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google cleavage at Arg1545 AR2 and FVa that not bind TFPIα or and is not at the of prothrombinase by these (Fig. is functional and binding The with is that it is to which of FV or binds TFPIα as FV that or We that FV-short is constitutively procoagulant in the of as it functions in the prothrombinase complex like FVa (Fig. FV-short binds to and blocks FV-short procoagulant that FV-short, the is the physiologic of TFPIα. with for the a of the forms of that bind to TFPIα proteolytic by thrombin (Fig. The with FV as the show that cleavage at Arg709 and Arg1018 is necessary and sufficient for is that or charged bind FV and the FV FV to with TFPIα. Our with FV and TFPIα with However, the of FV-short and for of removal of FV and TFPIα with FV not for FV-short in the as it these J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar, M. The C-terminus of factor pathway inhibitor is for its with V and Thromb. Haemost. PubMed Scopus Google Scholar). FV-short, to at the of TFPIα in B. the regulation of coagulation by factor V factor pathway and Thromb. Haemost. PubMed Scopus Google in these as a the J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar, M. binding to is for of cofactor for activated PubMed Google or a M. The C-terminus of factor pathway inhibitor is for its with V and Thromb. Haemost. PubMed Scopus Google Scholar). with FV and TFPIα a of J. of factor pathway inhibitor in with factor V PubMed Scopus Google the the binding affinity in we to binding between FV and TFPIα in (Fig. or (Fig. We that the affinity the that the of a of FV which FV-short or cleaved forms of FV or forms of FV to binding a as TFPIα is to to FV-short in the with associated with FV Dahlback B. Coagulation factor via PubMed Scopus Google Scholar). that TFPIα binds with a affinity to FV-short with FV, with binding However, it not these how FV associated with TFPIα and it with its BR or the of the FV BR with AR2 it that TFPIα FV BR. is that the or is to that the of FV FV-short and TFPIα and the of the TFPIα to FV-short to However, TFPIα is associated with the these and is to with FV and which the of factor pathway Thromb. Haemost. PubMed Scopus Google Scholar). FV is associated with of and the of these to in the J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar). is a as it the J. of factor pathway inhibitor in with factor V PubMed Scopus Google Scholar, R.M. factor V Thromb. PubMed Scopus Google Scholar). to that FV and TFPIα in the of We that the between FV and FV-short is that FV in FV-short a for the to it the to an to FV-short in the TFPIα thrombin by binding cleaved forms of FV with an AR2 and prothrombinase R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar). FV, cleavage at Arg709 and Arg1018 the of FV is and from in a of factor V and its by factor Xa and PubMed Google Scholar, K.G. of factor V in and PubMed Google it by TFPIα. the of FV, FV-short, and it is that cleaved is by TFPIα in to TFPIα FV-short and The is to the that TFPIα is from of factor pathway PubMed Scopus Google Scholar, a coagulation inhibitor and to the coagulation PubMed Google Scholar, factor pathway inhibitor PubMed Scopus Google Scholar). We the binding and regulation of FV-short by TFPIα are it has procoagulant of FV-short at Arg1545 is from thrombin to the with AR2 J. The C-terminus of factor pathway factor V by the cleavage Thromb. Haemost. PubMed Scopus Google Scholar). the it is that in or the effects of thrombin are R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, J. The C-terminus of factor pathway factor V by the cleavage Thromb. Haemost. PubMed Scopus Google Scholar). to is TFPIα regulation of is physiologically relevant in is an to as it to these to thrombin relevant as are in for that which or not these molecular factor pathway inhibitor A to the of J. PubMed Scopus Google Scholar). The has been to the of by TFPIα binding to M. R.M. J. Factor V has an anticoagulant cofactor that the of PubMed Scopus Google Scholar, B. the regulation of coagulation by factor V factor pathway and Thromb. Haemost. PubMed Scopus Google Scholar, B. B. functional for the cofactor of in with Thromb. Haemost. PubMed Scopus Google Scholar). it that FV the of TFPIα of factor pathway inhibitor do not PubMed Google Scholar, M. J. of binding in the of by Haemost. PubMed Google that it is the of FV and that is M. R.M. J. Factor V has an anticoagulant cofactor that the of PubMed Scopus Google Scholar). that FV not M. R.M. J. Factor V has an anticoagulant cofactor that the of PubMed Scopus Google Scholar, B. Factor and as factor pathway inhibitor Thromb. Haemost. PubMed Scopus Google Scholar). FV-short not TFPIα but in the of and a cofactor for TFPIα with FV B. Factor and as factor pathway inhibitor Thromb. Haemost. PubMed Scopus Google Scholar). are and that a key function of FV-short to the of TFPIα in the regulation of the of coagulation by At the role of cleaved FV or FV in is not we the by which TFPIα via its C-terminal BR with and forms of FV including FV not bind binds to TFPIα and is not by FV-short is the key physiologic for TFPIα. serves including FV-short procoagulant complex formation in and of TFPIα The FV-short is cleaved at However, it to role has in is is that the of AR2 forms of FV in the blood FV, FV-short, FVa, FV, and TFPIα binding and regulation. We that the of TFPIα with these forms of FV an to or the coagulation from and its in for PubMed Scopus Google Scholar). from and from for the of coagulation a from the of the of the of FV and the and the of FV from The and from and from from from TFPIα in of an C-terminal region by not The the of TFPIα from and from and the from from and the to once in of as of and and as K.G. The of factor V and factor with PubMed Google Scholar). functional at in and FV from of by by as of in its to factor PubMed Scopus Google Scholar, K.G. of functional coagulation factor V by a PubMed Scopus Google Scholar, anticoagulant a factor Xa that is for binding to PubMed Scopus Google Scholar). and from as anticoagulant a factor Xa that is for binding to PubMed Scopus Google Scholar, of the in factor by the PubMed Scopus Google Scholar, R.M. and the of by at the PubMed Scopus Google and and thrombin of the and of in its to factor PubMed Scopus Google Scholar, R.M. and the of by at the PubMed Scopus Google Scholar, K.G. PubMed Scopus Google Scholar). FV-short and in which with in and as R.M. of B-domain from factor V the by which cofactor function is PubMed Scopus Google Scholar, R.M. the B-domain factor V in an inactive PubMed Scopus Google Scholar). FVa of FV-short with thrombin and as R.M. of B-domain from factor V the by which cofactor function is PubMed Scopus Google Scholar, M. K.G. Factor PubMed Scopus Google Scholar). TFPIα-BR, and an N-terminal in and as Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar). by to as Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google and its and of the as well as and been M.H. R.M. A region the B-domain function in factor PubMed Scopus Google Scholar, Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar). FV-short and for and an R.M. of B-domain from factor V the by which cofactor function is PubMed Scopus Google Scholar). nM) in with thrombin as and nM) in with as for at The with thrombin with by to the or by with the and the of FV with and with as of in its to factor PubMed Scopus Google Scholar). and in for at and the by the of from by in FV nM) in to in with The at in with FV in the or of BR and with as R.M. Kalafatis M. Proteolysis of factor V by and that cleavage at cofactor function by factor Xa PubMed Scopus Google Scholar). with a in the a thrombin in Haemost. Thromb. 2003; PubMed Scopus Google Scholar, R.M. factor Xa thrombin and the pathway in PubMed Scopus Google Scholar). with FV or or for with of by of or or and and by the in at the and of and for a of in at as R.M. of B-domain from factor V the by which cofactor function is PubMed Scopus Google Scholar, Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. and the of by at the PubMed Scopus Google Scholar, by PubMed Scopus Google Scholar). The and the and with FVa or FV-short nM) in and the with At of the in and thrombin the The of BR and TFPIα the prothrombinase the and FVa or FV-short nM) with BR or TFPIα nM) with a of The with A with and with of TFPIα or FV or with FV at for and by at for and for to the TFPIα for the and TFPIα from the as FV-short and TFPIα not and not in a and of and with in the in a at in as Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, R.M. Tissue factor pathway prothrombinase the of blood PubMed Scopus Google Scholar, from the of cleavage by the prothrombinase PubMed Scopus Google Scholar). binding of FV nM) to the or or TFPIα the and in which FV of thrombin for to a in which with FV and in from the for or FV-short nM) or with at for nM) and by FV-short cleavage by thrombin in the of of or TFPIα. and at for and to in the with in and for in a of the with and the with for and for of the by to the the for and the in at FV for the between and FV from the of the with of FV and for the in anticoagulant that PubMed Scopus Google Scholar, complex of and complex PubMed Google Scholar). in which or TFPIα of and to as Bos M.H. R.M. the procofactor of factor by with B-domain PubMed Scopus Google Scholar, from the of cleavage by the prothrombinase PubMed Scopus Google Scholar). and of the A to with Google Scholar, binding by its to prothrombinase for PubMed Scopus Google Scholar). for are the the in the are from the is a for and from and We of for and of the and the and M. the and the and M. the and the the by of and The is the of the and not the of the of

Topics & Concepts