(Received for publication, May 1, 1995)
From the
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 -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.
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 -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
-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
-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.
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
, 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. The eluate was dialyzed against 10 mM Tris, 100 mM NaCl before use.
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.
For some
studies, platelets were preincubated with purified intact or
F(ab`) 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.
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.
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.
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.
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
-granules (
1,
2,
3, and
4).
Multimerin (goat anti-rabbit gold-5; arrowheads) colocalizes
with factor V (goat anti-mouse gold-10; arrows) in two of the
-granules (
1 and
2), but as observed
in this inset, some
-granules contain factor V alone (
3) or multimerin alone (
4) (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
-granules supports the existence of factor V-multimerin complexes
during storage in resting platelets.
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.
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
-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 -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
-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`) 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. ()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 -granular proteins. (
)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 -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.