Peptide Bond Cleavages and Loss of Functional Activity during Inactivation of Factor Va and Factor VaR506Q by Activated Protein C

Abstract

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506→ Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (<5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 × 107M−1s−1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 × 106M−1s−1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 × 106M−1s−1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s−1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km= 196 nM and kcat = 0.37 s−1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent. This indicates that the binding of factor Va to phospholipids changes the accessibility of the cleavage sites and/or the sequence of peptide bond cleavage by APC. Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506→ Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (<5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 × 107M−1s−1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 × 106M−1s−1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 × 106M−1s−1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s−1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km= 196 nM and kcat = 0.37 s−1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent. This indicates that the binding of factor Va to phospholipids changes the accessibility of the cleavage sites and/or the sequence of peptide bond cleavage by APC. INTRODUCTIONHuman blood coagulation factor Va is a that of a heavy chain = associated via a with a chain = Factor Va is during from the V Factor Va is an cofactor of the which further the factor and a the reaction factor Va prothrombin activation inactivation of factor Va by activated protein C protein factor Va in which Arg506 is by factor from is of the in the of thrombin APC-catalyzed cleavage of factor Va is by the presence of and by the protein protein The of cofactor activity of factor Va is associated with peptide bond cleavages in the heavy chain of factor Va at Arg306, Arg506, and The of the of factor Va activity by APC is by the of in that in protein C protein a by associated the of in a of with a to APC APC is at factors and an of in the The in was to to a mutation in the factor V that an at a that is cleaved during APC-catalyzed inactivation of factor a kinetic analysis of APC-catalyzed inactivation of factor Va that was from normal plasma from the plasma of a patient who is homozygous for the mutation were in which changes of activity of factor Va during inactivation by APC were correlated with the cleavage of peptide in the factor Va This was in to the of the in factor V for the of in prothrombin and were from and were from The and were by was from and was from protein were from protein and for protein were from coagulation factors in were from prothrombin and factor were purified to thrombin was from prothrombin activation mixtures as by and factor Xa was from purified factor activation with and from the activation by was purified from to C was purified and activated as by The of the preparation of factor V and factor VR506Q were as by with The factor V was to a and with of a in from were for with nM thrombin and factor were Factor Va was by factor V for with nM thrombin in and activation was to Factor Va was further to protein a at The factor Va to the was with of a in Factor Va activity from the in protein factor Va with of = and The factor Va were and into by a a Factor V and factor Va from an patient were purified by the same The patient was a with a of and from the patient was by The was The patient was as homozygous for the in the factor V Gln in factor by analysis in from concentrations were with the protein thrombin and factor Xa concentrations were by with concentrations were activation of prothrombin with and of thrombin with APC concentrations were with kinetic parameters by Factor V and factor Va were as phospholipid vesicles were as concentrations were by and Immunoblot of protein was by and with The inactivation of factor Va were of from the to and with a the heavy chain of factor Va and with as by Va Va cofactor activity was by the rate of factor prothrombin activation in reaction mixtures that contained a of factor Va, a concentration factor Xa (5 phospholipid vesicles and of prothrombin activation were with the The factor Va concentration in the was from the rate of prothrombin activation a of of prothrombin activated of factor Factor V was by the same activation with nM of a membrane-bound factor was by of prothrombin activation in the presence of phospholipid vesicles at a concentration of factor Va and of factor Xa. Factor factor Va, and phospholipid vesicles were for at in and activation was by the of prothrombin at in the same and of prothrombin activation were with the The for of the membrane-bound factor was from of the rate of prothrombin activation as a of the factor Xa concentration that were to the for a binding via kinetic parameters and for factor prothrombin activation were by the rate of thrombin at prothrombin concentrations in the presence of a phospholipid a of factor and a concentration of factor Va as in the The kinetic parameters were by the to the Michaelis-Menten of of Factor Va of factor Va factor VaR506Q inactivation by APC were by the of cofactor activity of factor Va as a of in the and in that the of factor Va cofactor activity via Factor Factor in which factor is a reaction intermediate with cofactor factor is a reaction that no cofactor is the rate for cleavage of peptide bond is the rate for cleavage of peptide bond in factor and is the rate for cleavage of peptide bond in native factor at which the inactivation rate is to the factor Va and APC the of cofactor activity is by and and in which is the cofactor activity at is the cofactor activity APC is is the cofactor activity of factor as of the cofactor activity of native factor and and the rate constants The rate constants and the cofactor activity of factor were by the to the of normal factor Va to associated with cleavage of the peptide bond at Arg306. of normal factor Va inactivation were with a from kinetic analysis of factor VaR506Q (factor Va inactivation in was a slow of factor Va cofactor activity in the absence of APC. these the of cofactor activity was to in Factor Va to by was that APC peptide in the heavy chain of factor Va that at Arg306, Arg506, and a of the heavy chain of factor Va in which the sites that to APC and the of cleavage the to the rate constants associated with the indicated peptide bond cleavages as and and of Factor Va from and from V and factor Va were purified from normal and homozygous plasma as The factor Va and factor VaR506Q had with the heavy chain with = and a chain at = and Va and factor VaR506Q had cofactor in prothrombin analysis showed that prothrombin was activated in the presence of phospholipid vesicles with the kinetic prothrombin = kcat = and of = nM for with normal factor Va and Km for prothrombin = kcat = and of = nM for with factor VaR506Q means that the factor Va that which the cofactor activity of factor Va in prothrombin of factor Va and factor of APC-catalyzed Inactivation of Factor Va and Factor of APC-catalyzed inactivation of factor Va and factor VaR506Q in the presence of phospholipid vesicles in a low APC concentration nM) normal factor Va was inactivated via a biphasic reaction during which of the factor Va activity was Factor VaR506Q is inactivated at a rate for of cofactor activity When a was at a high APC concentration nM) inactivation of factor VaR506Q to via a monophasic reaction with a the inactivation of normal factor Va proceeded via a biphasic reaction of factor Va and factor VaR506Q by APC in the presence of nM purified factor Va factor VaR506Q was with 0.15 nM APC nM APC and phospholipid vesicles in and at the indicated factor Va activity was as The the The rate constants and activity of the reaction intermediate were by of the of factor Va and VaR506Q inactivation factor Va activity were by the concentrations of factor Va VaR506Q between and the of factor Va inactivation were also to the of APC nM) that factor Va and VaR506Q inactivation were in factor and APC. This of rate constants for factor Va inactivation from as in the of and that the biphasic of inactivation of normal factor Va in from rapid cleavage at Arg506 yielding a reaction intermediate that cofactor activity in prothrombin This reaction intermediate is fully inactivated by subsequent slow cleavage at Arg306. Inactivation of factor VaR506Q via a monophasic reaction that is the of slow cleavage at of inactivation an that the of cofactor activity factor Va is inactivated via the reaction The of the in the rate = × = 1.7 × and 40% cofactor activity for the reaction intermediate (factor Va cleaved at in the of normal factor Va inactivation and = × and a reaction that virtually its cofactor activity in the of factor VaR506Q of APC-catalyzed Inactivation of Factor Va and Factor of heavy chain during APC-catalyzed factor Va and factor VaR506Q inactivation was by to and of the reaction with a the heavy chain of factor Va the of the the of reaction was associated with peptide bond cleavages at Arg306, Arg506, Arg679 the of of heavy chain indicated in analysis of factor Va and factor VaR506Q inactivation by APC in the presence of nM purified factor Va and nM nM factor VaR506Q and nM APC were with phospholipid vesicles in and at the of normal factor Va the APC concentration was to nM the indicated from the inactivation were to to the heavy chain and were with a the heavy chain of factor Va. The of the indicated at the of and The cofactor activity at the indicated were and and and the phase of inactivation of normal factor Va, reaction with of and were which is of cleavage at Arg506 of factor Va with a high APC concentration resulted in the of the = reaction intermediate at and of reaction with of and The with = with and was the the of the inactivation reaction the peptide bond at Arg679 was also as is indicated by the of the = and the of low with the in the of the of APC-catalyzed inactivation of membrane-bound factor VaR506Q from that of normal factor Va. Inactivation of factor VaR506Q correlated with the of reaction with = and = which with cleavage at Arg306. The further demonstrates that cleavage at Arg679 to factor VaR506Q inactivation no in the heavy chain = of the This cleavage at Arg679 had analysis the for APC-catalyzed of factor Va in the The rapid phase of the of inactivation of normal factor Va is associated with cleavage at Arg506 and the subsequent slow phase is to cleavage at Arg306. Inactivation of factor VaR506Q via a monophasic reaction which is by cleavage at Inactivation of Factor Va and Factor the of to the of the the peptide bond cleavages during factor Va and factor VaR506Q inactivation by APC also of factor Va inactivation in the absence of phospholipids was to these of inactivation with a For factor Va and factor VaR506Q was to that inactivation proceeded via a biphasic reaction with a reaction of factor Va and factor VaR506Q by APC in the absence of nM purified factor Va factor VaR506Q was with nM APC in and at the indicated factor Va activity was as The the The rate constants and activity of the reaction intermediate were by analysis of the the of cofactor activity with peptide bond cleavages in the heavy chain the by analysis of from the inactivation at Compared with the peptide bond cleavage of membrane-bound factor Va a was the absence of phospholipids cleavage at Arg679 occurred at in factor VaR506Q This is from the of an in the heavy chain of the analysis of factor Va and factor VaR506Q inactivation by APC in the absence of nM purified factor Va and nM nM factor VaR506Q and nM APC were in and at the of normal factor Va the APC concentration was to nM APC the indicated from the inactivation were to to the heavy chain and were with a the heavy chain of factor Va. The of the indicated at the of and The cofactor activity at the indicated were and and and that in the of factor VaR506Q inactivation at Arg679 and Arg306 contributed to factor Va inactivation and that cleavage at Arg679 cleavage at Arg306. the of APC-catalyzed factor VaR506Q inactivation to the for a biphasic reaction with a intermediate rate constants of × and × for and and cofactor activity for the reaction intermediate that cleaved at analysis indicates that during the of normal factor Va inactivation peptide bond cleavages at Arg506 and Arg306 contributed to the of factor Va cofactor of the that inactivation of normal factor Va that cleavage at Arg679 had a to the of factor Va cofactor a also with a biphasic This rate constants of × and × for cleavage at Arg506 and Arg306, and a reaction intermediate with 40% cofactor that the of the rate constants to the that cleavage at Arg679 to for APC-catalyzed Cleavage of during Inactivation of and Factor Va and Factor rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in factor Va and VaR506Q in was to rate constants for cleavage at Arg679 in normal factor Va from the of inactivation in from the of the = between and that cleavage at Arg679 in factor Va occurred with × at nM and that the same peptide bond in membrane-bound factor Va was cleaved with × at nM in a The in that phospholipids the APC-catalyzed peptide bond cleavages in factor Va to a different extent. to no is that cleavage at Arg306 in factor VaR506Q occurred at a rate in normal factor Va. For membrane-bound factor VaR506Q and factor Va was for of phospholipids and/or cleavage at Arg506 the cleavages at Arg306 and Arg679 for APC-catalyzed Cleavage at Factor Va and Factor differences in rate constants for cleavage at Arg506 and Arg306 in membrane-bound factor Va from differences between the Michaelis-Menten kinetic parameters and/or for these peptide bond to the kinetic of the differences the kinetic Km and by the of factor Va inactivation and of cofactor at factor Va factor VaR506Q concentrations. The with normal factor Va the kcat and Km for cleavage at Arg506 peptide bond is cleaved during the phase of inactivation. Michaelis-Menten kinetic analysis of factor VaR506Q inactivation kinetic parameters for cleavage at in that cleavage at Arg506 was characterized by a Km of nM and a kcat of 0.96 s−1. Inactivation of factor which with cleavage at Arg306, had a high Km nM) and a kcat of 0.37 s−1. the by Michaelis-Menten analysis the rate from a of inactivation at low factor Va concentrations the for cleavage at Arg506 × and Arg306 × the rate constants at reaction This that the kinetic Km and associated with the indicated peptide bond The further that the differences between the rate constants of cleavage at Arg506 and Arg306 to differences between the Km for these peptide bond for inactivation of factor Va and factor VaR506Q by APC in the presence of of factor Va inactivation were at concentrations of factor Va factor VaR506Q that were with nM APC nM APC and phospholipid vesicles in and at different the factor Va activity was as and of factor Va inactivation were as nM factor Va APC that normal factor Va is converted into an intermediate with 40% cofactor The a of the the Michaelis-Menten with Km = nM and = nM factor Va APC = 0.96 for factor Va and Km = 196 nM and = nM factor VaR506Q APC = 0.37 for factor VaR506Q of Factor Va Cleavage at of inactivation of normal factor Va in a reaction that consisted of with the of a reaction intermediate (factor Va cleaved at that retained cofactor cofactor activity of factor Va is its with factor and its ability to the activity of factor which of these was impaired in factor Va that is cleaved at Arg506, characterized the of the inactivation normal factor Va was inactivated by APC in the absence of phospholipids and of the phase of inactivation the reaction was and a The reaction intermediate to and in a as an between and contained inactivation intermediate of factor Va and factor Va cleaved at factor Va nM) was with nM APC in and at the inactivation was and to a protein was with of a in Immunoblot analysis of factor Va and the between and is in the from the inactivation factor and from the were to of prothrombin activation at factor Va factor Va cleaved at Arg506 in a reaction phospholipid vesicles and concentrations of factor Xa in and at of prothrombin activation were as and were for thrombin in the absence of factor Va. The the with = nM and = of of factor Va = for factor Va and = nM and = of of protein = for the (factor Va cleaved at of the same the of factor Va and the purified inactivation intermediate to with factor Xa into a membrane-bound of prothrombin activation at of factor Va factor Va cleaved at Arg506 as a of the factor Xa concentration and of factor Xa were required for full of activity in the of normal factor Va = nM). factor Va that was cleaved at Arg506 much factor Xa concentrations = nM) were to The of prothrombin activation with the purified inactivation intermediate was that for native factor Va. The converted into kcat the of the protein of the This kcat of and for native and cleaved factor Va, of the membrane-bound factor with prothrombin was by cleavage at Arg506 the Km for prothrombin were virtually the same for with normal and cleaved factor Va these that APC-catalyzed cleavage at Arg506 of the heavy chain of factor Va in the of a reaction that of its cofactor activity of a of its for factor of the Factor Va and the of Inactivation of Factor Va and Factor in that differences in rate constants of cleavage at Arg506 and Arg306 to differences in the Km for these peptide bond This means that at high factor Va the of cleavage differences in of inactivation of factor Va and factor VaR506Q become less also that the cofactor of native factor Va and factor Va cleaved at Arg506 the factor Xa concentration in the factor Va was that factor Va that is cleaved at Arg506 much cofactor activity native factor Va at factor Xa concentrations that the of factor Va factor VaR506Q inactivation by APC the factor Va concentration in the inactivation and the at which the of cofactor activity of factor Va is Compared with factor normal factor Va inactivated at low at high factor Va concentrations. The of cofactor activity of normal factor Va rapid and of activity much from the inactivation at a factor Xa concentration at which the inactivation intermediate no nM were by an in which of factor Va inactivation were at low high concentrations of factor Va VaR506Q and in which from the same inactivation were for the of cofactor activity in mixtures that contained a low a high factor Xa concentration the differences in APC-catalyzed of cofactor activity of normal factor Va and factor VaR506Q were pronounced factor Va was inactivated at a low concentration and in a prothrombin activation that contained a low factor Xa concentration in of inactivation of factor Va and factor VaR506Q were reduced high factor Va concentrations were inactivated by APC and the of cofactor activity was in prothrombin activation mixtures that contained a high factor Xa concentration of and factor Va concentration the of cofactor activity during factor Va inactivation by APC. factor Va factor VaR506Q was with APC and phospholipid vesicles in and at the indicated factor Va activity was as in mixtures that contained nM nM factor Xa. inactivation nM factor Va nM factor VaR506Q 0.15 nM phospholipid vesicles cofactor nM factor Xa. inactivation nM factor Va nM factor VaR506Q nM phospholipid vesicles cofactor nM factor inactivation of membrane-bound factor Va by APC cleavage of peptide at Arg506 and Arg306 in the heavy chain of factor Va low factor Va concentrations cleavage at Arg506 at a rate that is the rate of cleavage at Arg306. This in the accumulation of a reaction intermediate that is cleaved at Arg506 and that cofactor activity in prothrombin activation Cleavage at Arg306, which in the reaction intermediate and in native factor Va in a of cofactor cofactor activity of the factor Va cleaved at Arg506 the factor Xa concentration in the prothrombin activation in which factor Va is activity of the reaction intermediate in prothrombin activation is at high factor Xa concentrations. Compared with native factor Va, full of cofactor activity of factor Va that is cleaved at Arg506 a factor Xa This indicates that cleavage at Arg506 in the heavy chain of factor Va its with factor Xa and in a of for factor VaR506Q from factor V purified from the plasma of a homozygous patient is inactivated by APC This factor Va the cleavage at Arg506, to of by of inactivation of low concentrations of membrane-bound factor VaR506Q with an for a cleavage at Arg306 during the inactivation of membrane-bound factor that APC factor VaR506Q by a cleavage at Arg306 which in the of a reaction that virtually its cofactor activity in prothrombin the of factor Va cleavage at Arg679 a to the of cofactor activity in the of APC-catalyzed inactivation of membrane-bound factor Va factor This is from the that cleavage at Arg679 of the = of in the heavy chain the = of is at which of the cofactor activity of factor Va is factor cofactor activity and factor is a reaction that no cofactor that APC-catalyzed cleavage at Arg506 and Arg306 in membrane-bound factor Va in a and that cleavage at Arg306 is by cleavage at This is from the that the rate constants for cleavage at Arg306 from of inactivation of normal and factor Va the same is 2.3 × and 1.7 × for normal Va and factor analysis the that for the between and The kinetic parameters for cleavage at Arg506 in normal factor Va Km for factor Va = nM and kcat = 0.96 s−1. The peptide bond at Arg306 in factor VaR506Q was cleaved with Km = 196 nM and kcat = 0.37 s−1. The that the the same as the rate constants for cleavage at Arg506 in normal factor Va and for cleavage at Arg306 in factor VaR506Q that the Michaelis-Menten kinetic parameters associated with the indicated peptide bond that the differences in rate constants for cleavage at Arg506 and Arg306 from differences in the Km and at high factor Va concentrations differences between of factor Va and factor VaR506Q inactivation much less pronounced also fully factor Va and factor VaR506Q in the absence of a these cleavage at Arg679 also to the of factor Va cofactor This is for factor in which the peptide bond at Arg679 is cleaved at a rate the at Arg306. Cleavage at Arg679 cleavage at Arg506 Arg306 analysis the of a in the = of the which indicates that cleavage at Arg679 in the heavy of rate constants for and membrane-bound factor Va that phospholipids APC-catalyzed factor Va inactivation. of to the that the (factor and the to the a that and reaction in a to that of coagulation factor in the of APC-catalyzed factor Va phospholipids the peptide bond cleavages to the same Cleavage at Arg506 and Arg306 in normal factor Va is cleavage at Arg306 in factor VaR506Q is by This is of a of phospholipids APC-catalyzed cleavage at Arg306 and that is in membrane-bound factor Va. This demonstrates that binding of factor Va to phospholipids the peptide bond at Arg679 is to cleavage by membrane-bound to of who that APC-catalyzed cleavages in the heavy chain of membrane-bound factor Va in an in which cleavage at Arg506 cleavage at Arg306 and also that cleavage at Arg506 the cofactor activity of normal factor Va and that cleavage at Arg679 is required for full inactivation of factor Va. by the of peptide bond cleavages in the heavy chain of factor Va the factor Va concentration in the inactivation and that the cofactor activity of reaction the reaction at which the activity of factor Va and its is is to that at high factor Va concentrations nM) and a factor Va with high concentrations of coagulation at low factor Va concentrations nM) and of cofactor activity of factor Va in prothrombin activation mixtures that contained concentrations of coagulation the of reaction and that the of factor Va cofactor activity during in activation by APC the concentration at which factor Va is inactivated and the at which the of cofactor activity is of factor Va and factor VaR506Q inactivation at a high factor Va in which the of cofactor activity of factor Va is in activation mixtures that a high factor Xa and factor Va is inactivated at a low concentration and its cofactor activity is in a prothrombin activation that a low of factor the differences between factor Va and factor VaR506Q This from coagulation at low concentrations of factor Xa and factor Va plasma factor V concentration is of the cofactor activity of factor Va by APC in the of normal factor Va and impaired in of factor This the for in as a of a factor V in which Arg506 is by Gln INTRODUCTIONHuman blood coagulation factor Va is a that of a heavy chain = associated via a with a chain = Factor Va is during from the V Factor Va is an cofactor of the which further the factor and a the reaction factor Va prothrombin activation inactivation of factor Va by activated protein C protein factor Va in which Arg506 is by factor from is of the in the of thrombin APC-catalyzed cleavage of factor Va is by the presence of and by the protein protein The of cofactor activity of factor Va is associated with peptide bond cleavages in the heavy chain of factor Va at Arg306, Arg506, and The of the of factor Va activity by APC is by the of in that in protein C protein a by associated the of in a of with a to APC APC is at factors and an of in the The in was to to a mutation in the factor V that an at a that is cleaved during APC-catalyzed inactivation of factor a kinetic analysis of APC-catalyzed inactivation of factor Va that was from normal plasma from the plasma of a patient who is homozygous for the mutation were in which changes of activity of factor Va during inactivation by APC were correlated with the cleavage of peptide in the factor Va This was in to the of the in factor V for the of in prothrombin