Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.

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.

Human blood coagulation factor Va is a heterodimeric glycoprotein (1) that consists of a heavy chain (M r ϭ 105,000) associated via a single Ca 2ϩ ion with a light chain (M r ϭ 74,000 or 72,000, cf. Ref 2). Factor Va is formed during hemostasis from the inactive procofactor V after limited proteolysis. Factor Va is an essential nonenzymatic cofactor of the prothrombin-activating complex, which further comprises the serine protease factor Xa, calcium ions, and a procoagulant membrane surface. Depending on the reaction conditions factor Va accelerates prothrombin activation 10 3 -10 5 -fold (3)(4)(5).
Proteolytic inactivation of factor Va by activated protein C (APC), 1 is one of the key reactions in the regulation of thrombin formation. APC-catalyzed cleavage of factor Va is stimulated by the presence of negatively charged membrane surfaces (6 -8) and by the protein cofactor, protein S (8 -10). The loss of cofactor activity of factor Va is associated with peptide bond cleavages in the heavy chain of factor Va at Arg 306 , Arg 506 , and Arg 679 (11,12). The physiologic importance of the down-regulation of factor Va activity by APC is underscored by the observation of recurrent thromboembolic events in individuals that are deficient in either protein C or protein S (13)(14)(15).
Since a first publication by Dahlbäck et al. (16), several laboratories have associated the occurrence of familial thrombophilia in a large group of patients with a poor anticoagulant response to APC (APC resistance). APC resistance is at least 10 times more common than all other known genetic thrombosis risk factors together and has an allelic frequency of ϳ2% in the Dutch population (17). The molecular defect in APC-resistant patients was recently demonstrated (18 -21) to be linked to a single-point mutation in the factor V gene that causes an amino acid substitution (Arg 506 3 Gln) at a site that is cleaved during APC-catalyzed inactivation of factor Va (11).
In this paper we present a kinetic analysis of APC-catalyzed inactivation of human factor Va that was either obtained from normal plasma or from the plasma of a patient who is homozygous for the mutation (Arg 506 3 Gln). Parallel experiments were performed in which changes of functional activity of factor Va during inactivation by APC were correlated with the cleavage of peptide bonds in the factor Va molecule. This study was undertaken in order to elucidate the consequences of the molecular defect in factor V for the regulation of in vivo prothrombin activation.

EXPERIMENTAL PROCEDURES
Materials-Hepes, Tris, bovine serum albumin, and Russel's viper venom were purchased from Sigma. DOPC and DOPS were obtained from Avanti Polar Lipids. The chromogenic substrates S2238 and S2366 were supplied by Chromogenix, Mölndal, Sweden. D-Pro-Phe-Arg-CH 2 Cl was obtained from Calbiochem and p-NPGB was from Nutritional Biochemicals. Micro BCA protein assay kits were from Pierce. Fast protein liquid chromatography equipment and column materials used for protein purification were purchased from Pharmacia, Uppsala, Sweden.
Proteins-Human coagulation factors used in this study were isolated from fresh frozen plasma. Human prothrombin and factor X were purified according to DiScipio et al. (22). Human thrombin was prepared from prothrombin activation mixtures as described by Pletcher and Nelsestuen (23). Human factor Xa was obtained from purified factor X after activation with RVV-X and isolation from the activation mixture by affinity chromatography on soybean trypsin inhibitor-Sepharose (24). RVV-X was purified from Russel's viper venom according to Schiffman et al. (25). Protein C was purified and activated as described by Gruber et al. (26). The first steps of the preparation of human factor V and factor V R506Q were essentially as described by Dahlbä ck (27) with minor modifications (28). The factor V (in 25 mM Hepes, pH 7.5, 50 mM NaCl, 5 mM CaCl 2 ) thus obtained was applied to a Resource S column and eluted with 20 ml of a linear gradient (50 -1000 mM NH 4 Cl) in 25 mM Hepes (pH 6.5), 5 mM CaCl 2 . Aliquots from column fractions were incubated for 15 min with 5 nM thrombin and factor Va-containing fractions were pooled. Factor Va was prepared by incubating factor V (33 g/ml) for 20 min with 5 nM thrombin in 25 mM Hepes (pH 7.5), 175 mM NaCl, and 5 mM CaCl 2 . After activation 15 nM D-Pro-Phe-Arg-CH 2 Cl was added to inhibit thrombin. Factor Va was further subjected to fast protein liquid chromatography on a Mono S column (HR 5/5) at room temperature (2). The factor Va bound to the Mono S column was eluted with 15 ml of a linear gradient (50 -1000 mM NH 4 Cl) in 25 mM Hepes (pH 7.5), 5 mM CaCl 2 . Factor Va activity eluted from the column in two well-separated protein peaks containing factor Va with light chains of M r ϭ 74,000 and 71,000, respectively (2). The two factor Va containing peaks were pooled and brought into 25 mM Hepes (pH 7.5), 175 mM NaCl, 5 mM CaCl 2 by a buffer exchange on a PD-10 column. Factor V and factor Va from an APC-resistant patient were purified by the same methods. The patient was a female with a history of deep vein thrombosis and pulmonary embolism. Plasma from the patient was obtained by plasmapheresis. The APC-resistance ratio was 1.31 (Coatest APC-resistance kit, Chromogenix, Mölndal, Sweden). The patient was identified as homozygous for the substitution G 1691 3 A in the factor V gene (amino acid substitution Arg 509 3 Gln in factor V) by genetic analysis in genomic DNA samples isolated from peripheral blood (29).
Protein Concentrations-Protein concentrations were determined with the micro BCA protein assay (30). Molar thrombin and factor Xa concentrations were determined by active-site titration with p-NPGB (31,32). Prothrombin concentrations were determined after complete activation of prothrombin with Echis carinatus venom and quantitation of thrombin with p-NPGB. APC concentrations were determined with S2366 using kinetic parameters reported by Sala et al. (33). Factor V and factor Va were quantitated as described below.
Gel Electrophoretic and Immunoblot Analysis-Purity of protein preparations was analyzed by SDS-PAGE on 8% acrylamide slab gels (35) and staining with Coomassie Brilliant Blue R-250. The inactivation patterns of factor Va were analyzed after SDS-PAGE, electrophoretic transfer of proteins from the gel to Immobilon-P membranes, and visualization with a polyclonal rabbit-antibody directed against the heavy chain of factor Va and goat-anti-rabbit IgG conjugated with alkaline phosphatase as described by Blake et al. (36).
Factor Va Assay-Factor Va cofactor activity was quantitated by determining the rate of factor Xa-catalyzed prothrombin activation in reaction mixtures that contained a limiting amount of factor Va, a saturating concentration factor Xa (5 nM), 50 M phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol), and 0.5 M prothrombin (28). Rates of prothrombin activation were determined with the chromogenic substrate S2238 (4). The molar factor Va concentration in the assay mixture was calculated from the rate of prothrombin activation using a turnover number of 6000 mol of prothrombin activated per min/mol of factor Xa-Va complex (37). Factor V was quantitated by the same method after activation with 5 nM thrombin.
Kinetic Data Analysis-The formation of a membrane-bound factor Xa-Va complex was determined by measuring rates of prothrombin activation in the presence of phospholipid vesicles at a fixed (limiting) concentration of factor Va and varying amounts of factor Xa. Factor Xa, factor Va, and phospholipid vesicles were preincubated for 5 min at 37°C in 25 mM Hepes (pH 7.5), 175 mM NaCl, 2 mM CaCl 2 , and 5 mg/ml BSA. Prothrombin activation was started by the addition of prothrombin (preincubated at 37°C in the same buffer) and rates of prothrombin activation were determined with the chromogenic substrate S2238 (4). The apparent K d for dissociation of the membrane-bound factor Xa-Va complex (K1 ⁄2Xa ) was obtained from plots of the rate of prothrombin activation as a function of the factor Xa concentration that were fitted to the equation for a single-site binding isotherm (hyperbola) via nonlinear least squares regression analysis.
The kinetic parameters (K m and V max ) for factor Xa-catalyzed prothrombin activation were determined by measuring the rate of thrombin formation at varying prothrombin concentrations in the presence of a fixed phospholipid concentration, a limiting amount of factor Xa, and a saturating concentration of factor Va as described in the previous paragraph. The kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation using non-linear least squares regression analysis.
Curve Fitting of Pseudo-first Order Time Courses of Factor Va Inactivation-Time courses of factor Va or factor Va R506Q inactivation by APC were determined by following the loss of cofactor activity of factor Va as a function of time. Data reported in the literature (11,12) and in this paper demonstrate that the loss of factor Va cofactor activity can proceed via 2 pathways: in which factor Va int is a reaction intermediate with partial cofactor activity, factor Va i is a reaction product that has no cofactor activity, k 1 is the second-order rate constant for cleavage of peptide bond 1, k 2 is the second-order rate constant for cleavage of peptide bond 2 in factor Va int , and k 3 is the second-order rate constant for cleavage of peptide bond 2 in native factor Va. Under first order conditions, i.e. conditions at which the inactivation rate is directly proportional to the factor Va and APC concentrations, the loss of cofactor activity is described by Equation 3 (38): in which Va t is the cofactor activity determined at time t, Va o is the cofactor activity determined before APC is added, B is the cofactor activity of factor Va int (expressed as fraction of the cofactor activity of native factor Va), and k 1 , k 2 , and k 3 are the rate constants defined above. The rate constants and the cofactor activity of factor Va int were obtained by fitting the data to Equation 3 using non-linear least squares regression analysis.
In the case of normal factor Va inactivation, k 3 appeared to be associated with cleavage of the peptide bond at Arg 306 . Therefore, time courses of normal factor Va inactivation were fitted with a fixed k 3 , obtained from kinetic analysis of factor Va R506Q inactivation.
In some experiments (factor Va inactivation in free solution) there was a slow loss of factor Va cofactor activity in the absence of APC. In these cases the spontaneous loss of cofactor activity (0.4%/min) was added to k 3 .

RESULTS
Peptide Bonds in Factor Va Susceptible to Proteolysis by APC-Recently it was reported (11) that APC can cleave 3 peptide bonds in the heavy chain of human factor Va that are located at Arg 306 , Arg 506 , and Arg 679 . Fig. 1 represents a schematic diagram of the heavy chain of factor Va in which we have depicted the sites that are susceptible to APC and the molecular weights of possible cleavage products. In the present paper we will refer to the rate constants associated with the indicated peptide bond cleavages as k 306 , k 506 , and k 679 , respectively.
Structural and Functional Properties of Factor Va Purified from Normal and from APC-resistant Plasma-Factor V and thrombin-activated factor Va were purified from normal and homozygous APC-resistant plasma as described under "Experimental Procedures." The thrombin-activated factor Va and factor Va R506Q preparations had identical gel electrophoretic properties with the characteristic heavy chain with M r ϭ 105,000 and a light chain doublet at M r ϭ 74,000 and 71,000 (data not shown).
Factor Va and factor Va R506Q had similar cofactor activities in prothrombin activation. Kinetic analysis showed that prothrombin was activated in the presence of 25 M phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) with the following kinetic parameters: K m for prothrombin ϭ 0.13 M, k cat ϭ 8570 min Ϫ1 and K1 ⁄2Xa of Xa-Va complex formation ϭ 0.083 nM for prothrombinase complexes with normal factor Va and K m for prothrombin ϭ 0.12 M, k cat ϭ 8450 min Ϫ1 and K1 ⁄2Xa of Xa-Va complex formation ϭ 0.105 nM for prothrombinase complexes with factor Va R506Q (data not shown).
This means that the factor Va assay that we used, which measures the cofactor activity of factor Va in prothrombin activation, allows quantitative comparison of factor Va and factor Va R506Q .
Kinetic Analysis of APC-catalyzed Inactivation of Membranebound Factor Va and Factor Va R506Q -Time courses of APCcatalyzed inactivation of factor Va and factor Va R506Q in the presence of phospholipid vesicles are shown in Fig. 2. At a low APC concentration (0.15 nM) normal factor Va was inactivated via a biphasic reaction during which 50% of the factor Va activity was lost within 3 min. Factor Va R506Q is inactivated at a considerably lower rate requiring more than 45 min for 50% loss of cofactor activity ( Fig. 2A). When a similar experiment was performed at a high APC concentration (1.5 nM) inactivation of factor Va R506Q appeared to occur via a monophasic reaction (the data points could be fitted with a single exponential), whereas the inactivation of normal factor Va still proceeded via a biphasic reaction (Fig. 2B).
The time courses of factor Va and Va R506Q inactivation (% factor Va activity versus time) were not affected by varying the concentrations of factor Va or Va R506Q between 0 and 5 nM. Since the rates of factor Va inactivation were also directly proportional to the amount of APC present (0.05-5 nM) we concluded that factor Va and Va R506Q inactivation were firstorder in both factor Va(Va R506Q ) and APC. This allows calculation of apparent second-order rate constants for factor Va inactivation from time courses such as shown in Fig. 2.
On the basis of literature data (11,12) and experiments presented below it can be postulated that the biphasic time courses of inactivation of normal factor Va presented in Fig. 2 result from rapid cleavage at Arg 506 yielding a reaction intermediate that possesses partial cofactor activity in prothrombin activation. This reaction intermediate is fully inactivated by subsequent slow cleavage at Arg 306 . Inactivation of factor Va R506Q likely proceeds via a monophasic reaction that is the result of slow cleavage at Arg 306 .
The time courses of inactivation could indeed be fitted ( Fig.  2, solid lines) using an equation that describes the time-dependent loss of cofactor activity when factor Va is inactivated via the random ordered two-step reaction model described under "Experimental Procedures." The fits of the particular experiment presented in Fig. 2 yielded the following second-order rate constants: k 506 ϭ 4.7 ϫ 10 7 M Ϫ1 s Ϫ1 , k 306 ϭ 1.7 ϫ 10 6 M Ϫ1 s Ϫ1 , and 40% cofactor activity for the reaction intermediate (factor Va cleaved at Arg 506 ) in the case of normal factor Va inactivation and k 306 ϭ 1.4 ϫ 10 6 M Ϫ1 s Ϫ1 and a reaction product that has virtually lost its cofactor activity in the case of factor Va R506Q inactivation.
Immunoblot Analysis of APC-catalyzed Inactivation of Membrane-bound Factor Va and Factor Va R506Q -Generation of heavy chain fragments during APC-catalyzed factor Va and FIG. 1. Schematic representation of APC cleavage sites in the heavy chain of factor Va. The heavy chain of human factor Va contains three peptide bonds that can be cleaved by APC and that are located at Arg 306 , Arg 506 , and Arg 679 . The molecular weights of possible cleavage products correspond to those given in other publications (11,12). factor Va R506Q inactivation was analyzed by SDS-PAGE, transfer to Immobilon-P membranes, and visualization of the reaction products with a polyclonal antibody directed against the heavy chain domain of human factor Va (Fig. 3). In the description of the gel patterns presented below, the formation of reaction products was associated with peptide bond cleavages at Arg 306 , Arg 506 , or Arg 679 on the basis of molecular weights of heavy chain fragments indicated in Fig. 1.
During the initial phase of inactivation of normal factor Va, reaction products with M r of 75,000 and 26,000/28,000 were formed, which is indicative of cleavage at Arg 506 (Fig. 3A). Prolonged incubation of factor Va with a high APC concentration resulted in the disappearance of the M r ϭ 75,000 reaction intermediate (cleavage at Arg 306 ) and formation of reaction products with M r of 45,000 and 30,000, respectively. The fragment with M r ϭ 30,000 stains poorly with our polyclonal antibody and was hardly visible on the gel. During the final stage of the inactivation reaction the peptide bond at Arg 679 was also cleaved, as is indicated by the disappearance of the M r ϭ 26,000/28,000 doublet and the formation of low molecular weight products migrating with the dye front in the gel.
The immunoblot of the time course of APC-catalyzed inactivation of membrane-bound factor Va R506Q (Fig. 3B) differs from that of normal factor Va. Inactivation of factor Va R506Q correlated with the appearance of only two reaction products with M r ϭ 60,000/62,000 and M r ϭ 45,000, which corresponds with cleavage at Arg 306 . The immunoblot further demonstrates that cleavage at Arg 679 did not significantly contribute to factor Va R506Q inactivation since no additional bands became visible in the heavy chain or M r ϭ 54,000/56,000 region of the immunoblot. This would have been expected if cleavage at Arg 679 had occurred.
The immunoblot analysis supports the model for APC-catalyzed proteolysis of factor Va presented in the previous section. The rapid phase of the time course of inactivation of normal factor Va is associated with cleavage at Arg 506 and the subsequent slow phase is due to cleavage at Arg 306 . Inactivation of factor Va R506Q proceeds via a monophasic reaction which is explained by cleavage at Arg 306 .
APC-catalyzed Inactivation of Factor Va and Factor Va R506Q in the Absence of Phospholipids-In order to quantify the effect of the membrane on the peptide bond cleavages during factor Va and factor Va R506Q inactivation by APC we have also determined time courses of factor Va inactivation in the absence of phospholipids under pseudo-first order conditions (Fig. 4). It was not possible to fit these time courses of inactivation with a single exponential. For both factor Va and factor Va R506Q it was necessary to postulate that inactivation proceeded via a biphasic reaction with a partially active reaction intermediate.
To correlate the loss of cofactor activity with peptide bond cleavages in the heavy chain we analyzed the product generation pattern by immunoblot analysis of samples taken from the inactivation mixture at selected time intervals (Fig. 5). Compared with the peptide bond cleavage pattern of membranebound factor Va a striking difference was observed. In the absence of phospholipids cleavage at Arg 679 occurred at appreciable rates in factor Va R506Q (Fig. 5B). This is concluded from the time-dependent appearance of an additional band in the heavy chain region of the gel.
Thus, it appears that in the case of factor Va R506Q inactivation proteolysis at Arg 679 and Arg 306 contributed to factor Va inactivation and that cleavage at Arg 679 preceded cleavage at Arg 306 . Fitting the time course of APC-catalyzed factor Va R506Q inactivation (Fig. 4, solid lines) to the equation for a biphasic reaction with a partially active intermediate yields rate constants of 8.0 ϫ 10 3 M Ϫ1 s Ϫ1 and 1.0 ϫ 10 3 M Ϫ1 s Ϫ1 for k 679 and k 306 , respectively, and 70% cofactor activity for the reaction intermediate that has been cleaved at Arg 679 .
Immunoblot analysis (Fig. 5A) indicates that during the time courses of normal factor Va inactivation (Fig. 4) only peptide bond cleavages at Arg 506 and Arg 306 significantly contributed to the loss of factor Va cofactor activity. Inspection of the immunoblot that represents inactivation of normal factor Va reveals that cleavage at Arg 679 had a minor contribution to the loss of factor Va cofactor activity. As a first approximation we have, therefore, also fitted this time course with a biphasic exponential. This fit (Fig. 4, solid line)   assumption that cleavage at Arg 679 did not contribute to inactivation.
Rate Constants for APC-catalyzed Cleavage of Peptide Bonds during Inactivation of Free and Membrane-bound Factor Va and Factor Va R506Q -The rate constants for APC-catalyzed cleavage at Arg 306 , Arg 506 , and Arg 679 in factor Va and Va R506Q are summarized in Table I. It was not possible to calculate rate constants for cleavage at Arg 679 in normal factor Va from the time courses of inactivation presented in Figs. 2 and 4. However, from the disappearance of the M r ϭ 26,000/28,000 doublet on immunoblots (time points between 10 and 60 min, data not shown) it could be estimated that cleavage at Arg 679 in free factor Va occurred with k 679 Ϸ 5.9 ϫ 10 3 M Ϫ1 s Ϫ1 (t1 ⁄2 Ϸ 16 min at 156 nM APC) and that the same peptide bond in membranebound factor Va was cleaved with k 679 Ϸ 7.6 ϫ 10 3 M Ϫ1 s Ϫ1 (t1 ⁄2 Ϸ 30 min at 50 nM APC).
The data presented in Table I show that phospholipids stimulated the APC-catalyzed peptide bond cleavages in factor Va to a different extent. Phospholipids appeared to have no effect on k 679 . Furthermore, it is clear that cleavage at Arg 306 in free factor Va R506Q occurred at a lower rate than in normal factor Va. For membrane-bound factor Va R506Q and factor Va this difference was annulated. These observations are indicative for specific effects of phospholipids and/or prior cleavage at Arg 506 on the cleavages at Arg 306 and Arg 679 (see "Discussion").

Kinetic Parameters for APC-catalyzed Cleavage at Arg 506 and Arg 306 in Membrane-bound Factor Va and Factor
Va R506Q -The differences in rate constants for cleavage at Arg 506 and Arg 306 in membrane-bound factor Va must result from differences between the Michaelis-Menten kinetic parameters (k cat and/or K m ) for these peptide bond cleavages. In order to obtain information on the kinetic basis of the differences we have determined the kinetic parameters, K m and k cat , by measuring the initial rates of factor Va inactivation (between 0 and 20% loss of cofactor activity) at varying factor Va or factor Va R506Q concentrations. The experiment with normal factor Va yields the k cat and K m for cleavage at Arg 506 since this peptide bond is preferentially cleaved during the initial phase of inac-   tivation. Michaelis-Menten kinetic analysis of factor Va R506Q inactivation will give kinetic parameters for cleavage at Arg 306 .
The Lineweaver-Burk plots presented in Fig. 6 show that cleavage at Arg 506 was characterized by a K m of 20 nM and a k cat of 0.96 s Ϫ1 . Inactivation of factor Va R506Q , which corresponds with cleavage at Arg 306 , had a high K m (196 nM) and a k cat of 0.37 s Ϫ1 . Theoretically the k cat /K m obtained by Michaelis-Menten analysis should equal the second-order rate constant calculated from a time course of inactivation determined at low factor Va concentrations (Fig. 2). Indeed the k cat /K m values determined for cleavage at Arg 506 (4.8 ϫ 10 7 M Ϫ1 s Ϫ1 ) and Arg 306 (1.9 ϫ 10 6 M Ϫ1 s Ϫ1 ) closely resemble the rate constants obtained at pseudo-first order reaction conditions (Table I). This shows that the kinetic parameters, K m and k cat , are indeed associated with the indicated peptide bond cleavages. The data further demonstrate that the differences between the rate constants of cleavage at Arg 506 and Arg 306 are mainly due to differences between the K m values for these peptide bond cleavages.
Cofactor Activity of Factor Va after Cleavage at Arg 506 by APC-Pseudo-first order time courses of inactivation of normal factor Va (Figs. 2 and 4) could only be fitted in a reaction model that consisted of two steps with the formation of a reaction intermediate (factor Va cleaved at Arg 506 ) that retained partial cofactor activity.
The actual cofactor activity of factor Va is dependent on its interaction with procoagulant membranes, factor Xa, prothrombin, and on its ability to increase the catalytic activity (k cat ) of factor Xa (2)(3)(4)(5)40). To examine which of these functions was impaired in factor Va that is cleaved at Arg 506 , we have characterized the functional properties of the inactivation intermediate. To this end normal factor Va was inactivated by APC in the absence of phospholipids (cf. Fig. 4) and after completion of the first phase of inactivation (Arg 506 cleavage) the reaction mixture was diluted and chromatographed on a Mono S column. The reaction intermediate bound to Mono S and eluted in a NH 4 Cl gradient as an overlapping doublet between 700 and 800 mM NH 4 Cl. These column fractions contained almost pure inactivation intermediate (Fig. 7B).
To compare the abilities of factor Va and the purified inactivation intermediate to assemble with factor Xa into a membrane-bound prothrombinase complex we determined initial steady-state rates of prothrombin activation at limiting amounts of factor Va or factor Va cleaved at Arg 506 as a function of the factor Xa concentration (Fig. 7, A and B). Low amounts of factor Xa were required for full expression of prothrombinase activity in the case of normal factor Va (K1 ⁄2Xa ϭ 0.088 nM). With factor Va that was cleaved at Arg 506 much higher factor Xa concentrations (K1 ⁄2Xa ϭ 3.9 nM) were needed to obtain maximal prothrombinase activity. The V max of prothrombin activation obtained with the purified inactivation intermediate was lower than that determined for native factor Va. The V max values could be converted into k cat values on the basis of the protein contents of the titrated fractions. This yielded k cat values of 153 and 96 s Ϫ1 for native and cleaved factor Va, respectively.
The interaction of the membrane-bound factor Xa-Va complex with prothrombin was not affected by cleavage at Arg 506 FIG. 6. Lineweaver-Burk plots for inactivation of factor Va and factor Va R506Q by APC in the presence of phospholipids. Initial rates of factor Va inactivation were determined at varying concentrations of factor Va ( ) or factor Va R506Q (f) that were incubated with 0.28 nM APC ( ) or 1 nM APC (f) and 25 M phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl 2 , and 5 mg/ml BSA at 37°C. After different time intervals the factor Va activity was determined as described under "Experimental Procedures" and initial rates of factor Va inactivation were expressed as nM factor Va inactivated/min/nM APC (assuming that normal factor Va is converted into an intermediate with 40% cofactor activity). Immunoblot analysis of factor Va (A) and the fraction eluting between 700 and 800 mM NH 4 Cl (B) is shown in the inset. Aliquots taken from the inactivation mixture after 0 min (native factor Va) and from the column fraction were used to determine rates of prothrombin activation at (A) 1.1 ng/ml factor Va ( ) or (B) 1.2 ng/ml factor Va cleaved at Arg 506 (f) in a reaction mixture containing 25 M phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol), 0.5 M prothrombin, and varying concentrations of factor Xa in 25 mM Hepes (pH 7.5), 175 mM NaCl, 2 mM CaCl 2 , and 5 mg/ml BSA at 37°C. Rates of prothrombin activation were determined as described under "Experimental Procedures" and were corrected for thrombin formed in the absence of factor Va. The solid lines represent hyperbolas obtained after fitting the data with K1 ⁄2Xa ϭ 0.088 nM and V max ϭ 50.2 nmol of IIa/min/g of factor Va (k cat ϭ 151 s Ϫ1 ) for factor Va ( ) and K1 ⁄2Xa ϭ 3.9 nM and V max ϭ 28.7 nmol of IIa/min/g of protein (k cat ϭ 72 s Ϫ1 ) for the column fraction (factor Va cleaved at Arg 506 ) (f). Inset, double reciprocal plots of the same data. since the K m values for prothrombin were virtually the same for prothrombinase complexes with normal and cleaved factor Va (data not shown).
From these data we conclude that APC-catalyzed cleavage at Arg 506 of the heavy chain of factor Va results in the formation of a reaction product that has lost part of its cofactor activity mainly because of a reduction of its affinity for factor Xa.

The Effect of the Factor Va Concentration and the Assay Conditions on Observed Time Courses of Inactivation of Membrane-bound Factor Va and Factor Va R506Q by APC-
The data presented in this paper demonstrate that differences in rate constants of cleavage at Arg 506 and Arg 306 are primarily due to differences in the K m for these peptide bond cleavages. This means that at high factor Va concentrations, where the rates of cleavage approach V max , differences in time courses of inactivation of factor Va and factor Va R506Q should become less pronounced.
We have also shown (Fig. 7) that the relative cofactor activities of native factor Va and factor Va cleaved at Arg 506 strongly depend on the factor Xa concentration present in the factor Va assay. It was demonstrated that factor Va that is cleaved at Arg 506 will exhibit much lower cofactor activity than native factor Va when assayed at factor Xa concentrations below K1 ⁄2Xa (3.9 nM).
These observations predict that the observed time courses of factor Va or factor Va R506Q inactivation by APC will depend: 1) on the factor Va concentration in the inactivation mixture and 2) on the assay conditions at which the loss of cofactor activity of factor Va is followed. Compared with factor Va R506Q , normal factor Va will be more rapidly inactivated at low than at high factor Va concentrations. The apparent loss of cofactor activity of normal factor Va will be even more rapid and approach complete loss of activity much faster when samples from the inactivation mixture are assayed at a factor Xa concentration at which the inactivation intermediate has no activity, i.e.
[factor Xa] Ͻ Ͻ 3.9 nM (K1 ⁄2Xa ). These predictions were confirmed by an experiment in which time courses of factor Va inactivation were determined at low (Fig. 8A) or high concentrations of factor Va or Va R506Q (Fig.  8B) and in which samples from the same inactivation mixture were assayed for the loss of cofactor activity in assay mixtures that contained a low (Fig. 8A) or a high factor Xa concentration (Fig. 8B). As predicted, the differences in APC-catalyzed loss of cofactor activity of normal factor Va and factor Va R506Q were most pronounced when factor Va was inactivated at a low concentration and assayed in a prothrombin activation mixture that contained a low factor Xa concentration (Fig. 8A). Differ-ences in time courses of inactivation of factor Va and factor Va R506Q were indeed greatly reduced when high factor Va concentrations were inactivated by APC and the loss of cofactor activity was monitored in prothrombin activation mixtures that contained a high factor Xa concentration (Fig. 8B).

DISCUSSION
Proteolytic inactivation of membrane-bound factor Va by APC requires cleavage of two peptide bonds located at Arg 506 and Arg 306 in the heavy chain domain of factor Va ( Fig. 1). At low factor Va concentrations cleavage at Arg 506 occurs at a rate that is approximately 20-fold higher than the rate of cleavage at Arg 306 . This results in the accumulation of a reaction intermediate that is cleaved at Arg 506 and that exhibits partial cofactor activity in prothrombin activation (Fig. 2). Cleavage at Arg 306 , which may occur both in the reaction intermediate and in native factor Va (see below), results in a complete loss of cofactor activity.
The cofactor activity of the factor Va derivative cleaved at Arg 506 strongly depends on the factor Xa concentration in the prothrombin activation mixture in which factor Va is assayed. Cofactor activity of the reaction intermediate in prothrombin activation is only observed at high factor Xa concentrations. Compared with native factor Va, full expression of cofactor activity of factor Va that is cleaved at Arg 506 requires a 45-fold higher factor Xa concentration. This indicates that cleavage at Arg 506 in the heavy chain of factor Va affects its interaction with factor Xa and results in a considerable loss of affinity for factor Xa.
Factor Va R506Q obtained from factor V purified from the plasma of a homozygous APC-resistant patient is slowly inactivated by APC (cf. Ref 12). This factor Va molecule lacks the cleavage site at Arg 506 , due to replacement of Arg by Gln (18 -21). Time courses of inactivation of low concentrations of membrane-bound factor Va R506Q could be fitted with an equation for a single exponential curve. Immunoblotting experiments (Fig. 3B) show preferential cleavage at Arg 306 during the inactivation of membrane-bound factor Va R506Q . These data indicate that APC inactivates factor Va R506Q by a single cleavage at position Arg 306 which results in the formation of a reaction product that has virtually lost its cofactor activity in prothrombin activation.
Under the conditions of our experiments (low factor Va concentrations) cleavage at Arg 679 has a negligible contribution to the loss of cofactor activity in the case of APC-catalyzed inactivation of membrane-bound factor Va or factor Va R506Q . This is concluded from the fact that cleavage at Arg 679 (i.e. disappear- ance of the M r ϭ 26,000/28,000 fragment or appearance of doublet bands in the heavy chain or the M r ϭ 54,000/56,000 region of immunoblots) is only observed after time intervals at which more than 90% of the cofactor activity of factor Va is already lost.
These data suggest that inactivation of membrane-bound factor Va and factor Va R506Q by APC proceeds via the pathways depicted below. In this scheme factor Va int exhibits partial cofactor activity and factor Va i is a reaction product that has no detectable cofactor activity. We propose that APC-catalyzed cleavage at Arg 506 and Arg 306 in membrane-bound factor Va occurs in a random fashion and that cleavage at Arg 306 is not affected by prior cleavage at Arg 506 . This is inferred from the fact that the rate constants for cleavage at Arg 306 calculated from time courses of inactivation of normal and APC-resistant factor Va are approximately the same (k 306 is 2.3 ϫ 10 6 M Ϫ1 s Ϫ1 and 1.7 ϫ 10 6 M Ϫ1 s Ϫ1 for normal Va and factor Va R506Q , respectively).
Michaelis-Menten analysis provided additional information regarding the mechanism that accounts for the observed difference between k 506 and k 306 . The kinetic parameters determined for cleavage at Arg 506 in normal factor Va were: K m for factor Va ϭ 20 nM and k cat ϭ 0.96 s Ϫ1 . The peptide bond at Arg 306 in factor Va R506Q was cleaved with K m ϭ 196 nM and k cat ϭ 0.37 s Ϫ1 . The fact that the calculated catalytic efficiencies (k cat /K m ) have the same values as the second-order rate constants calculated for cleavage at Arg 506 in normal factor Va and for cleavage at Arg 306 in factor Va R506Q verifies that the Michaelis-Menten kinetic parameters are indeed associated with the indicated peptide bond cleavages. These data suggest that the differences in rate constants for cleavage at Arg 506 and Arg 306 mainly result from differences in the K m and explain why at high factor Va concentrations (ϾK m ) differences between time courses of factor Va and factor Va R506Q inactivation are much less pronounced (Fig. 8B).
APC can also fully inactivate factor Va and factor Va R506Q in the absence of a membrane surface (Fig. 4). Under these conditions cleavage at Arg 679 also contributes to the loss of factor Va cofactor activity. This is particularly observed for factor Va R506Q , in which the peptide bond at Arg 679 is even cleaved at a 8-fold higher rate than the site at Arg 306 . Cleavage at Arg 679 does not require prior cleavage at Arg 506 or Arg 306 since immunoblot analysis shows the appearance of a doublet band in the M r ϭ 105,000 region of the gel, which indicates that cleavage at position Arg 679 can already occur in the intact heavy chain.
Comparison of rate constants obtained for free and membrane-bound factor Va shows that phospholipids greatly accelerate APC-catalyzed factor Va inactivation. Part of this stimulation will presumably be due to the fact that both the substrate (factor Va) and the enzyme (APC) bind to the membrane surface, a condition that promotes their interaction and reaction in a way similar to that of phospholipid-dependent coagulation factor activations (4, 5, 8, 40 -42). However, in the case of APC-catalyzed factor Va inactivation, phospholipids do not accelerate the peptide bond cleavages to the same extent (Table I). Cleavage at Arg 506 and Arg 306 in normal factor Va is stimulated 300 -600-fold, whereas cleavage at Arg 306 in factor Va R506Q is enhanced 1800-fold by phospholipids. This is indicative of a specific effect of phospholipids on APC-catalyzed cleavage at Arg 306 (cf. Refs. 11 and 12). We observed that k 679 is not significantly increased in membrane-bound factor Va. This demonstrates that after binding of factor Va to phospholipids the peptide bond at position Arg 679 is hardly accessible to cleavage by membrane-bound APC.
Taken together our conclusions seem to contradict earlier observations of Kalafatis et al. (11,12) who reported that APCcatalyzed cleavages in the heavy chain of membrane-bound factor Va occur in an ordered, sequential fashion in which cleavage at Arg 506 promotes cleavage at Arg 306 and Arg 679 . They also suggested that cleavage at Arg 506 does not affect the cofactor activity of normal factor Va and that cleavage at position Arg 679 is required for full inactivation of factor Va. These apparent discrepancies may be explained by our observations that: 1) the relative rates of peptide bond cleavages in the heavy chain of factor Va depend on the factor Va concentration present in the inactivation mixture (Fig. 6) and 2) that the residual cofactor activity of partially active reaction intermediates strongly depends on the reaction conditions at which the functional activity of factor Va and its degradation products is assayed (Figs. 7 and 8). In this respect it is essential to mention that Kalafatis et al. (11,12) collected their data at high factor Va concentrations (Ͼ200 nM) and used a factor Va assay system with high concentrations of coagulation factors, while our conclusions are based on experiments at low factor Va concentrations (Ͻ10 nM) and assessment of cofactor activity of factor Va in prothrombin activation mixtures that contained varying concentrations of coagulation factors.
To emphasize the importance of reaction and assay conditions, we demonstrated that the apparent loss of factor Va cofactor activity during in activation by APC indeed strongly depends on the concentration at which factor Va is inactivated and on the prothrombinase conditions at which the loss of cofactor activity is monitored (Fig.8). Time courses of factor Va and factor Va R506Q inactivation at a high factor Va concentration, in which the loss of cofactor activity of factor Va is followed in prothombin activation mixtures that contain a high factor Xa concentration, show relatively little difference ( Fig.  8B and Ref. 12). However, when factor Va is inactivated at a low concentration and when its cofactor activity is assayed in a prothrombin activation mixture that contains a low amount of factor Xa, the differences between factor Va and factor Va R506Q are profound (Fig. 8A). This observation may have significant physiological consequences. If thrombotic events result from ongoing coagulation at low concentrations of factor Xa and factor Va (the plasma factor V concentration is ϳ25 nM), downregulation of the cofactor activity of factor Va by APC will be most efficient in the case of normal factor Va and will be maximally impaired in case of factor Va R506Q . This underscores the increased risk for venous thromboembolism in individuals whose plasmas are APC-resistant as a consequence of a factor V phenotype in which Arg 506 is substituted by Gln.