Activated protein C (APC) ()plays an important role in the down-regulation of thrombin formation (1) . The anticoagulant action of APC is associated with its ability to degrade the activated cofactors of the coagulation cascade, factors Va (2, 3) and VIIIa(4, 5) , by limited proteolysis. The mechanism of proteolytic inactivation of factor Va by APC is rather well documented. Human factor Va loses its cofactor activity in prothrombin activation after peptide bond cleavages in the heavy chain(6) . Sequential cleavage at Arg and Arg of the heavy chain appears to be the major pathway of APC-catalyzed factor Va inactivation(6, 7, 8) .

Optimal expression of anticoagulant activity of APC requires the presence of negatively charged phospholipids (3, 9, 10) and the protein cofactor, protein S(10, 11, 12) . The mechanism of action of protein S in factor Va inactivation is as yet not fully understood. In reaction systems with purified coagulation factors, protein S is a weak stimulator of APC-catalyzed factor Va inactivation(10, 12) . It has been suggested (12) that under physiological conditions protein S may act by annihilating the ability of factor Xa to protect factor Va from inactivation by APC(3, 9, 13, 14) .

The importance of protein C and protein S in the regulation of hemostatic plug formation is demonstrated by the association between thrombosis and protein C or protein S deficiency(15, 16, 17) . Recently, it has been reported (18, 19, 20, 21) that a hereditary defect that results in a poor anticoagulant response to APC (APC resistance) also is a basis for familial thrombophilia. APC resistance is associated with a single point mutation in the factor V gene that results in the replacement of Arg by Gln(22, 23, 24, 25) . This replacement affects the peptide bond in factor Va that has to be cleaved by APC in order to obtain a rapid loss of factor Va cofactor activity(6, 7, 8) .

In the present study we have determined the effects of protein S and factor Xa on apparent second order rate constants for APC-catalyzed inactivation of normal factor Va and of factor Va that was purified from the plasma of a patient who is homozygous for the mutation Arg Gln. The loss of functional activity of factor Va was correlated with the effects of protein S and factor Xa on the cleavage of APC-susceptible peptide bonds in the heavy chain of factor Va by immunoblot analysis.

EXPERIMENTAL PROCEDURES

Materials

Proteins

Protein Concentrations

Phospholipid Vesicle Preparations

Gel Electrophoretic and Immunoblot Analysis

Factor Va Assay

Kinetic Data Analysis of Pseudo-first Order Time Courses of Factor Va Inactivation

in which factor Va is a reaction intermediate with partial cofactor activity, factor Va is a form of factor Va that has completely lost its cofactor activity, k is the second order rate constant for cleavage at Arg, k is the second order rate constant for cleavage at Arg in factor Va, and k` is the second order rate constant for cleavage at Arg in native factor Va.

Under first order conditions, i.e. conditions at which the inactivation rate is directly proportional to the concentrations factor Va and APC, the loss of cofactor activity is described by (43) ,

in which Va is the cofactor activity determined at time t, Va is the cofactor activity determined before APC is added, B is the cofactor activity of factor Va (expressed as fraction of the cofactor activity of native factor Va) and k, k, and k` are the rate constants defined above. The rate constants and the cofactor activity of factor Va were obtained by fitting the data to using non-linear least squares regression analysis. Time courses of inactivation of normal factor Va were fitted with a fixed k`, that was obtained from kinetic analysis of factor Va inactivation (cf. (8) ).

In some experiments (factor Va inactivation in the presence of high factor Xa concentrations), there was a slow loss of factor Va cofactor activity in the absence of APC. In these cases the rate constant for the loss of cofactor activity in the absence of APC was determined in a separate experiment and added to k`.

RESULTS

Peptide Bonds in the Heavy Chain of Human Factor Va That Can Be Cleaved by APC

Figure 1: APC cleavage sites in the heavy chain of factor Va. The peptide bonds located at Arg, Arg, and Arg in the heavy chain of human factor Va are susceptible to proteolytic cleavage by APC. The molecular weights of possible cleavage products correspond to those given in other publications(6, 7) .

Effect of Protein S on APC-catalyzed Inactivation of Membrane-bound Factor Va

Figure 2: Effect of protein S on APC-catalyzed inactivation of membrane-bound factor Va. Purified human factor Va (1 nM) was incubated with 0.2 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA at 37 °C in the absence () or presence of 490 nM protein S (). At the indicated time points, factor Va activity was determined as described under ``Experimental Procedures.'' The rate constants and the activity of the reaction intermediate were obtained from the best fit of the data (solid lines) to given under ``Experimental Procedures'' using nonlinear least-squares regression analysis.

The proposed mode of action of protein S was confirmed by immunoblot analysis of heavy chain degradation during factor Va inactivation (Fig. 3). When factor Va was inactivated by APC in the absence of protein S, there was considerable accumulation of a transient reaction product with M = 75,000 and formation of a M = 26,000/28,000 doublet, which are generated as the result of rapid cleavage at Arg (Fig. 3A). The reaction intermediate with M = 75,000 was slowly converted into products with M = 45,000 and M = 30,000, which is indicative for cleavage at Arg. The reaction product with M = 30,000 was hardly visible on the gel since it stains poorly with our polyclonal antibody against the heavy chain.

Figure 3: Immunoblot analysis of APC-catalyzed inactivation of membrane-bound factor Va with and without protein S. Purified human factor Va (7 nM) was incubated with 1.1 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 0.2 mg/ml BSA at 37 °C in the absence (panel A) or presence of 1 µM protein S (panel B). At the indicated time intervals, samples from the inactivation mixture were subjected to SDS-PAGE. After transfer to Immobilon-P membranes, the heavy chain and derived fragments were visualized with a polyclonal antibody directed against the heavy chain of factor Va. The positions of the molecular weight markers are indicated at left of panels A and B. The percentages of cofactor activity at the indicated time points were 100, 73, 56, 45, 30, 25, 23, and 0% (A) and 100, 32, 12, 6, 3, 2, 1, and 0% (B). Further experimental details are described under ``Experimental Procedures''.

Immunoblot analysis of a time course of factor Va inactivation in the presence of protein S showed much less accumulation of the M = 75,000 intermediate (Fig. 3B). The fact that formation of the fragments with M = 26,000/28,000 occurred at the same rate as without protein S and that the fragments with M = 60,000/62,000 and M = 45,000 were already visible at early time points (<0.5 min) supports our conclusion that protein S does not affect cleavage at Arg but promotes cleavage at Arg. This results in 1) an increased rate of factor Va inactivation by direct cleavage at Arg and 2) accelerated processing of the partially active reaction intermediate that is formed when the peptide bond at Arg is cleaved first.

Effect of Factor Xa on APC-catalyzed Inactivation of Membrane-bound Factor Va

Figure 4: Effect of factor Xa and 1,5-DNS-GGACK-factor Xa on APC-catalyzed inactivation of membrane-bound factor Va. Purified human factor Va (1 nM) was incubated with 0.2 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA at 37 °C in the absence () or presence of 20 nM factor Xa () or 20 nM 1,5-DNS-GGACK-factor Xa (). At the indicated time points, factor Va activity was determined as described under ``Experimental Procedures.'' The rate constants and the activity of the reaction intermediate were obtained from the best fit of the data (solid lines) to given under ``Experimental Procedures'' using nonlinear least-squares regression analysis.

The selective inhibition of the cleavage at Arg was confirmed by immunoblot analysis of APC-catalyzed inactivation of membrane-bound factor Va in the absence (Fig. 5A) and presence (Fig. 5B) of factor Xa. Comparison of the immunoblots shows that factor Xa prevented formation of the M = 75,000 heavy chain degradation product, which is indicative for inhibition of the cleavage at Arg and which shows that APC converts factor Va directly, but slowly, into products with M = 62,000/60,000 and 45,000 by cleavage at Arg.

Figure 5: Immunoblot analysis of APC-catalyzed inactivation of membrane-bound factor Va in the absence or presence of 1,5-DNS-GGACK-factor Xa. Purified human factor Va (7 nM) was incubated with 1.4 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 0.2 mg/ml BSA at 37 °C in the absence (A) or presence of 20 nM 1,5-DNS-GGACK-factor Xa (B). At the indicated time intervals, samples from the inactivation mixture were subjected to SDS-PAGE. After transfer to Immobilon-P membranes the heavy chain and derived fragments were visualized with a polyclonal antibody directed against the heavy chain of factor Va. The positions of the molecular weight markers are indicated at left of panels A and B. The percentages of cofactor activity at the indicated time points were 100, 70, 51, 40, 25, 21, 18, and 0% (A) and 100, 92, 84, 70, 59, 49, 41 and 0% (B). Further details are described under ``Experimental Procedures.''

Effects of Protein S and Factor Xa on APC-catalyzed Inactivation of Membrane-bound Factor Va

Figure 6: Effect of protein S and factor Xa on APC-catalyzed inactivation of membrane-bound factor Va. Purified human factor Va (1 nM) was incubated with 1 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA at 37 °C in the absence () or presence of 490 nM protein S () or 20 nM factor Xa (). At the indicated time points, factor Va activity was determined as described under ``Experimental Procedures.'' The rate constants of inactivation were obtained from the best fit of the data (solid lines) to given under ``Experimental Procedures'' using nonlinear least-squares regression analysis.

If, as argued above, protein S accelerates inactivation by promoting cleavage at Arg and factor Xa blocks inactivation of normal factor Va by protection of cleavage at Arg, it is to be expected that protein S will considerably enhance the inactivation of this abnormal factor Va variant and that factor Xa will have no effect on APC-catalyzed inactivation of factor Va. The data presented in Fig. 6indeed show that protein S accelerated the rate of inactivation 14-fold (k = 2.2 10M s) and that the inactivation of factor Va was not affected by factor Xa.

In Table 1we have summarized the rate constants for APC-catalyzed cleavage at Arg and Arg of the heavy chain of membrane-bound factor Va and factor Va that were determined in the absence and presence of protein S or factor Xa.

Concentration Dependence of the Effects of Protein S and Factor Xa on APC-catalyzed Inactivation of Membrane-bound Factor Va

Figure 7: Effects of varying protein S and factor Xa concentrations on rate constants of APC-catalyzed cleavage at Arg and at Arg in membrane-bound factor Va. The rate constants k and k were obtained from time courses of inactivation of 1 nM factor Va by 0.2 nM APC on 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA (37 °C) in the presence of varying concentrations protein S and/or factor Xa as described at Fig. 2and Fig. 4. A, k as function of the concentration protein S. B, k as function of the concentration factor Xa.

We have also determined the ability of factor Xa to protect factor Va from APC inactivation at varying factor Xa concentrations (Fig. 7B). Factor Xa could completely block cleavage at Arg without having an effect on the slow phase of factor Va inactivation (k). Half-maximal protection of cleavage at Arg was observed at 1.9 nM factor Xa.

It appeared that protein S and factor Xa can exert their effects on APC-catalyzed factor Va inactivation independent of each other. At a concentration of factor Xa that completely blocked cleavage at Arg (20 nM), protein S was still able to accelerate factor Va inactivation 12-fold (Fig. 8). The fact that the time course of inactivation determined in the presence of factor Xa and protein S significantly differs from that obtained in the presence of protein S alone indicates that the factor Xa protection of cleavage at Arg was not annihilated by protein S.

Figure 8: Combined effects of protein S and factor Xa on APC-catalyzed inactivation of membrane-bound factor Va. Purified human factor Va (1 nM) was incubated with 0.2 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA at 37 °C in the presence of 20 nM factor Xa (), 490 nM protein S (), or 490 nM protein S plus 20 nM factor Xa (). At the indicated time points, factor Va activity was determined as described under ``Experimental Procedures.''

Collectively these data support our conclusion that the stimulatory effect of protein S and the protective effect of factor Xa are associated with the different steps in the pathway of APC-catalyzed factor Va inactivation, i.e. are confined to the peptide bond cleavages at Arg and Arg, respectively.

Inactivation of Factor Va and Factor Vaby APC in the Presence of Both Factor Xa and Protein S

Figure 9: APC-catalyzed inactivation of membrane-bound factor Va and factor Va in the presence of protein S and Factor Xa. 1 nM purified human factor Va or factor Va was incubated with 0.2 nM APC and 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol) in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA at 37 °C in the absence or presence of 200 nM protein S plus 20 nM factor Xa. At the indicated time points, factor Va activity was determined as described in the ``Experimental Procedures''. , factor Va; , normal factor Va; , factor Va plus protein S and factor Xa; , normal factor Va plus protein S and factor Xa.

Protection of Factor Va against APC Inactivation by Incorporation in the Prothrombinase Complex

Therefore, we have determined in the same experiment the amounts of factor Xa required to protect factor Va from APC cleavage and to express the cofactor activity of factor Va in prothrombin activation (Fig. 10). Under these conditions (low concentration of factor Va and prothrombin present), similar amounts of factor Xa were required to protect factor Va from inactivation by APC and to express factor Va cofactor activity in prothrombin activation (K = 0.03 nM for protection, K = 0.08 nM for the expression of cofactor activity). These data indicate that factor Va is optimally protected from inactivation by APC when it is incorporated into the prothrombinase complex during ongoing catalysis and that complex formation between factor Xa and factor Va at the same time expresses the cofactor activity of factor Va and protects it from inactivation by APC.

Figure 10: Effects of factor Xa on factor Va activity in prothrombin activation and on the protection of factor Va from inactivation by APC. The rate constant k was obtained from time courses of inactivation of 6 pM factor Va by 0.2 nM APC determined in the presence of 25 µM phospholipid vesicles (DOPS/DOPC, 10/90, mol/mol), 1 µM prothrombin, and varying concentrations of factor Xa in 25 mM Hepes (pH 7.5), 175 mM NaCl, 3 mM CaCl, and 5 mg/ml BSA (37 °C). Plotted is the value of k as function of the concentration of factor Xa (). The factor Xa dependence of expression of factor Va cofactor activity () was determined in the same reaction mixtures by measuring the rate of thrombin formation before APC was added.

DISCUSSION

Recently it has been reported that APC-catalyzed inactivation of factor Va proceeds via a biphasic reaction(6, 7, 8) . The first step of the inactivation process is a rapid cleavage at Arg in the heavy chain domain of factor Va that results in the formation of a reaction intermediate that exhibits partial cofactor activity in prothrombin activation. This reaction intermediate is subsequently fully inactivated by slow cleavage at Arg. Here we report that protein S stimulates the inactivation of membrane-bound factor Va 20-fold by specific acceleration of the cleavage at Arg and that factor Xa protects factor Va from inactivation by APC by blocking the cleavage site at Arg. However, factor Xa does not completely inhibit factor Va inactivation by APC. At saturating factor Xa concentrations, factor Va inactivation still proceeds with a slow rate that is associated with APC-catalyzed cleavage at Arg. This means that the latter cleavage is apparently not affected by factor Xa.

These conclusions are supported by immunoblot analysis and by experiments with factor Va obtained from a homozygous APC-resistant patient. Immunoblots of time courses of inactivation of membrane-bound factor Va by APC, in which the heavy chain and its degradation products were visualized with a polyclonal antibody directed against the heavy chain, showed transient generation of a reaction product with M = 75,000 which is formed after rapid cleavage of the peptide bond at Arg by APC (Fig. 3A). With protein S present there is much less accumulation of the M = 75,000 fragment and increased formation of products with M of 60,000/62,000, and 45,000 (Fig. 3B). This is indicative for a protein S-dependent increase of the rate of cleavage at Arg, which either results in increased direct factor Va inactivation or in accelerated processing of the M = 75,000 intermediate.

In the presence of factor Xa, there is also no formation of the M = 75,000 reaction intermediate (Fig. 5B). In this situation there is a slow formation of products with M = 62,000/60,000 and 45,000, which shows that factor Xa blocks cleavage at Arg and that inactivation of factor Va proceeds via slow cleavage at Arg.

Similar information is obtained from kinetic analysis of inactivation of factor Va. This abnormal factor Va molecule lacks the cleavage site at Arg due to a substitution of Arg by Gln(22, 23, 24, 25) . In line with the proposed effects of protein S and factor Xa on APC-catalyzed peptide bond cleavages in the heavy chain of factor Va, we observed that protein S drastically stimulates inactivation of factor Va (Fig. 6), which has been reported to be primarily associated with cleavage at Arg(7, 8) . We further show that factor Xa does not protect factor Va from inactivation by APC. This observation is explained by the fact that factor Va lacks Arg, which is the target site for factor Xa.

It is the first time that a profound effect is reported for protein S in a reaction system with purified coagulation factors. In earlier publications(10, 12) , modest effects were described for protein S. The reason for this apparent discrepancy is that in the earlier studies the effect of protein S was determined on initial rates of APC-catalyzed factor Va inactivation that is a phase of the reaction that is primarily associated with cleavage at Arg and which, according to our data, is hardly affected by protein S. Here we show that protein S actually acts at the slower step of the inactivation process, that is the cleavage of the peptide bond at Arg. The presence of protein S thus ensures rapid inactivation of factor Va by accelerating direct cleavage at Arg or by promoting the conversion of a partially active reaction intermediate (factor Va cleaved at Arg) into a product with no detectable cofactor activity. Given the known clinical consequences of protein S deficiencies, it is likely that the above described contribution of protein S to factor Va inactivation is of prime physiological importance.

Titration experiments at low APC and factor Va concentrations on 25 µM DOPS/DOPC phospholipid vesicles show that protein S stimulates APC-catalyzed inactivation of factor Va with a K of 200 nM and that protection by factor Xa is characterized by a K of 1.9 nM. We have further shown that protein S and factor Xa can exert their effects on APC-catalyzed factor Va inactivation independent of each other. At a concentration of factor Xa that completely blocks cleavage at Arg, protein S was still able to accelerate factor Va inactivation 12-fold. These data support our conclusion that the stimulatory effect of protein S and the protective effect of factor Xa on inactivation of factor Va by APC result from different interactions with factor Va and are confined to steps that involve the peptide bond cleavages at Arg and Arg, respectively.

In terms of physiological relevance for regulation of factor Va activity, factor Xa actually has two (procoagulant) functions: 1) factor Xa protects factor Va from inactivation by APC and 2) factor Xa brings the cofactor activity of factor Va to expression by formation of the prothrombin-activating complex. It appeared that much higher concentrations of factor Xa were required for protection of factor Va from inactivation by APC (K = 1.9 nM) than for incorporation of factor Va into a catalytically active prothrombinase complex (K = 0.08 nM). However, prothrombinase complex formation is usually determined at low factor Va concentrations in reaction mixtures that contain prothrombin as additional component. We observed that much lower amounts of factor Xa (K = 0.03 nM) were required to protect factor Va from inactivation by APC when it was incorporated into a prothrombinase complex that was catalyzing prothrombin activation. These data demonstrate: 1) that factor Va is most effectively protected from inactivation by APC when incorporated into the prothrombinase complex during ongoing prothrombin activation and 2) that protection of factor Va from APC cleavage and prothrombinase complex formation likely involve the same interaction of factor Xa with factor Va.

Recently, it was recognized that APC resistance alone is only a mild risk factor for thrombosis(46, 47, 48) . Considering the large differences in inactivation rates of factor Va and factor Va by APC, this is a somewhat puzzling observation. In accordance with the proposed effects of protein S and factor Xa, we observed that the large differences in the rates of APC-catalyzed inactivation of factor Va and Va were almost annihilated when factor Va and factor Va were inactivated by APC in the presence of factor Xa and protein S (Fig. 9). Since it is likely that in vivo regulation of factor Va activity by APC occurs in the presence of protein S and factor Xa, our observations may explain why in the absence of other genetic or environmental risk factors APC resistance only results in a relatively weak prothrombotic condition.

FOOTNOTES

*

This work was supported by Program Grant 900-526-192 from the Dutch Organisation for Scientific Research (NWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Biochemistry, University of Limburg, P. O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: 31-43-3881678, Fax: 31-43-3670988.

()

The abbreviations used are: APC, activated protein C; BCA, bicinchoninic acid; BSA, bovine serum albumin; 1,5-DNS-GGACK-factor Xa, factor Xa inhibited with 1,5-dansyl-Glu-Gly-Arg chloromethylketone; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoserine; factor Va, abnormal factor Va in which Arg is substituted by Gln; p-NPGB, p-nitrophenyl-p`-guanidinobenzoate hydrochloride; RVV-X, purified factor X activator from Russel's viper venom; PAGE, polyacrylamide gel electrophoresis; S2238, D-Phe-(pipecolyl)-Arg-pNA; S2366, L-pyroGlu-Pro-Arg-pNA.

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