©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mechanism of Inactivation of Human Platelet Factor Va from Normal and Activated Protein C-resistant Individuals (*)

(Received for publication, May 23, 1995)

Rodney M. Camire (1) Michael Kalafatis (1) Mary Cushman (2)(§) Russell P. Tracy (1) (2) Kenneth G. Mann (1) Paula B. Tracy (1)(¶)

From the  (1)Department of Biochemistry and the (2)Department of Pathology, University of Vermont, Burlington, Vermont 05405

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg, with subsequent cleavages occurring at Arg and Arg. Alternatively, APC can cleave at Arg initially, with further cleavage at Arg then at Arg or at Arg followed by cleavage at Arg. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg Gln mutation (Factor V Leiden, Factor V). With respect to the mutant platelet factor Va in the presence of APC, >70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor Va, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg will occur in the absence of cleavage at Arg, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg or Arg and 2) the extent to which it is cleaved initially at Arg, in contrast to cleavage of Arg, will define both the extent and rate of inactivation.


INTRODUCTION

The delicate balance between normal hemostasis and thrombosis is mediated through the formation of alpha-thrombin, a potent procoagulant enzyme which also initiates anticoagulant events through its thrombomodulin-dependent activation of protein C(1, 2) . alpha-Thrombin is formed subsequent to the proteolytic conversion of prothrombin by the enzyme complex prothrombinase, which is composed of a vitamin-K-dependent serine protease factor Xa, and the cofactor factor Va, assembled on an appropriate membrane surface in the presence of Ca ions. The required cofactor is supplied subsequent to the thrombin or factor Xa-catalyzed(3, 4, 5) activation of plasma factor V, or by the release of the functional cofactor from platelet alpha-granules(6, 7, 8) . In addition to binding the substrate prothrombin(9) , factor Va fulfills its cofactor function by providing at least part of the receptor for factor Xa at the membrane surface(10, 11, 12) . Therefore, deletion of factor Va from the complex reduces the rate of the reaction by 4 orders of magnitude(13) . Since factor Va is central to the proper assembly of the catalyst on a membrane surface and profoundly influences the rate of thrombin formation, regulation of prothrombinase activity can be expressed through alterations in factor Va activity.

Human plasma factor Va is proteolytically inactivated by APC (^1)through a mechanism requiring ordered and sequential cleavages, only some of which are membrane-dependent (14, 15) (Fig. 1). Sequential cleavage of plasma factor Va occurs only in the heavy chain (105 kDa) of the cofactor at positions Arg, Arg, and Arg. Cleavage at Arg gives rise to a 75-kDa intermediate with cleavages at Arg and Arg required for complete loss of activity. Cleavage at Arg is membrane-dependent, whereas cleavages at Arg and Arg are not. Recently, it has been reported that an increased risk toward venous thrombosis is associated with a single point mutation in the factor V molecule (Factor V Leiden Arg Gln, Factor V)(16) . Termed APC Resistance, patients suffer from a poor anticoagulant response to APC, presumably because these individuals lack an APC cleavage site at position 506 of the factor V molecule(16, 17, 18, 19, 20) . Resistance to APC is the most common identifiable defect among patients with a thrombotic disorder(17, 18, 21) . Recently, our laboratory has characterized the molecular defect in plasma factor V(22) . In the absence of the APC cleavage site at position 506, inactivation of factor Va proceeded, but at a slower rate when compared to the normal cofactor. Western blot analysis revealed that cleavage at Arg results in the generation of a 45-kDa fragment and a 62/60-kDa doublet (Fig. 1). Complete loss of cofactor activity was accomplished and was correlated with the cleavage of the 62/60-kDa doublet at position 679, which generated a 56/54-kDa doublet.


Figure 1: Schematic representation of membrane-bound human plasma factor Va heavy chain inactivation by APC. The heavy chain of factor Va (105 kDa) is composed of two A domains (A1-A2) associated through a connecting region(47) . Normal plasma factor Va is inactivated following three ordered and sequential cleavages at Arg, Arg, and Arg. Cleavage at Arg, which gives rise to a 75-kDa fragment and a 28/26-kDa doublet, is necessary to optimally expose the site at Arg. Further cleavage at Arg yields a 45-kDa fragment and a 30-kDa fragment. Individuals with the Arg Gln mutation no longer have a cleavage site at position 506 which slows the rate of cleavage at Arg. Cleavage at Arg yields a 45-kDa fragment and a 62/60-kDa doublet. Further cleavage of the 62/60-kDa doublet at Arg yields a 56/54-kDa doublet. The rate of inactivation of the mutant cofactor is much slower than that of the normal cofactor (22) . Fragments that are recognized by the monoclonal antibody (alphaHFVa#6) used in this study are indicated by the shaded boxes.



In addition to the factor V which circulates in plasma, a significant amount of factor V/Va is contained within the alpha-granules of platelets. Of the total factor V/Va found in whole blood, 80% is contained in plasma and 20% is found in platelets(23) , yet at a site of vascular injury the concentration of platelet factor V/Va has been estimated to be about 600 times that of plasma factor V(24) . Platelet factor V clearly plays a preeminent role in hemostasis since individuals deficient in platelet but not plasma factor V exhibit a severe, life-threatening bleeding diathesis(25) . Previous studies from our laboratory indicate that the platelet membrane supports the APC-catalyzed inactivation of plasma factor Va. Kinetic parameters defining the reaction and obtained using platelets or phospholipid vesicles were nearly identical(26) . However, previous studies indicate also that platelet factor V and plasma factor V are separate and distinct substrates for many reactions(5, 6, 13, 27, 28, 29) . Therefore, this report details the APC-catalyzed inactivation of platelet factor Va from normal and APC-resistant individuals on the surface of both thrombin-activated platelets and phospholipid vesicles.


EXPERIMENTAL PROCEDURES

Materials and Reagents

Trizma (Tris base), L-alpha-phosphatidyl-L-serine (bovine brain) (PS), L-alpha-phosphatidylcholine (egg yolk) (PC), Arg-Gly-Asp-Ser (RGDS) peptide, Tween 20, and glycine were purchased from Sigma. HEPES was purchased from J. T. Baker. Crystallized bovine serum albumin was purchased from ICN ImmunoBiologicals. The alpha-thrombin inhibitor hirudin was obtained from Genentech. The fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was synthesized as described(30) . Phospholipid vesicles composed of 75% (% w/w) PC and 25% (% w/w) PS (PCPS) were prepared as described previously(31, 32) . The concentration of the phospholipid vesicles was determined by phosphorous assay(33) . Human protein C was purchased from Hematologic Technologies Inc., Essex Junction, VT.

Preparation of Proteins

Proteins were purified from human fresh frozen plasma. Factor V was isolated by immunoaffinity chromatography as described, and was activated to factor Va with 2 NIH units/ml of alpha-thrombin for 15 min at 37 °C(34, 35) . Factor X and prothrombin were purified by the method of Bajaj et al. (36) . Factor X was activated with the factor X activator purified from Russell's viper venom(37) . alpha-Thrombin was prepared by activation of prothrombin with taipan snake venom as described by Owen and Jackson (38) . Protein purity was assessed by SDS-polyacrylamide gel electrophoresis before and after disulfide bond reduction according to the method of Laemmli(39) . Proteins were visualized by Coomassie Brilliant Blue R-250 staining. Molecular weights and extinction coefficients, E1%, of the various proteins used were taken as follows: prothrombin, 72,000, 14.2 (40) ; thrombin, 37,000, 17.4(40, 41) ; factor V, 330,000, 9.6(23) ; factor Xa, 50,000, 11.6(36) .

Protein C (1 µM) was activated with alpha-thrombin (0.16 µM) at 37 °C for 1 h in 20 mM HEPES, 0.15 M NaCl, pH 7.4, as described previously (26) with slight modifications. Full activation of protein C to APC was confirmed by measuring the amidolytic activity toward the chromogenic substrate S-2238 (American Diagnostica, Inc.) after addition of 0.18 µM hirudin(42) . Aliquots of APC were stored at -80 °C and amidolytic activity measurements of APC were made prior to each experiment.

Isolation of Platelets

Platelets were isolated by the method of Mustard et al.(43) from human venous blood, obtained from consenting non-medicated normal or APC-resistant individuals. Modifications to this procedure included omission of apyrase from all washing steps and the use of 5 mM HEPES-Tyrode's, pH 7.4, as the final platelet suspension buffer. Platelets were counted on a Coulter counter (Coulter Electronics, LTD.) and brought to a final platelet concentration of 1 10^9/ml for all experiments.

APC Inactivation of Platelet Factor Va

Platelet factor Va release and activation was accomplished by platelet incubation (1 10^9/ml) with 20 nM alpha-thrombin (2 NIH units/ml) for 5 min at ambient temperature, followed by the addition of 24 nM hirudin. For experiments in which activated platelets provided the membrane surface required for factor Va inactivation, platelets were preincubated with RGDS peptide (1 mM) at 37 °C for 1-2 h to prevent platelet aggregation. In experiments in which PCPS vesicles provided the membrane surface required for inactivation, thrombin-activated platelets were removed from platelet factor Va by centrifugation at 1000 g for 5 min and PCPS vesicles (10 µM) were then added. In both protocols, the initial concentrations of platelet factor Va was donor dependent, and ranged from 2.3 to 5.3 nM. Inactivation of the platelet factor Va was initiated by APC addition (0.25-0.50 nM). Aliquots of the inactivation mixture were withdrawn at various time intervals. Where necessary, platelets were removed by centrifugation at 10,000 g for 5 s and assayed immediately for residual cofactor activity and prepared for SDS-gel electrophoresis. Factor Va cofactor activity was assessed by measuring the effect of factor Va on prothrombin activation through assembly and function of the prothrombinase complex(13) . Assay mixtures contained, 1.39 µM prothrombin, 20 µM PCPS, 3 µM DAPA, 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4, and platelet factor Va at less than 0.5 nM. Reactions were initiated by addition of 5 nM factor Xa. Under these conditions, the initial rate of the reaction was proportional to the concentration of factor Va. The increase in fluorescence intensity of DAPA as it binds to thrombin (30) was continuously monitored at an excitation wavelength of 335 nm and at an emission wavelength of 565 nm on a Perkin-Elmer LS-3b fluorescence spectrometer(35) .

Gel Electrophoresis/Electroblotting/Chemiluminescence

Following platelet removal if necessary, aliquots of the reaction mixture were prepared for SDS-PAGE by addition of 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% beta-mercaptoethanol, 0.001% bromphenol blue (final concentrations). Following heating at 95 °C for 3 min, SDS-PAGE analysis was performed on 5-15% gradient slab gels according to the method of Laemmli(39) . Following electrophoresis, the proteolytic fragments resulting from APC-catalyzed cleavage of factor Va were transferred to nitrocellulose using electroblotting techniques as described by Towbin et al.(44) . Transfer was performed at 500 mA for 2 h at 4 °C(5) . Nitrocellulose was blocked with 5% non-fat dry milk in 20 mM Tris, 0.15 M NaCl, 0.05% Tween 20 (TBS-T) at pH 7.4. The factor Va antigen was probed with a mouse anti-human factor V heavy chain IgG monoclonal antibody (alphaHFVa#6)(22, 45, 46) . The secondary antibody used was a horse anti-mouse IgG coupled to horseradish peroxidase (Southern Biotechnologies). Detection of factor Va was performed by enhanced chemiluminescence (Western Blot Chemiluminescence Detection Kit, DuPont NEN) by exposure of blots (5-30 s) to Kodak Scientific Imaging film (X-Omat) developed in a Kodak M35A X-Omat processor.


RESULTS

Inactivation of Platelet Factor Va on Platelets and PCPS Vesicles

Platelets (1 10^9/ml) were activated with thrombin as described under ``Experimental Procedures'' to both activate the platelet and to release and activate the platelet factor Va. Following the addition of hirudin to neutralize thrombin, APC (0.25-0.5 nM) was added to determine the rate and mechanism of inactivation of platelet factor Va on the activated platelet surface. Depending on the platelet donor, 2.3-5.3 nM factor Va was present in the inactivation mixtures (n = 9). The rates of platelet factor Va inactivation by APC on PCPS vesicles and thrombin-activated platelets are compared in Fig. 2A. When PCPS vesicles were used to provide the membrane surface (closed symbols), 50% of the initial factor Va cofactor activity was lost within 30 s of APC addition, and 20% of the cofactor activity remained at 15 min. No further loss of cofactor activity occurred even though the reaction was monitored for an additional 3 h. When thrombin-activated platelets were used to provide the membrane surface (open symbols), the rate of inactivation was significantly slower. Even after a 30-min incubation with APC, approximately 40% of the factor Va cofactor activity remained. Removal of platelets at this point by centrifugation, followed by PCPS vesicle addition (10 µM), resulted in rapid platelet factor Va inactivation such that 20% of the original cofactor activity remained. Again no additional loss in cofactor activity was observed with an additional 2-h incubation.


Figure 2: Inactivation of membrane-bound human platelet factor Va by APC. Human platelets were activated with thrombin as described under ``Experimental Procedures.'' In A, platelet factor Va (2.6 nM) in the presence of PCPS vesicles (10 µM, bullet) or thrombin-activated platelets (1 10^9/ml, ) was inactivated upon addition of APC (0.5 nM). At selected time intervals, aliquots were assayed for cofactor activity. The arrowhead indicates the point at which platelets were removed from the platelet inactivation mixture and PCPS vesicles (10 µM) were added (40-min time point). The inset allows comparison of the inactivation rates from 0 to 30 min. At the same time intervals aliquots of the reaction mixture were withdrawn and analyzed by SDS-PAGE. Following transfer to nitrocellulose proteolytic fragments were visualized using the monoclonal antibody alphaHFVa#6. Panel B represents the proteolytic fragments derived from the inactivation on PCPS vesicles. Lane 1, platelet factor Va, no APC; Lanes 2-10, membrane-bound platelet factor Va with APC at 30 s, 4, 8, 15, 30, 60, 90, and 120 min. The position of the molecular weight markers is indicated at the left of panel B. Nonspecific fragments appear at approximately 68 and 43 kDa, resulting from the reaction of bovine serum albumin and platelet IgG, respectively, with the secondary antibody.



Western blot analysis depicting the proteolytic fragments accompanying inactivation on phospholipid vesicles is shown in Fig. 2B. The monoclonal antibody used to detect the fragments is specific for an epitope located on the 30-kDa fragment (amino acid residues 307-506) resulting from APC-catalyzed cleavage at Arg and Arg and was used previously to confirm the mechanism of APC-catalyzed cleavage of normal plasma factor V and Va and to elucidate that of factor V/Va(22) . Lane 1 represents the zero time (no APC addition) and is mainly characterized by an intense band at 105 kDa, the factor Va heavy chain. Lanes 2-10 represent the inactivation time course shown in Fig. 2A and depict the appearance, and subsequent slow cleavage of the 75-kDa fragment (amino acids 1-506; a) to give rise to a 30-kDa fragment (amino acids 307-506; b) consistent with an initial cleavage at Arg followed by cleavage at Arg as has been observed for normal plasma factor Va(15) . Peptides of 62 and 56/54 kDa were observed also (c). The 62-kDa fragment most likely appears as a result of initial cleavage of the heavy chain at Arg (amino acids 307-709) followed by its subsequent cleavage at Arg to yield the 56/54-kDa fragment (amino acids 307-679). Further cleavage of the 62-kDa or 56/54-kDa fragments at position 506 will give rise to the accumulation of the 30-kDa fragment (amino acids 307-506). Nonspecific binding resulting from platelet protein interactions with the secondary antibody appear at 68 and 43 kDa.

Substantial differences in the initial rate of APC-catalyzed platelet factor Va inactivation on thrombin-activated platelets were observed with different platelet donors (n = 4). These differences are most evident by comparison of Fig. 2A and 3A. In marked contrast to the data shown in Fig. 2A, those shown in Fig. 3A indicate that as much as 50% of the initial platelet factor Va cofactor activity was lost within 1 min of APC addition. The cleavage products observed (Fig. 3B) were identical to those seen previously (Fig. 2B) as detailed above. In all experiments, the loss in platelet factor Va activity could be attributed completely to APC-catalyzed inactivation. Control experiments such as that shown in Fig. 3A (open triangles), where APC was omitted from the reaction mixture, indicated that platelet factor Va cofactor activity was unaltered over a 2-h incubation.


Figure 3: Inactivation of platelet factor Va by APC utilizing activated platelets as a membrane surface. A, platelets were treated with thrombin as described under ``Experimental Procedures.'' Normal platelet factor Va (3.8 nM) in the presence of thrombin-activated platelets (1 10^9/ml), was inactivated with APC (0.25 nM, ) and compared to a control with no APC added (). At various time intervals aliquots of the inactivation mixture were assayed for cofactor activity. Panel B represents the proteolytic fragments derived from the inactivation on thrombin-activated platelets. Following transfer to nitrocellulose, proteolytic fragments were visualized using the monoclonal antibody alphaHFVa#6. Lane 1, platelet factor Va, no APC; Lanes 2-11, platelet factor Va treated with APC at 30 s, 2, 4, 8.5, 15, 30, 45, 65, 135, and 200 min. The position of the molecular weight markers is indicated at the left of panel B.



APC-catalyzed Inactivation of Platelet Factor Va

A clinically significant thrombotic tendency is associated with ``APC resistance'' which is defined by an Arg to Gln mutation in the factor Va heavy chain at position 506 (factor Va), and designated Factor V Leiden(16) . The significance of this mutation is that Arg is an important APC cleavage site such that impaired APC-catalyzed factor Va inactivation would lead to an increased risk of thrombosis. Recent studies of the APC-catalyzed inactivation of purified plasma factor Va have shown that in the absence of cleavage at Arg the inactivation process proceeds at a significantly reduced rate, yet near complete inactivation is observed (<5% cofactor activity remaining) due to the APC-catalyzed cleavages at positions 306 and 679. To determine if these characteristics were manifested by platelet factor Va, experiments identical to those described above were done with the platelets of an individual who is heterozygous for this mutation as determined by DNA analysis(16) . Isolated, washed platelets were thrombin-activated as detailed previously to release and activate platelet factor Va. The APC-catalyzed inactivation of the mutant platelet factor Va was studied on both thrombin-activated platelets and PCPS vesicles (Fig. 4). As seen with normal platelet factor Va, the rate of cofactor inactivation was substantially faster in the presence of PCPS vesicles. However, a significant level of cofactor activity was clearly resistant to APC inactivation (>30%). Likewise, in the presence of platelets, 55% of the initial cofactor activity was still observed following a 10-min incubation with APC, with 30% of the activity remaining even though the incubation proceeded for an additional 2 h. The addition of PCPS vesicles (10 µM) did not result in further inactivation. Examination of platelet factor Va proteolytic fragments observed on PCPS vesicles (Fig. 4B) or thrombin-activated platelets (Fig. 4C) over time indicate that the mutant factor Va heavy chain from patients heterozygous for the Arg Gln mutation which cannot be cleaved at Arg, is cleaved at Arg to yield a 62-kDa fragment which is subsequently cleaved at Arg to yield a 56/54-kDa doublet which is resistant to further APC-catalyzed cleavage. The normal platelet factor Va heavy chain derived from the platelets of this heterozygous individual is cleaved as previously detailed in Fig. 2B and Fig. 3B to yield a 75-kDa fragment (amino acids 1-506) which is subsequently cleaved at Arg to yield the 30-kDa fragment (amino acids 307-506). The extent to which the normal factor Va heavy chain cleavage contributes to the appearance of the 62- and 56/54-kDa fragments cannot be assessed using this experimental protocol.


Figure 4: APC-catalyzed inactivation of membrane-bound platelet factor Va from an APC-resistant individual. Platelets from an APC-resistant patient (heterozygous for the Arg Gln mutation) were treated with thrombin as described under ``Experimental Procedures.'' In A, platelet factor Va from the APC-resistant patient (3.7 nM) was incubated with APC (0.25 nM) and PCPS vesicles (10 µM, bullet) or thrombin-activated platelets (1 10^9/ml, ). At selected time intervals aliquots of the reaction mixture were assayed for cofactor activity. At the same time intervals aliquots were withdrawn and analyzed on a 5-15% linear gradient SDS-PAGE. Following transfer to nitrocellulose, fragments were visualized using monoclonal antibody alphaHFVa#6. Panel B depicts APC-catalyzed platelet factor Va inactivation in the presence of PCPS vesicles. Lane 1, no APC; Lanes 2-8, 30 s, 2.5, 5, 10, 20, 40, and 60 min, respectively. Panel C, depicts APC-catalyzed platelet factor Va inactivation when thrombin-activated platelets provide the membrane surface. Lane 1, platelet factor Va, no APC; Lanes 2-11, platelet factor Va with APC at 1, 2.5, 6.5, 10, 30, 60, 90, 120, 127, and 140 min. The position of the molecular weight markers is indicated at the left of both panels B and C.




DISCUSSION

Recent studies have clearly indicated that the APC-catalyzed inactivation of plasma factor Va is mediated by three sequential cleavages in the factor Va heavy chain at Arg, Arg, and Arg(15) . The studies reported here indicate that the APC-catalyzed proteolysis of platelet factor Va occurs through the initial cleavage of the factor Va heavy chain at either Arg or Arg (Fig. 1). Consistent with observations made with plasma factor Va, initial cleavage at Arg appears to be preferred; however, depending upon the platelet factor Va donor, substantial initial cleavage at Arg occurs also. Initial cleavage at Arg is followed by cleavages at Arg and Arg (Fig. 1). In contrast, initial cleavage in platelet factor Va at Arg is followed by sequential cleavages at Arg and Arg. The initial cleavage in platelet factor Va at Arg is consistent with the sequential cleavages which occur in plasma and platelet factor Va where in the absence of an arginine at position 506, cleavage at Arg occurs followed by cleavage at Arg.

Studies with normal plasma factor Va indicate that its sequential cleavage at positions 506, 306, and 679 renders it completely inactive. Studies with plasma factor Va indicate that, even in the absence of a cleavage site at Arg, cleavage at positions 306 and 679 is sufficient to effect almost complete cofactor inactivation (<5% initial activity remaining), although the rate of inactivation is slowed(22) . In marked contrast, our present data indicate that APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. In the four normal platelet donors assayed, 10-25% of the initial cofactor activity remained even after prolonged incubation. In the two individuals assayed who were heterozygous for the Arg to Gln mutation at position 506 in the factor V molecule, 30-40% of their initial factor Va cofactor activity remained subsequent to a 2-h incubation. Since the extent of platelet factor Va inactivation is independent of the membrane surface to which the normal or mutant platelet factor Va is bound (PCPS vesicles or thrombin-activated platelets), it appears that the apparent APC resistance expressed by both normal platelet factor Va and platelet factor Va is inherent to the platelet factor Va molecule and its fragments. However, how much cofactor activity is associated with the various platelet factor Va proteolytic fragments cannot be determined at this time. Meaningful correlations between the proteolytic fragments observed by Western blot analysis and the cofactor activity measurements cannot be made at this time since only qualitative data can be derived from the Western blotting results. Even if the affinity of the antibody (alphaHFVa#6) for the various fragments was known, quantitating the various reactive fragments would be difficult. Similar studies as those detailed here with purified platelet factor Va and using protein stains to visualize the cleaved products will obviate this problem and must be done.

The observation that the APC-catalyzed inactivation of platelet factor Va is not completely analogous to that observed with plasma factor Va is not surprising. Several observations have been made previously which suggest that subtle differences exist between plasma and platelet factor Va which render them different substrates for many reactions, and thus somewhat different cofactors. For example, platelet factor V/Va is stored within the platelet as a partially proteolyzed molecule, ranging in molecular mass from 115 to 330 kDa(27) , and exhibits significant cofactor activity upon release from the platelet, demonstrating only a 2-3-fold increase in cofactor activity upon further activation with factor Xa or thrombin(6) . This is in direct contrast to plasma factor V, which expresses virtually no cofactor activity in the single-chain form(13) . Factor Xa- or thrombin-catalyzed activation of platelet factor V yields proteolytic fragments of 105 and 74 kDa(5, 6) . Thrombin-catalyzed activation of plasma factor V also yields proteolytic fragments of 105 and 74 kDa, whereas factor Xa-catalyzed activation yields fragments of molecular mass 220 and 105 kDa(5) . It has also been shown by our laboratory that factor Xa activates platelet factor V 50-100 times more effectively than thrombin, whereas activation of plasma factor V by factor Xa or thrombin is characterized by the same catalytic efficiency(6) . Additional data highlighting structural differences between platelet and plasma factor Va are their differential phosphorylation by platelet kinases. Platelet factor Va is phosphorylated exclusively on the light chain whereas plasma factor Va is phosphorylated on both the light and heavy chain(28, 29) . Collectively these data suggest that platelet factor V may be a different substrate than plasma factor V, but at present, the identity of these two cofactors in both primary structure and post-translational modifications has yet to be elucidated(29) .

These same differences which may make normal platelet factor Va somewhat of a different substrate may account for the data shown in Fig. 2and Fig. 3. The data (representative of four similar experiments) indicate that additional cofactor activity appears over time suggesting that APC, in some way, may have a positive effect on platelet factor Va function. Alternatively since these experiments were done with activated platelet suspensions or platelet releasates, enzymes, other than APC, may be affecting factor Va function and rendering it resistant to APC-catalyzed inactivation.

Similar mechanisms can be invoked to account for the substantial differences observed in the initial rate of APC-catalyzed platelet factor Va inactivation on thrombin-activated platelets from different platelet donors. Other hypotheses can be proposed as well. For example, since in every experiment both the platelet and platelet factor Va concentrations were nearly identical, the marked differences in inactivation rates may be due to different numbers of either factor Va and/or APC binding sites on the thrombin-activated platelets. In addition, even though platelet stores of protein S cannot be ruled out, previous results from our laboratory suggest that protein S has little, if any, effect on APC-catalyzed factor Va inactivation on platelets (26) .

In conclusion, our data demonstrate that the mechanism of platelet factor Va inactivation is not determined by the membrane to which it was bound (PCPS vesicles or thrombin-activated platelets). Furthermore, potential structural differences in platelet factor Va relative to plasma factor Va appear to define the extent to which the cofactor is cleaved initially at Arg first, resulting in a reduced rate of cofactor inactivation. This conclusion is consistent with our studies with a patient heterozygous for the Arg Gln mutation, as well as our previous results with plasma factor Va(22) , indicating that cleavage at Arg can occur in the absence of cleavage of Arg, but the rate of cofactor inactivation is slower. Consequently, a factor Va molecule that does not have a cleavage site at Arg (Factor Va) or is in a structural conformation such that APC cleavage at Arg is preferred over Arg, will be observed to be APC-resistant.


FOOTNOTES

*
This work was supported in part by Grants HL-P01-46703 (Project 4) (to P. B. T.) and Merit Award R37 HL34575 (to K. G. M.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, and the Department of Biochemistry, University of Vermont College of Medicine. 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.

§
Supported by Hemostasis and Thrombosis Training Grant HL 07594.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Vermont College of Medicine, Given Building C409, Burlington, VT 05405. Tel.: 802-656-1995; Fax: 802-862-8229.

(^1)
The abbreviations used are: APC, activated protein C; PS, L-alpha-phosphatidyl-L-serine; PC, L-alpha-phosphatidylcholine; RGDS, Arg-GlyAsp-Ser peptide; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; PAGE, polyacrylamide gel electrophoresis.


REFERENCES

  1. Mann, K. G., Jenny, R. J., and Krishnaswamy, S. (1988) Annu. Rev. Biochem. 57,915-956 [CrossRef][Medline] [Order article via Infotrieve]
  2. Mann, K. G., Nesheim, M. E., Chruch, W. R., Haley, P., and Krishnaswamy, S. (1990) Blood 76,1-16 [Abstract]
  3. Nesheim, M. E., and Mann, K. G. (1979) J. Biol. Chem. 254,1326-1334 [Abstract]
  4. Foster, W. B., Nesheim, M. E., and Mann, K. G. (1983) J. Biol. Chem. 258,13970-13977 [Abstract/Free Full Text]
  5. Monkovic, D. D., and Tracy, P. B. (1990) Biochemistry 29,1118-1128 [Medline] [Order article via Infotrieve]
  6. Monkovic, D., and Tracy, P. B. (1990) J. Biol. Chem. 265,17132-17140 [Abstract/Free Full Text]
  7. Osterud, B., Rapaport, S. I., and Lavine, K. K. (1977) Blood 49,819-834 [Abstract]
  8. Wencel-Drake, J. D., Dahlback, B., White, J. G., and Ginsberg, M. H. (1986) Blood 68,244-249 [Abstract]
  9. Guinto, E. R., and Esmon, C. T. (1984) J. Biol. Chem. 259,13986-13992 [Abstract/Free Full Text]
  10. Kane, W. H., Lindhout, M. J., Jackson, C. M., and Majerus, P. W. (1980) J. Biol. Chem. 255,1170-1174 [Free Full Text]
  11. Tracy, P. B., Nesheim, M. E., and Mann, K. G. (1981) J. Biol. Chem. 256,743-751 [Abstract/Free Full Text]
  12. Tracy, P. B., and Mann, K. G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,2380-2386 [Abstract]
  13. Nesheim, M. E., Taswell, J. B., and Mann, K. G. (1979) J. Biol. Chem. 254,10952-10962 [Abstract]
  14. Kalafatis, M., and Mann, K. G. (1993) J. Biol. Chem. 268,27246-27257 [Abstract/Free Full Text]
  15. Kalafatis, M., Rand, M. D., and Mann, K. G. (1994) J. Biol. Chem. 269,31869-31880 [Abstract/Free Full Text]
  16. Bertina, R. M., Koeleman, B. P. C., Koster, T., Rosendaal, F. R., Dirven, R. J., de Ronde, H., van der Velden, P. A., and Reitsman, P. H. (1994) Nature 369,64-67 [CrossRef][Medline] [Order article via Infotrieve]
  17. Griffin, J. H., Evatt, B., Wideman, C., and Fernandez, J. A. (1993) Blood 82,1989-1993 [Abstract]
  18. Koster, T., Rosendaal, F. R., de Ronde, H., Briet, E., Vandenbroucke, J. P., and Bertina, R. M. (1993) Lancet 342,1503-1507 [Medline] [Order article via Infotrieve]
  19. Svensson, P., and Dahlback, B. (1994) N. Engl. J. Med. 330,517-522 [Abstract/Free Full Text]
  20. Voorberg, J., Roelse, J., Koopman, R., Buller, H., Berendes, F., ten Cate, J. W., Mertens, K., and van Mourik, J. A. (1994) Lancet 343,1535-1536 [Medline] [Order article via Infotrieve]
  21. Dahlback, B., Carlsson, M., and Svensson, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,1004-1008 [Abstract]
  22. Kalafatis, M., Bertina, R. M., Rand, M. D., and Mann, K. G. (1995) J. Biol. Chem. 270,4053-4057 [Abstract/Free Full Text]
  23. Tracy, P. B., Eide, L. L., Bowie, E. J., and Mann, K. G. (1982) Blood 60,59-63 [Abstract]
  24. Nesheim, M. E., Nichols, W. L., Cole, T. L., Houston, J. G., Schenk, R. B., Mann, K. G., and Bowie, E. J. W. (1986) J. Clin. Invest. 77,405-415 [Medline] [Order article via Infotrieve]
  25. Tracy, P. B., Giles, A. R., Mann, K. G., Eide, L. L., Hoogendoorn, H., and Rivard, G. E. (1984) J. Clin. Invest. 74,1221-1228 [Medline] [Order article via Infotrieve]
  26. Solymoss, S., Tucker, M. M., and Tracy, P. B. (1988) J. Biol. Chem. 263,14884-14890 [Abstract/Free Full Text]
  27. Viskup, R. W., Tracy, P. B., and Mann, K. G. (1987) Blood 69,1188-1195 [Abstract]
  28. Kalafatis, M., Rand, M. D., Jenny, R. J., Ehrlich, Y. H., and Mann, K. G. (1993) Blood 81,704-719 [Abstract]
  29. Rand, M. D., Kalafatis, M., and Mann, K. G. (1994) Blood 83,2180-2190 [Abstract/Free Full Text]
  30. Nesheim, M. E., Prendergast, F. G., and Mann, K. G. (1979) Biochemistry 18,996-1003 [Medline] [Order article via Infotrieve]
  31. Barenholz, Y., Gibbs, D., Litmann, B. J., Goll, J., Thompson, E., and Carlson, F. D. (1977) Biochemistry 16,2806-2810 [Medline] [Order article via Infotrieve]
  32. Bloom, J. W., Nesheim, M. E., and Mann, K. G. (1979) Biochemistry 18,4419-4425 [Medline] [Order article via Infotrieve]
  33. Gomori, G. (1942) J. Lab. Clin. Med. 27,955-960
  34. Katzmann, J. A., Nesheim, M. E., Hibbard, L. S., and Mann, K. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,162-166 [Abstract]
  35. Nesheim, M. E., Katzmann, J. A., Tracy, P. B., and Mann, K. G. (1981) Methods Enzymol. 80,249-274 [Medline] [Order article via Infotrieve]
  36. Bajaj, S. P., Rapaport, S. I., and Prodanos, C. (1981) Prep. Biochem. 11,394-412
  37. Jesty, J., and Nemerson, J. (1976) Methods Enzymol. 45,95-107 [Medline] [Order article via Infotrieve]
  38. Owen, W. G., and Jackson, C. M. (1973) Thromb. Res. 3,705-714
  39. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  40. Mann, K. G. (1976) Methods Enzymol. 45,123-127 [Medline] [Order article via Infotrieve]
  41. Fenton, J. W., II, Landis, B. H., Walz, D. A., and Findlayson, J. S. (1977) in Chemistry and Biology of Thrombin (Lundblad, R. L., Fenton, J. W., II, and Mann, K. G., eds) pp. 43-70, Ann Arbor-Science Publishers Inc., Ann Arbor
  42. Kisiel, W., Ericsson, L. H., and Davie, E. W. (1976) Biochemistry 15,4893-4900 [Medline] [Order article via Infotrieve]
  43. Mustard, J. F., Perry, D. W., Ardlie, N. G., and Packham, M. A. (1972) Br. J. Haematol. 22,193-204 [Medline] [Order article via Infotrieve]
  44. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 [Abstract]
  45. Lawson, J. H., Kalafatis, M., Stram, S., and Mann, K. G. (1994) J. Biol. Chem. 269,23357-23366 [Abstract/Free Full Text]
  46. Church, W. R., Messier, T., Howard, P. R., Amiral, J., Meyer, D., and Mann, K. G. (1988) J. Biol. Chem. 263,6259-6267 [Abstract/Free Full Text]
  47. Kane, W. H., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,6800-6804 [Abstract]

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