©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Factor V Is Complexed with Multimerin in Resting Platelet Lysates and Colocalizes with Multimerin in Platelet -Granules (*)

(Received for publication, May 1, 1995)

Catherine P. M. Hayward (1)(§) Emilia Furmaniak-Kazmierczak (2) Anne-Marie Cieutat (3) Jane C. Moore (1) Dorothy Ford Bainton (3) Michael E. Nesheim (2) John G. Kelton (1)(¶) Graham Côté (2)

From the  (1)Department of Pathology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada, the (2)Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada, and the (3)Department of Pathology, University of California, San Francisco, California 94143-0400

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Factor V stored in platelets is an important source of factor Va for the prothrombinase complex. Investigations of potential platelet factor Va-binding proteins, using factor Va light chain affinity chromatography, identified a disulfide-linked multimeric protein with a reduced mobility of 155 kDa in the column eluate. Immunodepletion and immunoblotting indicated that this protein was multimerin. Multimerin specifically bound factors V and Va and the isolated factor Va light chain, but not the heavy chain of factor Va. Factor V stored in platelets, but not plasma factor V, was found to be complexed with multimerin. Multimerin immunodepletion of resting platelet lysates was associated with the removal of factor V and the loss of factor V coagulant activity. Immunoelectron microscopic studies colocalized factor V with multimerin in the alpha-granules of resting platelets. With thrombin-induced platelet activation, we observed dissociation of factor Va-multimerin complexes, multimerin-independent membrane binding of factor Va, and prothrombinase activity that was not inhibitable by multimerin antibodies. This study indicates that platelet factor V is stored as a complex with multimerin and suggests a possible role for multimerin as a carrier protein for factor V stored in platelets.


INTRODUCTION

Factor Va plays a pivotal role in coagulation. On the platelet surface, factor Va participates in the assembly of the prothrombinase complex by enhancing the binding of factor Xa(1, 2, 3, 4, 5, 6, 7, 8, 9) and accelerating the conversion of prothrombin to thrombin(5, 7, 8, 9, 10, 11, 12) . Two potential sources of factor Va for coagulation exist: factor V in plasma and factor V stored in platelets(13) . Platelets contain 25% of the circulating factor V(13) . This source of factor V is thought to be important as patients who have severe reductions in plasma (but not platelet) levels of factor V due to inhibitors may not bleed(16) .

Factor V stored in platelets is synthesized by megakaryocytes(17, 18) and stored in the alpha-granules(14, 19) . With platelet stimulation and granule secretion, platelet factor V is activated to factor Va and binds to the platelet membrane(12, 14, 15) . Unlike plasma factor V, which is a 330-kDa protein(20, 21) , the platelet form of factor V ranges in size from 115 to 330 kDa(22) . Despite these differences, platelet factor V is biologically active as a cofactor for prothrombinase(4, 15) .

The binding of factor Va to the platelet surface occurs during platelet activation(23, 24, 25, 26) . The platelet membrane receptors for factor Va have not been determined, although negatively charged phospholipids have been implicated(1, 2, 9, 27, 28, 29) . Differences in the binding of the cofactors Va and VIIIa to activated platelets suggest that factor Va receptors, distinct from phospholipids, may exist (reviewed in (1) ). However, the role of platelet proteins in regulating platelet factor V activity has not yet been determined.

The intent of our study was to identify factor V/Va-binding proteins in platelets. Using affinity chromatography, we identified a complex, disulfide-linked multimeric platelet protein that specifically bound factors V and Va and the light chain of factor Va. Immunochemical analyses indicated that this protein was multimerin, a novel soluble multimeric platelet protein that is stored in alpha-granules and is expressed on the surface of activated platelets(30, 31, 32, 33) . Factor V in platelet lysates was found to be complexed with multimerin, and immunoelectron microscopy colocalized factor V with multimerin in platelet alpha-granules. These studies identify multimerin as a factor V/Va-binding protein and suggest a possible role for multimerin as a carrier protein for factor V stored in platelets.


MATERIALS AND METHODS

Antibodies

Antibodies used included monoclonal (JS-1) and rabbit polyclonal antibodies to multimerin(30, 31, 32) , monoclonal antibodies to factor V (anti-light chain antibodies V231, V237, and V241 and anti-heavy chain antibody V771; provided by Dr. C. Esmon), burro polyclonal anti-human factor V (provided by Drs. Kenneth G. Mann and Paula Tracy), a monoclonal antibody specific for the factor Va light chain (Hematologic Technologies, Essex Junction, VT), and a monoclonal antibody against human Fc receptor II (IV.3; Medarex, New Lebanon, NH). Control antibodies included monoclonal antibodies against PECAM-1 (provided by Dr. Alexey V. Mazurov) and against protein C inhibitor (PCI-174; provided by Dr. A. Giles), normal mouse IgG, and normal rabbit IgG. Secondary antibodies included alkaline phosphatase-conjugated rabbit anti-horse IgG (for burro antibodies), goat anti-mouse IgG and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., Bio/Can Scientific, Inc., Mississauga, Ontario, Canada), horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad Laboratories, Mississauga), and horseradish peroxidase-conjugated rabbit anti-horse IgG (Sigma). Nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad) and ECL (Amersham, Oakville, Ontario) were used for immunoblot detection.

Protein Preparation

Factor V was purified from bovine plasma and converted to factor Va with thrombin as described(34) . The light and heavy chains of bovine factor Va were separated by QAE-Sepharose chromatography (Sigma) in the presence of EDTA. Human prothrombin(35) , thrombin(36) , and factor Xa (37) were purified as described. Proteins were quantitated by the method of Bradford(38) , using bovine serum albumin as the standard, and by absorbance at 280 nm. For samples containing Triton X-100, proteins were quantitated by the method of Lowry et al.(39) . Radiolabeled factor Va (100 µg) was prepared using 1 mCi of NaI and two IODO-BEADs (Pierce) in 1 ml of 200 mM Tris, pH 7.4, 0.1 M NaCl. The iodinated factor Va was isolated using a Sephadex G-25 column and had a specific activity of 5000 cpm/ng. Bovine factor Va (84-86% amino acid identity to human factor V in the heavy and light chain domains) (40, 41, 42, 43) was used for affinity chromatography and direct binding studies because of the quantities required. For some experiments, active site-blocked factor Xa was prepared using 1,5-dansyl-L-glutamyl-L-glycyl-L-arginine (^1)chloromethyl ketone (Calbiochem).

Platelet Preparation

All studies were approved by the University Ethics Committee. Resting and thrombin-stimulated platelets from healthy volunteers were prepared as described(30) . For some studies, platelet lysates were prepared from freshly outdated platelet concentrates from the Canadian Red Cross(30) . Lysates were prepared using 1% Triton X-100, 1% Triton X-114, or 1% CHAPS (30) and lysing buffer containing protease inhibitors (20 mM Tris, 100 mM NaCl, pH 7.4, with 0.1 mM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 0.02 mg/ml soybean trypsin inhibitor, and 5 mMN-ethylmaleimide, with or without 10 mM EDTA). The cytoskeleton was removed from lysates by centrifugation (100,000 g, 0.5 h, 4 °C) before gel filtration or immunoprecipitation. Surface-radiolabeled resting and thrombin-activated platelets were prepared using I and lactoperoxidase(30) . For some experiments, surface-labeled platelets (suspended in lysing buffer with EDTA and protease inhibitors) were subjected to freeze-thaw lysis and sonication (3 30 s; Biosonik IV, Bronwill VWR Scientific) followed by separation of the membrane fraction by ultracentrifugation (100,000 g, 2 h, 4 °C). Supernatant and membrane fractions (solubilized in 1% Triton X-100; cytoskeleton removed) were used for immunoprecipitation studies.

Immunoprecipitation and Immunoblotting

Radioimmunoprecipitation and immunoblotting studies were performed as described(30, 31, 44, 45) . Proteins were analyzed by nonreduced and reduced SDS-PAGE followed by immunoblotting or silver staining(46) . For studies of factor V-multimerin complexes, platelet lysate (200 µl of 1 10^9 platelets/ml) was incubated with monoclonal antibodies (20 µg) to factor V (V241 and V771) or to multimerin or with normal mouse IgG. Complexes were captured using protein A beads (50 µl). For some investigations, the bound proteins were treated with thrombin (1 unit/ml, 10 min, 37 °C) followed by incubation in buffer with or without 10 mM EDTA. The bead eluates and supernatant fractions were analyzed by reduced SDS-PAGE and immunoblotting.

Preparation of Multimerin-immunodepleted Lysate

Platelet lysate (2 ml of 5 10^9 platelets/ml; prepared using lysing buffer containing 1% Triton X-100, 10 mM Tris, 150 mM NaCl with a 5 µg/ml concentration of the calpain inhibitor E-64 (Boehringer Mannheim, Laval, Quebec, Canada) and 5 mMN-ethylmaleimide) was immunodepleted of multimerin as follows. Six serial immunodepletions were performed (45 min, 4 °C) using 100 µl of JS-1-Sepharose, followed by three immunodepletions using polyclonal anti-multimerin bound to protein A beads (40 µg of antibody and 100 µl of protein A beads/depletion) and a final depletion using protein A beads (100 µl) without antisera to remove any unbound IgG-multimerin complexes. Control samples were sham-depleted in parallel using identical volumes of Sepharose beads and protein A beads without antibody. The starting material and sham-depleted and multimerin-depleted fractions were analyzed by immunoblotting and tested for factor V activity. Comparison of standard platelet lysates with those prepared using E-64 and N-ethylmaleimide as inhibitors indicated an identical profile of factor V in immunoblot analyses.

Gel Filtration Studies

Gel filtration experiments were used to study endogenous platelet factor V and to evaluate complex formation between factor V and platelet proteins. Experiments were carried out using a 1.5 60-cm column of either Bio-Gel A-5m (Bio-Rad) or Ultrogel AcA44 (Spectrum Medical Industries Inc., Los Angeles, CA) equilibrated in 20 mM Tris, pH 7.4, 0.01% Triton X-100 containing either 0.1 or 1 M NaCl (final salt concentrations of 0.12 and 1.02 M, respectively). Platelet lysate (1 ml of 2 10 platelets/ml in lysing buffer containing 1% Triton X-100 without EDTA) was applied to the column. In some experiments, I-labeled factor Va (40 ng) was added to the buffer, platelet lysate, the aqueous and detergent phases of a Triton X-114 platelet lysate(31) , or purified multimerin (5 µg), and the radioactivity of the fractions (1 ml) was quantitated. Immunoblot analyses were used to determine which fractions contained factor V and multimerin. Purified myosin (660 kDa) and plasma factor V (330 kDa; 0.12 and 1.02 M salt separations) were run for comparison.

Affinity Chromatography

Affinity columns were prepared by incubating BSA (5 mg) or the isolated bovine factor Va light chain (1 mg) with Affi-Gel 15 (Bio-Rad) according to the manufacturer's instructions, and remaining active esters on the Affi-Gel were blocked (0.2 M Tris, pH 8.0). Protein assays indicated that >95% of the added protein was coupled to the resin.

Platelet extracts used for factor Va affinity chromatography were prepared by lysing fresh human platelets (1 10) at 4 °C in lysing buffer (20 mM Tris, 0.1 M NaCl, 2 mM CaCl(2), pH 7.4, with 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 2 mM phenylmethylsulfonyl fluoride) containing either 1% Triton X-100 or 1% CHAPS. Insoluble material was removed (100,000 g, 30 min), and the supernatant was incubated with 5 ml of BSA-Affi-Gel (1 h). The unbound fraction was incubated with 1 ml of factor Va light chain-Affi-Gel or BSA-Affi-Gel. The columns were washed with lysing buffer containing 0.1% Triton X-100 or 0.1% CHAPS, and bound proteins were eluted with lysing buffer containing 1 M NaCl and 0.1% detergent and concentrated 10-fold (Amicon Centricon-30) prior to analysis. Identical volumes of the eluates from the factor Va light chain and control BSA columns were used for SDS-PAGE and immunoblot analyses.

For some experiments, monoclonal anti-multimerin linked to Sepharose was used to affinity purify multimerin from resting platelet lysates (30) . The affinity column was washed with 10 column volumes of 1 M NaCl prior to elution of the bound multimerin with 3 M MgCl(2). The eluate was dialyzed against 10 mM Tris, 100 mM NaCl before use.

Binding Studies

Radiolabeled bovine factor Va (40 ng) was incubated with monoclonal antibody-purified multimerin (5 µg) or with the multimerin in the factor Va light chain eluate (20 µl). 20 µg of JS-1 and 50 µl of protein A beads were used to separate bound from free proteins. 100-Fold molar excesses of unlabeled intact factors V, Va, and Xa were tested for their ability to compete with labeled factor Va for binding to multimerin. To evaluate nonspecific binding, the binding of labeled factor Va to the capture beads in the absence of added multimerin was measured.

For other studies, multimerin (20 µl of the factor Va light chain eluate) was incubated with 10-µg quantities of purified bovine factor V or Va or factor Va light or heavy chain. Bound proteins were captured using monoclonal antibodies (V241 for factors V and Va and the factor Va light chain, V771 for the factor Va heavy chain, and PCI-174 as a control for nonspecific multimerin binding) and analyzed for multimerin content by SDS-PAGE and immunoblotting with monoclonal anti-multimerin.

Immunoelectron Microscopic Studies

Immunoelectron microscopic studies of frozen thin sections of resting and thrombin-stimulated platelets (fixed 5 min after activation with 2 units/ml thrombin in Tyrode's buffer containing 1 mM calcium) were performed as described(32) . Briefly, platelets were fixed with paraformaldehyde (4% in 0.1 M phosphate buffer, 3 h, 4 °C) and embedded in 2.3 M sucrose. Frozen thin sections were prepared from the cell block. Single and double labeling immunochemistries were performed as described(32, 47, 48, 49) . For double labeling studies, a pool of monoclonal anti-factor V antibodies (1:1 mixture of V231 and V241 and 1:1 mixture of V241 and V237; preparations used undiluted) and polyclonal antibodies to multimerin (1:25 dilution) were used. Secondary antibodies conjugated to different-sized gold particles (goat anti-mouse gold-10 and goat anti-rabbit gold-5, Amersham Corp.) were used for detection of factor V and multimerin(32) . Controls included primary incubations with normal mouse and normal rabbit sera and omission of the primary antibody.

Factor V Activity Assays

The factor V coagulant assays were used to determine the factor V biologic activity of sham- and multimerin-immunodepleted platelet lysates, prepared using the calpain inhibitors E-64 and N-ethylmaleimide to prevent degradation of platelet factor V by endogenous platelet calpains. Factor V activity was measured using a two-stage assay and factor V-deficient plasma prepared from normal human plasma(34, 50) . Briefly, control and sham- and multimerin-depleted lysates were incubated (1 min, room temperature) with human thrombin (20 units/ml) and then diluted 1:50 in 20 mM Tris-HCl, 0.15 M NaCl, pH 7.4. The diluted samples were assayed for factor V activity using factor V-deficient plasma and rabbit brain thromboplastin (Organon Teknika Corp., Durham, NC). Clotting times were compared with a standard curve using normal pooled plasma as the source of factor V. Dilutions of normal plasma were made in 20 mM Tris-HCl, 0.15 M NaCl, pH 7.4, containing the same final Triton X-100 concentration as the platelet lysates.

Prothrombinase Activity Assays

Prothrombinase assays were performed using dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA; 4 µM); freshly isolated human platelets (1 10^8; activated for 1 min with 1 unit/ml human thrombin in 20 mM Tris, 0.15 M NaCl, 2 mM CaCl(2), pH 7.5; final reaction volume 1.6 ml); and purified prothrombin (1.4 µM), factor Xa (5 nM), and factor Va (2 nM). The fluorescent thrombin inhibitor DAPA was prepared as described(52) . Factor Xa was added to initiate the reaction after all other components had been equilibrated to 22 °C in a thermostatted quartz cuvette with a microstirrer. Once all components were added, stirring was discontinued. The rate of conversion of prothrombin to thrombin was measured by monitoring the appearance of a DAPA-thrombin complex using an MPF-66 fluorescence spectrophotometer (Perkin-Elmer Canada Ltd.). An excitation wavelength of 355 nm, an emission wavelength of 545 nm, and a 430-nm cutoff filter were used. Direct plots of DAPA-thrombin formation (4, 8) were used to calculate the initial rate of thrombin generation.

For some studies, platelets were preincubated with purified intact or F(ab`)(2) monoclonal or polyclonal anti-multimerin. To prevent platelet activation via Fc receptor II, monoclonal antibody IV.3 was used to pretreat platelets in studies employing intact antibodies(53) . Control antibodies included preimmune rabbit IgG and normal mouse IgG.


RESULTS

Studies Investigating the Factor V/Va-Binding Proteins in Platelets

Bovine factor Va light chain affinity chromatography was used to identify potential platelet factor V/Va-binding proteins. Silver stain analyses identified a major band, with a reduced molecular mass of 155 kDa, in the eluate from the factor Va light chain column (Fig. 1, silverstain, arrow), but not in the eluate from the BSA column. Analyses using nonreduced SDS-PAGE indicated that the 155-kDa protein was a large, disulfide-linked multimeric protein (Fig. 1, silverstain, arrow). The mobility of the major protein in the factor Va light chain eluate resembled multimerin, a large, soluble, disulfide-linked multimeric protein stored in platelets(30, 31, 33) . Immunoblot analyses using both monoclonal (Fig. 1, multimerinimmunoblot) and polyclonal anti-multimerin antibodies confirmed the protein's identity as multimerin. No multimerin was detected in the eluate from the BSA column. Immunodepletion using monoclonal anti-multimerin removed all of the 155-kDa protein from the factor Va light chain eluate.


Figure 1: Analysis of the platelet factor Va light chain-binding proteins. Eluates (equal volumes) from the bovine factor Va light chain (fVaLC) affinity column and the control BSA column were compared using reduced and nonreduced SDS-PAGE and silver staining. Nonreduced lanes are indicated. A major band at 155 kDa (reduced) was observed in the eluate from the factor Va light chain column. Nonreduced separation indicated that the p155 protein was a multimeric protein, linked by interchain disulfide bonds. Additional bands of lower molecular mass were also observed in the factor Va light chain eluate. Immunoblot analyses of the eluates using monoclonal anti-multimerin indicated that the p155 protein was multimerin.



Investigations of Factor V-Multimerin Binding

The binding of multimerin to the factor Va light chain affinity column suggested that multimerin could be a specific factor V/Va-binding protein. Competitive binding studies using I-labeled bovine factor Va were therefore performed to study the interaction of factor V with multimerin. Radiolabeled factor Va bound to multimerin, and this interaction was inhibited by a 100-fold molar excess of unlabeled intact factor V or Va, but not by active site-blocked factor Xa (Fig. 2A), indicating that multimerin is a specific factor V/Va-binding protein.


Figure 2: Binding studies of multimerin and factor V. A, competitive binding studies of I-labeled factor Va and purified multimerin, measured in the absence or presence of a 100-fold molar excess of unlabeled factor V or Va or active site-blocked factor Xa. The data represent the percentage of counts bound (cpm test/cpm in the absence of competing proteins) using factor Va affinity-purified (blackbars) and monoclonal antibody-purified (whitebars) multimerin. The controlbar indicates the binding of labeled factor Va to the antibody capture beads in the absence of multimerin. B, immunoprecipitation studies comparing the binding of multimerin to bovine factor V (laneV), factor Va (laneVa), the light chain of factor Va (laneLC), the heavy chain of factor Va (laneHC), or buffer alone (laneC). The bound proteins were captured (using monoclonal antibodies to factor V or an irrelevant monoclonal antibody (lane C)) and analyzed by SDS-PAGE and immunoblotting with monoclonal anti-multimerin. An equivalent amount of multimerin (laneM) is shown for comparison. Bands attributable to immunoprecipitating mouse IgG (heavy and light chains, 53 and 23 kDa, respectively) were noted in the eluates (not shown).



Purified multimerin, bovine factors V and Va, and the isolated heavy and light chain domains were studied to determine the factor V domains involved in binding to multimerin. Multimerin bound to intact bovine factors V and Va and the factor Va light chain, but not to the factor Va heavy chain (Fig. 2B). Only trace quantities of multimerin were detected in the eluate of the control irrelevant monoclonal antibody beads. These data indicate that the light chain domain of factor V is involved in multimerin binding.

Investigations of Factor V-Multimerin Complexes

Following the demonstration that multimerin was a specific factor V/Va-binding protein, we investigated whether factor V stored in platelets is complexed with multimerin. Immunoprecipitates of platelet factor V were found to contain multimerin in addition to factor V, and similarly, platelet multimerin immunoprecipitates contained factor V in addition to multimerin (Fig. 3A). Control immunoprecipitates (precipitating antibody, normal mouse IgG) did not contain factor V or multimerin. Analyses of plasma indicated that plasma factor V was not complexed with multimerin, and no multimerin was detectable in the plasma (Fig. 3A).


Figure 3: Investigations of factor V-multimerin complexes in platelet lysates and plasma. A, plasma and platelet lysate (prepared from resting platelets) were immunoprecipitated using control normal mouse IgG, monoclonal anti-multimerin, and monoclonal anti-factor V antibodies (V241 + V771). Immunoprecipitates were eluted and analyzed using 7% SDS-PAGE (reduced) and immunoblotting with monoclonal anti-multimerin and polyclonal anti-factor V. Bands attributable to immunoprecipitating mouse IgG (heavy and light chains, 53 and 23 kDa, respectively) were noted in the eluates (not shown). B, immunodepleted platelet lysate was prepared using sham beads or multimerin antibody beads. The starting material and immunodepleted lysates were analyzed using reduced SDS-PAGE and immunoblotting with polyclonal anti-factor V and monoclonal anti-multimerin. These studies indicate that factor V and multimerin are present as a complex in platelet lysate, but not in plasma.



Multimerin immunodepletions were performed to determine the proportion of platelet factor V that was complexed with multimerin. Tests of the sham-depleted samples indicated an 50% reduction in the factor V activity of the platelet lysate (Table 1) and a corresponding loss of immunologically detectable factor V and multimerin (Fig. 3B), due to sample dilution. In contrast, multimerin immunodepletion removed all of the detectable platelet factor V activity (Table 1) with a corresponding loss of immunologically detectable factor V and multimerin (Fig. 3B). These findings indicate that platelet factor V is complexed with multimerin and that factor V bound to multimerin is biologically active.



Because the studies of factor Va-multimerin binding had been performed using bovine factor Va, we investigated whether human platelet factor Va and its light chain bound to multimerin (Fig. 4). Affinity-purified factor V-multimerin complexes (from resting platelet lysates) were incubated with thrombin, followed by SDS-PAGE and immunoblot analyses. No change in multimerin subunit mobility was observed after thrombin treatment, and the multimerin remained bound to the JS-1 affinity beads. Immunoblotting with polyclonal anti-factor V antibodies demonstrated cleavage of the bound factor V to Va following treatment with thrombin (Fig. 4)(54, 55, 56) . In the presence of EDTA, the factor Va heavy chain (but not the light chain) was eluted from the factor Va-multimerin complex (Fig. 4). These data indicate that multimerin binds both human and bovine factors V and Va and the factor Va light chain and that human factor Va remains bound to multimerin following treatment of factor V-multimerin complexes with thrombin.


Figure 4: Studies of human factor Va and multimerin. Affinity chromatography using monoclonal anti-multimerin was used to isolate factor V-multimerin complexes from resting platelet lysate. Samples were treated with buffer or thrombin, and then bound and released proteins were separated by centrifugation. The washed beads were incubated in buffer, with or without 10 mM EDTA, to allow dissociation of the factor Va heavy and light chains (HC and LC, respectively). The supernatants and bead eluates were analyzed by SDS-PAGE and immunoblotting with polyclonal anti-human factor V. This study indicates that human platelet factors V and Va and the factor Va light chain bind to multimerin and that factor Va remains bound to multimerin following treatment of the complex with thrombin. EDTA dissociated the factor Va heavy chain from the factor Va-multimerin complex.



Next, gel filtration studies were performed to study complex formation between factor V and multimerin. Multimerin was found in the high molecular mass fractions when platelet proteins were separated using 0.12 or 1.02 M salt (Fig. 5), consistent with its large nonreduced size. As shown in Fig. 5, platelet factor V comigrated with multimerin at 0.12 M salt and as a smaller protein in the presence of 1.02 M salt. 5). Although mobility shifts were observed with endogenous platelet factor V, purified bovine factor V had an identical migration (peak, fraction 22) in 0.12 or 1.02 M salt. Immunoprecipitation-immunoblot analyses of the high molecular mass fractions indicated that factor V and multimerin were complexed.


Figure 5: Gel filtration studies of factor V and multimerin in platelet lysates. Gel filtration studies of platelet proteins were performed using a Bio-Gel A-5m column in the presence of 0.12 or 1.02 M salt. The factor V and multimerin content of the 1-ml fractions was analyzed using immunoblotting with polyclonal anti-factor V and monoclonal anti-multimerin. Factor V migrated as a large protein complex that was dissociable by high salt concentrations. Multimerin was identified in the high molecular mass fractions that contained endogenous platelet factor V (0.12 M salt). Muscle myosin (660 kDa; peak, fraction 16) and purified bovine plasma factor V (330 kDa; peak, fraction 22) were run as controls. The mobility of bovine factor V was identical in 0.12 and 1.02 M salt.



Similar to the mobility of endogenous platelet factor V, radiolabeled bovine factors V and Va, added to human platelet lysate, migrated as a high molecular mass complex (Fig. 6, upperpanel), which was dissociated by high salt concentrations. Immunoblot analyses indicated that the peak factor Va binding activity was found in the fractions containing multimerin (Fig. 6, upper panel, inset). Comparison of the aqueous and detergent fractions of Triton X-114 platelet lysates indicated that the majority of the factor Va binding activity of platelet lysate was associated with the soluble protein fraction, which contained most of the multimerin.


Figure 6: Contribution of multimerin to the factor V binding properties of platelet lysates. AcA44 gel filtration studies were performed to compare the distribution of multimerin (insets; immunoblot analyses using monoclonal anti-multimerin) with the fractions containing I-labeled bovine factor Va binding activity. The mobility of labeled factor Va added to platelet lysate (upper panel) or to affinity-purified multimerin (lower panel), in the presence of 0.12 or 1.02 M salt, is shown. The radioactivities of the 1-ml fractions are indicated, and the mobility of I-labeled factor Va in buffer (0.12 M salt; indicated as buffer) is shown for comparison. These studies indicate that multimerin has factor Va binding activity.



Purified factor Va and multimerin were studied to determine if similar complexes could form in the absence of other platelet proteins. Affinity-purified multimerin complexed with radiolabeled bovine factor Va at physiologic salt concentrations, and this complex was dissociated at high salt concentrations (Fig. 6, lowerpanel). Immunoblotting (Fig. 6, lowerpanel, inset) confirmed the presence of multimerin in the gel filtration fractions that contained the factor Va complexes. These data confirm that endogenous platelet factor V exists as a complexed protein and also indicate that factor Va-multimerin complexes can form in the presence and absence of other platelet proteins.

Immunoelectron Microscopic Studies Investigating the Location of Factor V and Multimerin in Resting and Activated Platelets

The distribution of multimerin and factor V in frozen thin sections of platelets was studied to determine if multimerin and factor V were complexed in intact resting platelets or on the membrane of thrombin-activated platelets. Both factor V (14, 19) and multimerin (32) are known to be stored in the alpha-granules of human platelets. As previously reported, multimerin was found in the alpha-granules of resting platelets in an eccentric location (32) (Fig. 7A, arrowheads). Using monoclonal antibodies to factor V and immunogold-conjugated secondary antibodies, factor V was also found to be stored in alpha-granules in a similar eccentric distribution (Fig. 7A, arrows). Using double immunolabeling and quantitation of 120 labeled alpha-granules in resting platelets, factor V and multimerin colocalized in the 36 alpha-granules that contained label for both proteins (30%) (Fig. 7A, alpha1 and alpha2). The remaining 84 alpha-granules evaluated showed only single protein immunostaining. Factor V alone was found in 23 alpha-granules (20%) (Fig. 7A, alpha3), and multimerin alone was found in 61 alpha-granules (50%) (alpha4). The eccentric position of factor V and multimerin and the thinness of the sections may have contributed to the failure to visualize both proteins in every section of the alpha-granules.


Figure 7: Double immunogold labeling of multimerin and factor V in frozen thin sections of resting and activated platelets. A, a portion of a resting platelet containing four alpha-granules (alpha1, alpha2, alpha3, and alpha4). Multimerin (goat anti-rabbit gold-5; arrowheads) colocalizes with factor V (goat anti-mouse gold-10; arrows) in two of the alpha-granules (alpha1 and alpha2), but as observed in this inset, some alpha-granules contain factor V alone (alpha3) or multimerin alone (alpha4) (magnification 83,000). B, a portion of an activated platelet labeled using polyclonal antibodies against multimerin (m) and goat anti-rabbit gold-10 (arrowheads) and monoclonal antibodies against factor V (fV) and goat anti-mouse gold-5 (arrows). Both proteins are found in the large vacuoles of the canalicular system (sccs) and on the external plasma membrane (pm). While factor V is observed both in the SCCS and associated with smaller vacuoles (sv), multimerin is seen mainly in the larger vacuoles of the SCCS. In this field, only factor V is seen on the plasma membrane (arrows).



Immunoelectron microscopic studies of thrombin-activated platelets demonstrated the redistribution of factor V (Fig. 7B, arrows) and multimerin (arrowheads). Following activation, factor V was found within the surface-connected canalicular system (SCCS) and on the plasma membrane, whereas multimerin was found predominately in the SCCS. Quantitation of the immunogold particles associated with the plasma membrane identified 10% colocalization of factor V with multimerin (215 immunogold particles evaluated). The remainder of the activated plasma membrane showed isolated labeling for factor V (75%) or for multimerin (15%). The SCCS showed mainly multimerin immunostaining with occasional areas where multimerin and factor V were located in close proximity, but also other areas where the two proteins were widely dispersed (Fig. 7B). Additional smaller vacuoles that labeled for factor V only were observed (Fig. 7B).

The controls section of resting and activated platelets, processed with nonimmune serums or with omission of primary antibody, did not show significant background labeling. The codistribution of multimerin and factor V in platelet alpha-granules supports the existence of factor V-multimerin complexes during storage in resting platelets.

Investigation of Factor V-Multimerin Complexes on the Surface of Activated Platelets

The immunoelectron microscopic studies of activated platelet membranes indicated 10% colocalization of factor V with multimerin. To study further the association of multimerin and factor Va on activated platelets, radioimmunoprecipitation studies of I surface-radiolabeled, thrombin-stimulated platelets were performed.

Both multimerin and factor Va were expressed on the surface of thrombin-activated platelets (Fig. 8). However, only small amounts of a labeled p155 protein were detected in the reduced factor Va radioimmunoprecipitates (Fig. 8A). Multimerin immunodepletion of the lysate confirmed that the 155-kDa protein (reduced molecular mass) in the factor Va immunoprecipitates was multimerin (Fig. 8A). Multimerin immunodepletion had little impact on the quantity of factor Va detected (Fig. 8A), indicating that most of the factor Va was not complexed with multimerin. Only small quantities of radiolabeled multimerin and factor V were detected in immunoprecipitates prepared from surface-radiolabeled resting platelets.


Figure 8: Radioimmunoprecipitation studies of multimerin and factor Va expressed on the surface of thrombin-activated platelets. Radioimmunoprecipitation analyses of I surface-radiolabeled, thrombin-stimulated platelets were performed using control normal mouse IgG, monoclonal anti-multimerin, burro anti-factor V (factorV), a monoclonal antibody specific for the factor Va light chain (factorVaLC), and a monoclonal antibody to the membrane protein PECAM-1. The cell lysis and immunoprecipitation conditions were identical to those described in the legend to Fig. 3. A, the immunoprecipitates from control and sham-depleted (C) and multimerin-depleted (M) lysates were compared using reduced SDS-PAGE and autoradiography. Multimerin immunodepletion removed the small amount of multimerin (155 kDa) coprecipitated by the antibody to factor Va. Little factor Va light chain (doublet, 75 kDa) was detected in the multimerin immunoprecipitate. B, radioimmunoprecipitates were prepared from the soluble and membrane fractions. The membrane fraction contained PECAM-1, an integral membrane protein, and the factor Va light chain (LC). The factor V heavy chain (HC) was found in the soluble protein fraction. Multimerin was found mainly in the soluble protein fraction. These studies indicate that most of the multimerin and factor Va bound to thrombin-activated platelets are not complexed and that there is multimerin-independent binding of factor Va to the membrane of activated platelets.



In studies of the isolated membrane and soluble protein fractions of surface-labeled, thrombin-activated platelets (Fig. 8B), the factor Va light chain was found in the membrane fraction. Similar to the immunoelectron microscopic studies of activated platelets, multimerin was found mainly in the soluble protein fraction, with smaller amounts recovered from the membrane (Fig. 8B). A radiolabeled protein with the mobility of the factor V heavy chain was recovered from the soluble protein fraction with the polyclonal antibody to factor V (Fig. 8B). These results support the findings of the electron microscopic studies and indicate that most of the factor Va associated with thrombin-activated platelets is not bound to multimerin.

Studies Investigating the Effect of Multimerin Antibodies on the Prothrombinase Complex

Although the factor V activity of resting platelet lysates was associated with multimerin, studies of thrombin-activated platelets indicated that most of the factor Va was not bound to multimerin. Because small amounts of factor Va-multimerin complexes could be important for factor Va procoagulant function, we investigated a possible influence of multimerin on the rate of prothrombin conversion. The F(ab`)(2) fragment of monoclonal anti-multimerin and polyclonal anti-multimerin had no effect on the rate of prothrombin conversion to thrombin by thrombin-activated platelets. Intact monoclonal antibodies to multimerin accelerated the rate of prothrombin conversion, but this effect was prevented by preincubating platelets with an antibody (IV.3) that blocks the activation of platelets by Fc receptor II.


DISCUSSION

Differences in the platelet and plasma forms of factor V (22) are known to exist; however, the explanation(s) for these differences is not yet known. Despite these differences, both plasma factor V and platelet factor V are capable of generating factor Va and supporting coagulation. Because platelets contain a significant proportion of the circulating factor V(22) , this source of factor V may be important for prothrombinase assembly on activated platelets in the hemostatic plug. Due to differences in the procoagulant and factor Va binding properties of platelets and phospholipid vesicles, specific platelet receptors for factor Va have been postulated to exist(1) . However, no platelet receptors or binding proteins for factor Va have been identified. The purpose of the studies contained in this report was to determine if platelet factor V/Va-binding protein(s) exist and to study their interaction with factor V.

As a first step, we used the immobilized factor Va light chain to screen platelet lysates for potential factor V/Va-binding proteins. The column eluate contained a major protein (155 kDa, reduced) with disulfide-linked multimeric structure. The migration characteristics of this protein suggested that it could be multimerin, a large, complex multimeric protein of unknown function that is stored in platelets and expressed on their surface following activation(30, 31, 32, 33) . Immunodepletion and immunoblot analyses confirmed the identity of the protein as multimerin (Fig. 1).

We next studied the binding of multimerin and factor V. Competition binding studies (Fig. 2) using bovine factor V indicated that the interaction between factor Va and multimerin was due to specific binding, and it was inhibited by intact factors V and Va. Multimerin also bound to the isolated factor Va light chain. Similarly, multimerin binding was observed with human factors V and Va and the factor Va light chain (Fig. 3-5).

Multimerin is stored in platelets and is expressed only on the external membrane following platelet activation(30, 31, 32, 33) . This led us to investigate three different postulates for multimerin's interaction with factor V: it could serve as a receptor for factor Va on activated platelets, it could function as a component of the prothrombinase complex, or it could function as a carrier protein. Because both proteins are stored in platelet alpha-granules(14, 19, 32) , we first investigated whether platelet factor V and multimerin were complexed in resting platelets.

Initial studies using lysates prepared from resting platelets indicated that platelet factor V was noncovalently complexed with multimerin. Immunodepletion of platelet lysates using antisera specific for multimerin was associated with the removal of platelet factor V and the loss of all measurable factor V activity ( Fig. 3and Table 1). In contrast, plasma factor V was not complexed with multimerin, consistent with previous reports that plasma factor V is not complexed with other proteins (20) and that multimerin is not found in plasma(30, 33) .

Gel filtration studies confirmed comigration of platelet factor V with multimerin (Fig. 5). Similar high molecular mass complexes were observed when radiolabeled factor Va was added to multimerin in the presence and absence of other platelet proteins (Fig. 6).

Because the lysis of platelets by detergents could potentially lead to complex formation between proteins that are not associated in intact platelets, we studied the distribution of multimerin and factor V in intact platelets. In our previous studies, we demonstrated that multimerin is stored in alpha-granules in an unusual eccentric position (32) , colocalizing with von Willebrand factor(57, 58, 59) . In the experiments described in this report, we demonstrated that factor V colocalizes with multimerin in platelet alpha-granules (Fig. 7A).

Multimerin and factor V were determined to be complexed in resting platelet lysates and stored in similar locations; however, differences were observed in the distribution of these proteins following platelet activation (Fig. 7B). Factor Va was found on the membrane of activated platelets, whereas multimerin was found mainly in the open canalicular system. Studies of surface-radiolabeled platelets indicated that only small amounts of factor Va bound to activated platelets were associated with multimerin, and multimerin immunodepletion failed to deplete activated platelet lysates of factor Va (Fig. 8). Although factor Va remained bound to multimerin when isolated factor V-multimerin complexes were treated with thrombin, only small amounts of factor Va remained bound to multimerin when whole platelets were treated with thrombin. These results suggest that the process of platelet activation is important for dissociation of factor Va-multimerin complexes. Possible explanations for these differences include the exposure of higher affinity factor Va-binding sites or alterations in factor V and/or multimerin by factors other than thrombin during platelet activation.

Although the number of binding sites on activated platelets for factor Va and for the multimerin monoclonal antibody JS-1 is similar(11, 30) , our data indicate that the majority of the factor V released by thrombin-activated platelets binds to the platelet membrane independent of multimerin. Because small amounts of factor Va-multimerin complexes could nonetheless be important for the function of factor Va on activated platelets, the effect of multimerin antisera on the platelet prothrombinase complex was investigated. Neither the F(ab`)(2) fragment of the multimerin monoclonal antibody JS-1 nor the polyclonal antibodies to multimerin altered the rate of thrombin generation on thrombin-stimulated platelets. These findings indicate that multimerin is not the major platelet factor Va membrane receptor and suggest that multimerin is not part of the prothrombinase complex.

The existence of factor V-multimerin complexes in resting platelets suggests that multimerin functions as a carrier protein for factor V, analogous to the role of von Willebrand factor in binding factor VIII (60) . Factors V and VIII are unique coagulation cofactors with many similarities(61) . In plasma, factor VIII is bound to its carrier protein, von Willebrand factor(60) . The investigations contained in this report indicate that, similar to factor VIII, factor V also forms complexes with a large, complex multimeric protein. However, unlike factor VIII and von Willebrand factor, factor V and multimerin exist as a complex in platelets, but not in plasma. The reason for this difference may relate to the regulation of factor V stored in platelets and the absence of multimerin in plasma. Despite similarities between factors V and VIII, our studies indicate that there is no significant homology between the multimeric proteins that bind these cofactors. (^2)Although the biologically active factor V stored in platelets was found to be complexed with multimerin, the precise role of multimerin in regulating platelet factor V was not determined.

Plasma factor V and platelet factor V differ in their cells of origin, storage, and proteolytic processing, indicating differences in their regulation. Indeed, an autosomal dominant bleeding disorder, factor V (Quebec), has been described that is associated with a marked deficiency of functional platelet factor V despite adequate levels of plasma factor V(51) . Platelets from these individuals support prothrombinase normally when exogenous factor V is supplied, and their platelets possess normal numbers of factor Xa-binding sites(51) . Because these individuals have a severe defect in platelet (but not plasma) factor V function, we wondered if multimerin abnormalities could contribute to the platelet factor V abnormalities in this disorder. Our investigations indicate that these individuals are deficient in platelet multimerin with normal levels of other alpha-granular proteins. (^3)These findings suggest that the factor V binding function of multimerin may be important for the processing, stabilization, and/or storage of platelet factor V. As the platelets from these individuals support factor Va function normally when exogenous factor Va is supplied(51) , these data also indicate that multimerin is not directly involved in factor Va procoagulant function on activated platelets.

Our investigations identify multimerin as a specific factor V/Va-binding protein. Multimerin and factor V are complexed in resting platelets, but not in plasma. Factor V bound to multimerin is biologically active, and the removal of multimerin from resting platelet lysates results in the loss of detectable factor V activity. Although multimerin binds both human and bovine factor Va, the majority of the platelet factor Va expressed on the external membrane of thrombin-stimulated platelets is not bound to multimerin. Based on these observations, we postulate that multimerin may serve as a carrier protein for factor V during storage in platelet alpha-granules. Further investigations are required to define the precise function of multimerin in regulating platelet factor V and to identify the factors that cause dissociation of factor Va-multimerin complexes when platelets are activated by thrombin.


FOOTNOTES

*
This study was supported by grants from the Medical Research Council of Canada (to J. G. K.) and the Heart and Stroke Foundation of Ontario (to G. C. and M. E. N.) and by National Institutes of Health Grant HL-31610 (to D. F. B.). 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.

§
Centennial Fellow of the Medical Research Council of Canada. To whom correspondence should be addressed: HSC Rm. 2N32, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-521-2100 (ext. 3373); Fax: 905-577-0198.

Career Investigator of the Heart and Stroke Foundation of Ontario.

(^1)
The abbreviations used are: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; SCCS, surface-connected canalicular system.

(^2)
Hayward, C. P. M., Hassell, J. A., Denomme, G. A., Rachubinski, R. A., Brown, C., and Kelton, J. G.(1995) J. Biol. Chem.270, in press.

(^3)
C. P. M. Hayward, G. E. Rivard, J. C. Moore, J. Drouin, and J. G. Kelton, manuscript in preparation.


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