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(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
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
Factor V stored in platelets
is synthesized by megakaryocytes(17, 18) and stored
in the 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
Platelet extracts used for factor Va affinity chromatography were
prepared by lysing fresh human platelets (1 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
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`)
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
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
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
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
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 ( 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
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
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
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 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`) 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. ( 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 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
Volume 270,
Number 33,
Issue of August 18, pp. 19217-19224, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Granules (*)
-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.
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) .
-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) .-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.
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 (
)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
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
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. 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.
. 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.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
; activated for 1 min with 1 unit/ml human thrombin in 20
mM Tris, 0.15 M NaCl, 2 mM CaCl,
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. 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.
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.
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.
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).
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).
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.
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.
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 -granules of human platelets. As previously
reported, multimerin was found in the -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 -granules in a
similar eccentric distribution (Fig. 7A, arrows). Using double immunolabeling and quantitation of 120
labeled -granules in resting platelets, factor V and multimerin
colocalized in the 36 -granules that contained label for both
proteins (30%) (Fig. 7A,
1 and 2). The remaining 84 -granules evaluated showed only
single protein immunostaining. Factor V alone was found in 23
-granules (20%) (Fig. 7A,
3),
and multimerin alone was found in 61 -granules (50%) (
4). 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 -granules.
-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).
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).
-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.
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.
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`) 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.
-granules(14, 19, 32) , we first
investigated whether platelet factor V and multimerin were complexed in
resting platelets.-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). 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. )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.-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.-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.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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