(Received for publication, May 23, 1995)
From the
The inactivation of human platelet factor Va by activated
protein C (APC) was analyzed by functional assessment of cofactor
activity and Western blotting analysis to visualize the factor Va
fragments accompanying proteolysis. Platelets were treated with
thrombin to facilitate both their activation as well as the release and
further activation of platelet factor Va, followed by APC addition. The
rates of inactivation were donor-dependent such that 15-60% of
the initial cofactor activity was lost within 5 min of APC addition
with as much as 10-20% of the activity still remaining after 2 h
of incubation. Western blot analysis using a monoclonal antibody that
recognizes an epitope between amino acid residues 307 and 506 of the
factor V molecule suggested that the factor Va activity resistant to
APC inactivation was due to residual heavy chain. Furthermore, in
contrast to studies with normal plasma-derived factor Va, two possible
cleavage mechanisms could explain the platelet factor Va fragments
observed. APC can cleave platelet factor Va initially at
Arg, with subsequent cleavages occurring at Arg
and Arg
. Alternatively, APC can cleave at
Arg
initially, with further cleavage at Arg
then at Arg
or at Arg
followed by
cleavage at Arg
. Similar results were obtained if
platelets were removed from the inactivation mixtures and phospholipid
vesicles were used to supply the membrane surface required for
inactivation, suggesting that the order of platelet factor Va peptide
bond cleavage or the amount of cofactor activity remaining was not
altered by either of these surfaces. Thus, APC is unable to effect the
complete inactivation of platelet factor Va, even though it would
appear that the same cleavages which render the plasma cofactor
inactive are occurring in the platelet cofactor. Analogous protocols
were used to study an individual heterozygous for the Arg
Gln
mutation (Factor V Leiden, Factor
V
). With respect to the mutant platelet factor Va in
the presence of APC, >70% of the initial cofactor activity remained
after 1 min, with 30% activity still remaining after 2 h. As seen in
studies of the APC-catalyzed inactivation of plasma factor
Va
, proteolysis of the mutant platelet factor Va
confirms that even though cleavage at Arg
will occur in
the absence of cleavage at Arg
, the rate of inactivation
is slower. Collectively these data suggest that when compared to normal
plasma factor Va, differences in normal platelet factor Va
which define: 1) whether the heavy chain is susceptible to cleavage at
Arg
or Arg
and 2) the extent to which it is
cleaved initially at Arg
, in contrast to cleavage of
Arg
, will define both the extent and rate of
inactivation.
The delicate balance between normal hemostasis and thrombosis is
mediated through the formation of -thrombin, a potent procoagulant
enzyme which also initiates anticoagulant events through its
thrombomodulin-dependent activation of protein
C(1, 2) .
-Thrombin is formed subsequent to the
proteolytic conversion of prothrombin by the enzyme complex prothrombinase, which is composed of a vitamin-K-dependent
serine protease factor Xa, and the cofactor factor Va, assembled on an
appropriate membrane surface in the presence of Ca
ions. The required cofactor is supplied subsequent to the
thrombin or factor Xa-catalyzed(3, 4, 5) activation of plasma factor V, or by the release of the
functional cofactor from platelet
-granules(6, 7, 8) . In addition to
binding the substrate prothrombin(9) , factor Va fulfills its
cofactor function by providing at least part of the receptor for factor
Xa at the membrane surface(10, 11, 12) .
Therefore, deletion of factor Va from the complex reduces the rate of
the reaction by 4 orders of magnitude(13) . Since factor Va is
central to the proper assembly of the catalyst on a membrane surface
and profoundly influences the rate of thrombin formation, regulation of
prothrombinase activity can be expressed through alterations in factor
Va activity.
Human plasma factor Va is proteolytically inactivated
by APC ()through a mechanism requiring ordered and
sequential cleavages, only some of which are membrane-dependent (14, 15) (Fig. 1). Sequential cleavage of
plasma factor Va occurs only in the heavy chain (105 kDa) of the
cofactor at positions Arg
, Arg
, and
Arg
. Cleavage at Arg
gives rise to a 75-kDa
intermediate with cleavages at Arg
and Arg
required for complete loss of activity. Cleavage at Arg
is membrane-dependent, whereas cleavages at Arg
and
Arg
are not. Recently, it has been reported that an
increased risk toward venous thrombosis is associated with a single
point mutation in the factor V molecule (Factor V Leiden Arg
Gln, Factor V
)(16) . Termed APC
Resistance, patients suffer from a poor anticoagulant response to APC,
presumably because these individuals lack an APC cleavage site at
position 506 of the factor V
molecule(16, 17, 18, 19, 20) .
Resistance to APC is the most common identifiable defect among patients
with a thrombotic disorder(17, 18, 21) .
Recently, our laboratory has characterized the molecular defect in
plasma factor V
(22) . In the absence of the APC
cleavage site at position 506, inactivation of factor Va proceeded, but
at a slower rate when compared to the normal cofactor. Western blot
analysis revealed that cleavage at Arg
results in the
generation of a 45-kDa fragment and a 62/60-kDa doublet (Fig. 1). Complete loss of cofactor activity was accomplished
and was correlated with the cleavage of the 62/60-kDa doublet at
position 679, which generated a 56/54-kDa doublet.
Figure 1:
Schematic representation of
membrane-bound human plasma factor Va heavy chain inactivation by APC.
The heavy chain of factor Va (105 kDa) is composed of two A domains
(A1-A2) associated through a connecting region(47) . Normal
plasma factor Va is inactivated following three ordered and sequential
cleavages at Arg, Arg
, and
Arg
. Cleavage at Arg
, which gives rise to a
75-kDa fragment and a 28/26-kDa doublet, is necessary to optimally
expose the site at Arg
. Further cleavage at Arg
yields a 45-kDa fragment and a 30-kDa fragment. Individuals with
the Arg
Gln mutation no longer have a cleavage
site at position 506 which slows the rate of cleavage at
Arg
. Cleavage at Arg
yields a 45-kDa
fragment and a 62/60-kDa doublet. Further cleavage of the 62/60-kDa
doublet at Arg
yields a 56/54-kDa doublet. The rate of
inactivation of the mutant cofactor is much slower than that of the
normal cofactor (22) . Fragments that are recognized by the
monoclonal antibody (
HFVa
#6) used in this study are
indicated by the shaded boxes.
In addition to
the factor V which circulates in plasma, a significant amount of factor
V/Va is contained within the -granules of platelets. Of the total
factor V/Va found in whole blood, 80% is contained in plasma and 20% is
found in platelets(23) , yet at a site of vascular injury the
concentration of platelet factor V/Va has been estimated to be about
600 times that of plasma factor V(24) . Platelet factor V
clearly plays a preeminent role in hemostasis since individuals
deficient in platelet but not plasma factor V exhibit a severe,
life-threatening bleeding diathesis(25) . Previous studies from
our laboratory indicate that the platelet membrane supports the
APC-catalyzed inactivation of plasma factor Va. Kinetic parameters
defining the reaction and obtained using platelets or phospholipid
vesicles were nearly identical(26) . However, previous studies
indicate also that platelet factor V and plasma factor V are separate
and distinct substrates for many
reactions(5, 6, 13, 27, 28, 29) .
Therefore, this report details the APC-catalyzed inactivation of
platelet factor Va from normal and APC-resistant individuals on the
surface of both thrombin-activated platelets and phospholipid vesicles.
Protein C (1 µM) was activated with -thrombin
(0.16 µM) at 37 °C for 1 h in 20 mM HEPES,
0.15 M NaCl, pH 7.4, as described previously (26) with
slight modifications. Full activation of protein C to APC was confirmed
by measuring the amidolytic activity toward the chromogenic substrate
S-2238 (American Diagnostica, Inc.) after addition of 0.18
µM hirudin(42) . Aliquots of APC were stored at
-80 °C and amidolytic activity measurements of APC were made
prior to each experiment.
Figure 2:
Inactivation of membrane-bound human
platelet factor Va by APC. Human platelets were activated with thrombin
as described under ``Experimental Procedures.'' In A, platelet factor Va (2.6 nM) in the presence of
PCPS vesicles (10 µM, ) or thrombin-activated
platelets (1
10
/ml,
) was inactivated upon
addition of APC (0.5 nM). At selected time intervals, aliquots
were assayed for cofactor activity. The arrowhead indicates
the point at which platelets were removed from the platelet
inactivation mixture and PCPS vesicles (10 µM) were added
(40-min time point). The inset allows comparison of the
inactivation rates from 0 to 30 min. At the same time intervals
aliquots of the reaction mixture were withdrawn and analyzed by
SDS-PAGE. Following transfer to nitrocellulose proteolytic fragments
were visualized using the monoclonal antibody
HFVa
#6. Panel B represents the proteolytic fragments derived from the
inactivation on PCPS vesicles. Lane 1, platelet factor Va, no
APC; Lanes 2-10, membrane-bound platelet factor Va with
APC at 30 s, 4, 8, 15, 30, 60, 90, and 120 min. The position of the
molecular weight markers is indicated at the left of panel
B. Nonspecific fragments appear at approximately 68 and 43 kDa,
resulting from the reaction of bovine serum albumin and platelet IgG,
respectively, with the secondary antibody.
Western blot analysis depicting the proteolytic fragments
accompanying inactivation on phospholipid vesicles is shown in Fig. 2B. The monoclonal antibody used to detect the
fragments is specific for an epitope located on the 30-kDa fragment
(amino acid residues 307-506) resulting from APC-catalyzed
cleavage at Arg and Arg
and was used
previously to confirm the mechanism of APC-catalyzed cleavage of normal
plasma factor V and Va and to elucidate that of factor
V
/Va
(22) . Lane 1 represents the zero time (no APC addition) and is mainly
characterized by an intense band at 105 kDa, the factor Va heavy chain. Lanes 2-10 represent the inactivation time course shown
in Fig. 2A and depict the appearance, and subsequent
slow cleavage of the 75-kDa fragment (amino acids 1-506; a) to give rise to a 30-kDa fragment (amino acids
307-506; b) consistent with an initial cleavage at
Arg
followed by cleavage at Arg
as has been
observed for normal plasma factor Va(15) . Peptides of 62 and
56/54 kDa were observed also (c). The 62-kDa fragment most
likely appears as a result of initial cleavage of the heavy chain at
Arg
(amino acids 307-709) followed by its
subsequent cleavage at Arg
to yield the 56/54-kDa
fragment (amino acids 307-679). Further cleavage of the 62-kDa or
56/54-kDa fragments at position 506 will give rise to the accumulation
of the 30-kDa fragment (amino acids 307-506). Nonspecific binding
resulting from platelet protein interactions with the secondary
antibody appear at 68 and 43 kDa.
Substantial differences in the initial rate of APC-catalyzed platelet factor Va inactivation on thrombin-activated platelets were observed with different platelet donors (n = 4). These differences are most evident by comparison of Fig. 2A and 3A. In marked contrast to the data shown in Fig. 2A, those shown in Fig. 3A indicate that as much as 50% of the initial platelet factor Va cofactor activity was lost within 1 min of APC addition. The cleavage products observed (Fig. 3B) were identical to those seen previously (Fig. 2B) as detailed above. In all experiments, the loss in platelet factor Va activity could be attributed completely to APC-catalyzed inactivation. Control experiments such as that shown in Fig. 3A (open triangles), where APC was omitted from the reaction mixture, indicated that platelet factor Va cofactor activity was unaltered over a 2-h incubation.
Figure 3:
Inactivation of platelet factor Va by
APC utilizing activated platelets as a membrane surface. A,
platelets were treated with thrombin as described under
``Experimental Procedures.'' Normal platelet factor Va (3.8
nM) in the presence of thrombin-activated platelets (1
10
/ml), was inactivated with APC (0.25 nM,
)
and compared to a control with no APC added (
). At various time
intervals aliquots of the inactivation mixture were assayed for
cofactor activity. Panel B represents the proteolytic
fragments derived from the inactivation on thrombin-activated
platelets. Following transfer to nitrocellulose, proteolytic fragments
were visualized using the monoclonal antibody
HFVa
#6. Lane 1, platelet factor Va, no APC; Lanes 2-11,
platelet factor Va treated with APC at 30 s, 2, 4, 8.5, 15, 30, 45, 65,
135, and 200 min. The position of the molecular weight markers is
indicated at the left of panel
B.
Figure 4:
APC-catalyzed inactivation of
membrane-bound platelet factor Va from an APC-resistant individual.
Platelets from an APC-resistant patient (heterozygous for the
Arg
Gln mutation) were treated with thrombin as
described under ``Experimental Procedures.'' In A,
platelet factor Va from the APC-resistant patient (3.7 nM) was
incubated with APC (0.25 nM) and PCPS vesicles (10
µM,
) or thrombin-activated platelets (1
10
/ml,
). At selected time intervals aliquots of the
reaction mixture were assayed for cofactor activity. At the same time
intervals aliquots were withdrawn and analyzed on a 5-15% linear
gradient SDS-PAGE. Following transfer to nitrocellulose, fragments were
visualized using monoclonal antibody
HFVa
#6. Panel B depicts APC-catalyzed platelet factor Va inactivation
in the presence of PCPS vesicles. Lane 1, no APC; Lanes
2-8, 30 s, 2.5, 5, 10, 20, 40, and 60 min, respectively. Panel C, depicts APC-catalyzed platelet factor Va inactivation
when thrombin-activated platelets provide the membrane surface. Lane 1, platelet factor Va, no APC; Lanes 2-11,
platelet factor Va with APC at 1, 2.5, 6.5, 10, 30, 60, 90, 120, 127,
and 140 min. The position of the molecular weight markers is indicated
at the left of both panels B and C.
Recent studies have clearly indicated that the APC-catalyzed
inactivation of plasma factor Va is mediated by three sequential
cleavages in the factor Va heavy chain at Arg,
Arg
, and Arg
(15) . The studies
reported here indicate that the APC-catalyzed proteolysis of platelet
factor Va occurs through the initial cleavage of the factor Va heavy
chain at either Arg
or Arg
(Fig. 1).
Consistent with observations made with plasma factor Va, initial
cleavage at Arg
appears to be preferred; however,
depending upon the platelet factor Va donor, substantial initial
cleavage at Arg
occurs also. Initial cleavage at
Arg
is followed by cleavages at Arg
and
Arg
(Fig. 1). In contrast, initial cleavage in
platelet factor Va at Arg
is followed by sequential
cleavages at Arg
and Arg
. The initial
cleavage in platelet factor Va at Arg
is consistent with
the sequential cleavages which occur in plasma and platelet factor
Va
where in the absence of an arginine at position 506,
cleavage at Arg
occurs followed by cleavage at
Arg
.
Studies with normal plasma factor Va indicate
that its sequential cleavage at positions 506, 306, and 679 renders it
completely inactive. Studies with plasma factor Va indicate that, even in the absence of a cleavage site at
Arg
, cleavage at positions 306 and 679 is sufficient to
effect almost complete cofactor inactivation (<5% initial activity
remaining), although the rate of inactivation is slowed(22) .
In marked contrast, our present data indicate that APC is unable to
effect the complete inactivation of platelet factor Va, even though it
would appear that the same cleavages which render the plasma cofactor
inactive are occurring in the platelet cofactor. In the four normal
platelet donors assayed, 10-25% of the initial cofactor activity
remained even after prolonged incubation. In the two individuals
assayed who were heterozygous for the Arg to Gln mutation at position
506 in the factor V molecule, 30-40% of their initial factor Va
cofactor activity remained subsequent to a 2-h incubation. Since the
extent of platelet factor Va inactivation is independent of the
membrane surface to which the normal or mutant platelet factor Va is
bound (PCPS vesicles or thrombin-activated platelets), it appears that
the apparent APC resistance expressed by both normal platelet factor Va
and platelet factor Va
is inherent to the platelet
factor Va molecule and its fragments. However, how much cofactor
activity is associated with the various platelet factor Va proteolytic
fragments cannot be determined at this time. Meaningful correlations
between the proteolytic fragments observed by Western blot analysis and
the cofactor activity measurements cannot be made at this time since
only qualitative data can be derived from the Western blotting results.
Even if the affinity of the antibody (
HFVa
#6) for the
various fragments was known, quantitating the various reactive
fragments would be difficult. Similar studies as those detailed here
with purified platelet factor Va and using protein stains to visualize
the cleaved products will obviate this problem and must be done.
The observation that the APC-catalyzed inactivation of platelet factor Va is not completely analogous to that observed with plasma factor Va is not surprising. Several observations have been made previously which suggest that subtle differences exist between plasma and platelet factor Va which render them different substrates for many reactions, and thus somewhat different cofactors. For example, platelet factor V/Va is stored within the platelet as a partially proteolyzed molecule, ranging in molecular mass from 115 to 330 kDa(27) , and exhibits significant cofactor activity upon release from the platelet, demonstrating only a 2-3-fold increase in cofactor activity upon further activation with factor Xa or thrombin(6) . This is in direct contrast to plasma factor V, which expresses virtually no cofactor activity in the single-chain form(13) . Factor Xa- or thrombin-catalyzed activation of platelet factor V yields proteolytic fragments of 105 and 74 kDa(5, 6) . Thrombin-catalyzed activation of plasma factor V also yields proteolytic fragments of 105 and 74 kDa, whereas factor Xa-catalyzed activation yields fragments of molecular mass 220 and 105 kDa(5) . It has also been shown by our laboratory that factor Xa activates platelet factor V 50-100 times more effectively than thrombin, whereas activation of plasma factor V by factor Xa or thrombin is characterized by the same catalytic efficiency(6) . Additional data highlighting structural differences between platelet and plasma factor Va are their differential phosphorylation by platelet kinases. Platelet factor Va is phosphorylated exclusively on the light chain whereas plasma factor Va is phosphorylated on both the light and heavy chain(28, 29) . Collectively these data suggest that platelet factor V may be a different substrate than plasma factor V, but at present, the identity of these two cofactors in both primary structure and post-translational modifications has yet to be elucidated(29) .
These same differences which may make normal platelet factor Va somewhat of a different substrate may account for the data shown in Fig. 2and Fig. 3. The data (representative of four similar experiments) indicate that additional cofactor activity appears over time suggesting that APC, in some way, may have a positive effect on platelet factor Va function. Alternatively since these experiments were done with activated platelet suspensions or platelet releasates, enzymes, other than APC, may be affecting factor Va function and rendering it resistant to APC-catalyzed inactivation.
Similar mechanisms can be invoked to account for the substantial differences observed in the initial rate of APC-catalyzed platelet factor Va inactivation on thrombin-activated platelets from different platelet donors. Other hypotheses can be proposed as well. For example, since in every experiment both the platelet and platelet factor Va concentrations were nearly identical, the marked differences in inactivation rates may be due to different numbers of either factor Va and/or APC binding sites on the thrombin-activated platelets. In addition, even though platelet stores of protein S cannot be ruled out, previous results from our laboratory suggest that protein S has little, if any, effect on APC-catalyzed factor Va inactivation on platelets (26) .
In conclusion,
our data demonstrate that the mechanism of platelet factor Va
inactivation is not determined by the membrane to which it was bound
(PCPS vesicles or thrombin-activated platelets). Furthermore, potential
structural differences in platelet factor Va relative to plasma factor
Va appear to define the extent to which the cofactor is cleaved
initially at Arg first, resulting in a reduced rate of
cofactor inactivation. This conclusion is consistent with our studies
with a patient heterozygous for the Arg
Gln
mutation, as well as our previous results with plasma factor
Va
(22) , indicating that cleavage at
Arg
can occur in the absence of cleavage of
Arg
, but the rate of cofactor inactivation is slower.
Consequently, a factor Va molecule that does not have a cleavage site
at Arg
(Factor Va
) or is in a structural
conformation such that APC cleavage at Arg
is preferred
over Arg
, will be observed to be APC-resistant.