From the Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Factor Va, the essential cofactor
for prothrombinase, is phosphorylated on the acidic COOH terminus of
the heavy chain of the cofactor, at Ser692, by a
platelet membrane-associated casein kinase II (CKII). Consistent with
this observation, phosphorylation of the factor Va heavy chain by the
platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was
monitored using a polyclonal antibody to the -subunit, and kinase
activity in the various fractions was confirmed using human factor Va
as a substrate. Immunoblotting experiments using polyclonal antibodies
raised against synthetic peptides mimicking a portion of the deduced
amino acid sequence of the
-,
'-, and
-subunits of human CKII
demonstrated the coexistence of both
- and
'-subunits in
platelets and suggested that the platelet CKII kinase may exist in part
as an
'
2 complex. It is also possible that there
are two distinct populations of CKII in platelets, one that is
/
and one that is
'
'/
. In the presence of the
purified platelet-derived CKII, human factor Va incorporates between
0.8 and 1.3 mol of phosphate/mol of factor Va depending on the
concentration of the
-subunit in the kinase preparation. A peptide
mimicking the sequence 687-705 of the human factor V molecule
incorporates radioactivity in the presence of purified CKII and
inhibits factor Va heavy chain phosphorylation by the platelet CKII. In
contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation
by the platelet CKII. Human factor Va is inactivated by activated
protein C following three cleavages of the heavy chain at
Arg506, Arg306, and Arg679.
Cleavage at Arg506 is necessary for efficient exposure of
the inactivating cleavage site at Arg306. The
phosphorylated cofactor has increased susceptibility to inactivation by
activated protein C, since phosphorylated factor Va was found to be
inactivated approximately 3-fold faster than its native counterpart.
Acceleration of the inactivation process of the phosphorylated cofactor
occurs because of acceleration of the rate of cleavage at
Arg506. These data suggest a critical role for factor Va
phosphorylation on the surface of platelets in regulating cofactor
activity.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Coagulation factor Va is the cofactor for prothrombinase (1, 2).
Factor Va combines with the serine protease factor Xa on a phospholipid
or platelet surface in the presence of divalent cations to catalyze the
conversion of prothrombin to thrombin. Factor Xa alone can activate
prothrombin to -thrombin. However, the binding of factor Va to
factor Xa on a membrane surface in the presence of Ca2+
increases the efficiency of the complex by 5 orders of magnitude as
compared with factor Xa alone (3).
Most of the factor V is found in the plasma, where it circulates at a
concentration of 7 µg/ml (20 nM). Approximately 20% of
the total human factor V found in whole human blood originates from the
platelets' -granules (4). Factor V circulates in plasma as a large
single chain protein with a Mr of 330,000 (5). The cDNA and the deduced amino acid sequences for human and bovine factor V have been already determined (6, 7). Thrombin cleaves single
chain factor V to give rise to the active cofactor (factor Va), which
participates in the activation of prothrombin (5-7). The factor Va
resulting from
-thrombin cleavage is composed of a heavy chain of
Mr 105,000 containing the
NH2-terminal part of the factor V molecule (A1-A2 domains)
and a light chain of Mr 74,000 that derives from
the COOH-terminal end of the cofactor (A3-C1-C2 domains). Factor Va is
inactivated by activated protein C
(APC)1 in the presence of a
membrane surface (8, 9). APC inactivates human factor Va by cleavage at
Arg506, Arg306, and Arg679.
Cleavage at Arg506 is necessary for efficient exposure of
the inactivating, lipid-dependent cleavage site at
Arg306 (9).
The expression of the cofactor's catalytic activity requires the presence of a phospholipid or cell surface. Factor Va interaction with the lipid bilayer is promoted by two regions located on the light chain of the cofactor: a binding site located on the carboxyl-terminal part of the A3 domain, which involves hydrophobic interactions, and a binding site located on the C2 domain involving Ca2+-independent electrostatic interactions (10). In addition, the catalytic behavior of the prothrombinase complex on activated platelets is kinetically equivalent to the same phenomenon using synthetic phospholipid vesicles in the bovine system. Factor Va binds to platelets in a specific manner, and the binding involves high and low affinity binding sites with dissociation constants of 0.3 and 3 nM, respectively (11). Factor V binds to platelets with a Kd of 3 nM. The number of binding sites and their affinity are not affected by the state of platelet activation.
Early data have shown that platelet membranes possess protein kinase activity, which can phosphorylate endogenous platelet membrane proteins as well as exogenous proteins (12). Phosphorylation of proteins has been previously shown to occur upon activation of platelets. It has been thus proposed that protein phosphorylation could be involved in hemostasis and thrombosis during blood clotting (13). Further, it has been demonstrated that thrombin stimulation of platelets results in the activation of 14 new electrophoretically distinct, serine/threonine protein kinases (14). Interestingly, the activation of seven of the 14 new kinases was correlated with platelet secretion and aggregation. On the other hand, it has been repeatedly shown that human platelets contain a protein possessing casein kinase II (CKII) activity (15-19). It has thus been proposed that the plasma protein kinases may originate from platelets, since no kinase activity seems to be present in human blood plasma. Finally, immunoelectron microscopy (20) recently provided evidence for the presence of CKII in platelets.
Recent data have demonstrated that bovine coagulation factor Va
incorporates 32PO4 into the heavy chain in the
presence of [-32P]ATP and a platelet kinase contained
in the activated platelet soluble fraction (APSF) following activation
of human platelets by
-thrombin (21) and that human plasma factor Va
is stoichiometrically phosphorylated on both the heavy and light chains
in the presence of whole platelets (platelet membranes plus APSF) (22).
Phosphorylation of factor Va on the heavy chain implicates a platelet
protein-kinase that has many similarities with CKII (20), whereas
factor Va light chain is phosphorylated by a platelet protein kinase C
isoform (21). The present study was undertaken to identify and
characterize the platelet kinase that phosphorylates the heavy
chain of factor Va.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials and Reagents--
HEPES, ATP, diisopropyl
phosphofluoridate, bovine serum albumin, 1-palmitoyl-2-oleoyl
phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidyl serine,
2-mercaptoethanol, heparin-agarose resin, heparin salt, and sodium
pyrophosphate were purchased from Sigma. [-32P]ATP was
from ICN Biomedicals, Inc. (Costa Mesa, CA). Heparin-agarose and
Q-Sepharose fast flow resins were obtained from Amersham Pharmacia Biotech, and phosphocellulose was obtained from Whatman. The
-thrombin inhibitor, hirudin, was obtained from Genentech (South San
Francisco, CA). Bio-Beads SM-2 adsorbent was purchased from Bio-Rad.
Thromboplastin was purchased from Organon Teknika Corp. (Durham, NC).
Purified casein kinase II from sea star (Ptilocerus
ochraceus) as well as the polyclonal antibody against the
-subunit of human CKII were purchased from Upstate Biotechnology,
Inc. (Lake Placid, NY). Human prothrombin, thrombin, factor V, bovine
factor Va, and bovine factor Va heavy and light chains were purified
according to previously described methods (23-26). Platelet membranes,
the fluorescent thrombin inhibitor
dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide, human
factor Xa, and human APC were gifts from Drs. Paul Haley and Richard
Jenny (Hematologic Technologies Inc, Essex Junction, VT). Rabbit
polyclonal antibodies against specific epitopes of the
-,
'-, and
-subunits of human casein kinase II were prepared as described (27)
and were provided by Dr. David Litchfield (University of Western
Ontario, London, Ontario, Canada).
Peptide Synthesis-- Two peptides were synthesized (peptides 67 and 73). These peptides correspond to the region 687-705 of human factor V (6). Peptide 67 has the sequence PEDEESDADYDYQNRLAAA, whereas peptide 73 has an alanine instead of a serine at position 692 (underlined within the sequence of peptide 67). The peptides were prepared following a previously described method (28).
Purification of Platelet Casein Kinase II--
Single donor
platelet packs from 50-150 donors were pooled, and platelets were
washed and isolated as described (29) and resuspended in 200 ml of
Tyrode's solution. Platelets were activated by thrombin (1 unit/ml).
Following the addition of 25 µM Phe-Pro-Arg-chloromethyl ketone, the solution was centrifuged, the supernatant was collected, and the platelet membranes were stored at 20 °C. Frozen platelet membranes were thawed at 37 °C and solubilized in HEPES (20 mM), NaCl (0.15 M), CaCl2 (2 mM), MgCl2 (1 mM), pH 7.4 (HBS++), containing 0.5% Triton X-100 and diisopropyl
phosphofluoridate (2 mM). The solution was mixed
frequently, pulse-sonicated on ice (3 × 5 min), and centrifuged
for 30 min (10,000 × g). Further steps of purification
were carried out (at 4 °C) on the supernatant, which contains the
solubilized platelet membrane CKII. All of the chromatography columns
were preequilibrated with HBS++ buffer containing 0.5%
Triton X-100. After eluting each column, fractions were analyzed on an
8-18% linear gradient vertical slab SDS-PAGE as described by Laemmli
(30) under reducing conditions. Transfer to a nitrocellulose membrane
was performed as described (31), and the presence of the
-subunit of
CKII was confirmed by using a commercially available rabbit polyclonal
antibody raised against a synthetic peptide spanning region 70-91 of
the
-subunit of human CKII using chemiluminescence. Fractions
containing the CKII antigen were pooled and dialyzed overnight
versus HBS++, containing 0.5% Triton X-100.
Heparin-Agarose Chromatography-- The supernatant obtained following centrifugation of activated platelets was loaded onto a heparin-agarose column (100 ml). The column was washed with the equilibration buffer (HBS++), and elution was performed with a continuous gradient (0.15-1.0 M NaCl in HBS++, 0.5% Triton X-100). Fractions of 5 ml were collected. The positive fractions were pooled, and the pool was assessed for the presence of the CKII-like kinase activity using human factor Va or bovine factor Va heavy chain as a substrate.
Phosphocellulose Ion Exchange Chromatography Column-- Following dialysis, the heparin-agarose eluate was loaded onto a phosphocellulose column (65 ml). The column was washed with HBS++ and then eluted with a NaCl gradient (0.15-0.5 M NaCl in HBS++, 0.5% Triton X-100). Fractions of 3 ml were collected. Fractions containing the CKII-like kinase antigen and activity were pooled and dialyzed overnight as described above in HBS++.
Q-Sepharose Ion Exchange Chromatography--
The dialyzed pooled
fractions obtained from the phosphocellulose column were loaded on a
Q-Sepharose column (3 ml). Washing and eluting the column was performed
as described above (using a NaCl gradient of 0.15-2.0 M
NaCl in HBS++, 0.5% Triton X-100). Since the platelet CKII
was still eluted in a large volume, the pooled fractions containing
CKII antigen and activity were dialyzed against HBS++ and
loaded onto a 3-ml heparin-agarose column. Elution was performed with 1 M NaCl, and 300-400 µl fractions were collected.
Following dialysis, the kinase was kept at 80 °C in small aliquots
(100 µl). Based on activity as compared with the commercially
available CKII, a total of approximately 20-40 µg of the partially
purified platelet CKII was obtained from 100 units of platelets. Under these conditions, the kinase activity was stable for 4-5 months.
Phosphorylation of Human Factor V by the Purified Platelet
CKII--
Purified human factor V, bovine factor Va, and bovine factor
Va heavy chain were used as substrates for the platelet CKII. To
demonstrate that kinase activity was present in the pooled fractions, a
control phosphorylation reaction was performed in which a fraction that
did not show immunoreactivity with the anti-CKII antibody was used as a
source of kinase to phosphorylate the substrate. Human factor V at a
final concentration varying from 50 to 250 µg/ml (100-750
nM) was incubated with an aliquot of the purified CKII-like
kinase (representing approximately 1 µg of protein) and a mixture of
unlabeled ATP (2 mM) and [-32P]ATP in a
total reaction volume of 400 ml in HBS++ for 1 h at
30 °C. The reaction mixture was loaded on an Excellulose GF-5 gel
filtration desalting column (Pierce) at room temperature to remove
excess [
-32P]ATP. The column was previously saturated
with bovine serum albumin (0.5%) and equilibrated with
HBS++. A small column (approximately 0.5 cm) composed of
Bio-Beads SM-2 adsorbent (Bio-Rad) was poured on the top of the
Excellulose column to remove the Triton X-100 and thus to allow APC
inactivation of factor Va, which is a
phospholipid-dependent mechanism. The column was eluted
manually in the equilibrating buffer (400-µl fractions were
collected). The final concentration of phosphorylated factor V was
determined spectrophotometrically using an extinction coefficient
(
280 nm1%) of 0.9 for factor V and
1.74 for bovine factor Va (32) or 1.2 for bovine factor Va heavy
chain.
Stoichiometry of Phosphorylation-- The stoichiometry of the phosphorylation catalyzed by the platelet CKII was determined by direct precipitation of the phosphorylated protein with 2.5% trichloroacetic acid in 1% sodium pyrophosphate. The disintegrations/min obtained from the liquid scintillation counter (Beckman LS 6000IC) were compared with the specific activity of the ATP solution used and the known protein concentration of the phosphorylated factor Va molecule to calculate the stoichiometry of the phosphorylation reaction.
Inactivation of Phosphorylated Human Factor Va by
APC--
Phosphorylated human factor V was activated to factor Va by
-thrombin (1 unit/ml) for 5 min at 37 °C. Following the addition of hirudin (20 nM), phosphorylated factor Va was assessed
for its susceptibility to inactivation by APC. APC (at a 1:100
enzyme/substrate ratio) was added to the reaction mixture containing
native or phosphorylated factor Va at 37 °C in the presence of PCPS
vesicles (20 µM). Cofactor activity was monitored by
either a clotting assay using factor V-deficient plasma or an assay
measuring thrombin generation and employing a fluorescent thrombin
inhibitor and purified prothrombinase reagents as described by Nesheim
et al. (32). At each time point, a sample was also withdrawn
for SDS-PAGE analysis (5-15% linear gradient). A control reaction was
performed under similar experimental conditions but in the absence of
ATP. In some experiments, the degradation pattern of factor Va by APC was analyzed by using a monoclonal antibody that recognizes an epitope
between amino acid residues 307 and 506 of human factor V as described
(34, 35).
Phosphorylation by APSF and CKII-- Phosphorylation of bovine and human factor Va and bovine factor Va heavy chain by APSF was performed as described previously (21). The mol of phosphate incorporated into the proteins were calculated from the specific activity of the ATP mixture (21, 36). In some experiments, inhibition of bovine factor Va phosphorylation (3.4 µM) by the platelet kinase, by heparin (final concentration of 40 µg/ml) and the two synthetic peptides (at a final concentration of 80 µM) was assessed using APSF and ATP (100 µM, 0.5 µCi/µl). Following gel filtration, the proteins were analyzed on a 5-15% linear SDS-PAGE and visualized by Coomassie Blue staining. Radioactive proteins were identified by autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specificity of the Platelet Kinase
Previous data have demonstrated that while bovine factor Va heavy
chain incorporates radioactivity in the presence of APSF and
[-32P]ATP (Fig. 1,
lanes 1 and 5), a fragment containing the major portion of the heavy chain (Mr 90,000) but
lacking the acidic COOH terminus segment does not incorporate phosphate
in the presence of either APSF or purified CKII (21). Further, human
factor V is phosphorylated by purified CKII exclusively on the heavy chain portion of the molecule (21, 22). Since CKII is known to be
inhibited by heparin (37), inhibition of factor Va heavy chain
phosphorylation by the platelet kinase was evaluated. Fig. 1,
lane 2, shows that factor Va heavy chain phosphorylation by APSF was completely inhibited by an excess of heparin. Thus, heparin binds and inhibits the platelet CKII.
|
Previous data have shown that the heavy chain of factor Va (human or bovine) is phosphorylated within the acidic COOH terminus on a serine (21, 22). APC cleaves human factor Va heavy chain sequentially at Arg506, Arg306, and Arg679 (8). Following cleavage of phosphorylated human factor Va by APC at Arg506, radioactivity was found in the COOH-terminal fragment of the heavy chain (amino acid residues 507-709). Subsequent cleavage of this fragment at Arg679 resulted in the disappearance of radioactivity. Since phosphoserine was the only phosphoamino acid detected (21, 22), it was concluded that amino acid sequence 680-709 of human factor Va heavy chain must contain a serine that is phosphorylated by the platelet CKII. Examination of the human factor V deduced amino acid sequence (6, 7) revealed that the carboxyl-terminal part of factor Va heavy chain that would result after cleavage at Arg679 (amino acid residues 680-709) contains only one serine residue, Ser692. Further, Ser692 is a potential phosphorylation site for CKII because of the presence of acidic amino acids at positions +1 (Asp693), +3 (Asp695), and +5 (Asp697) (40). Additionally, if Tyr696 is sulfated in vivo (41) as suggested, more negative charges will be added to an already negative amino acid region, which will favor phosphorylation of this serine by CKII in vivo. Thus, based upon the known NH2-terminal sequence of the fragments derived from the heavy chain of factor Va following APC digestion (8), the deduced amino acid sequence of the human factor V molecule (6), and the substrate specificity for CKII (38-40), two peptides were synthesized. Both contained the sequence 687-705 of the human factor V molecule. Peptide 67, which has the same sequence as human factor V with a serine at position 692, showed a net incorporation of 32PO4 in the presence of purified CKII (Fig. 2, filled circles). In contrast, peptide 73, which has an alanine at position 692, did not show any incorporation of radioactivity under similar experimental conditions (Fig. 2, filled triangles). It has been demonstrated that synthetic peptides that are substrates for CKII are also inhibitors of the phosphorylation of the corresponding protein substrate (38). Thus, the inhibition of factor Va phosphorylation by the two synthetic peptides was evaluated next. Peptide 67 at a concentration of 80 µM almost completely inhibits factor Va heavy chain phosphorylation (Fig. 1, lane 4), whereas under similar experimental conditions peptide 73 does not have any inhibitory effect on the platelet-mediated factor Va phosphorylation (Fig. 1, lane 3). Overall, these data demonstrate that Ser692 is responsible for the incorporation of radioactivity in the human factor V molecule in the presence of CKII.
|
Localization of the Platelet Kinase
We have previously shown that bovine factor Va incorporates
32PO4 in the presence of APSF and
[-32P]ATP (0.03-0.05 mol of
32PO4/mol of factor Va) (21). However, in the
presence of a purified preparation of CKII from bovine testis, the
extent of phosphorylation was significantly higher and approaching
molar equivalency (21). Further, in the presence of unstimulated
platelets or in the presence of collagen-stimulated platelets, factor
Va was phosphorylated exclusively on the heavy chain (22). It has been
repeatedly shown that APSF contains a kinase activity associated with
platelet microparticles. Both the kinase from APSF and the kinase from the membrane fraction were tested in the present work as well as in the
previous publications and were found to phosphorylate the heavy chain
of human factor Va at a unique site (i.e.
Ser692). In the presence of whole platelets,
phosphorylation of the factor Va heavy chain occurs at a unique site
(Ser692). Together, these data suggest that most of the
kinase remains associated with the platelet membranes following
activation of platelets by
-thrombin. To verify this hypothesis,
factor Va heavy chain was phosphorylated by both APSF and the
corresponding platelet membranes resulting from
-thrombin activation
of platelets (Fig. 3). The presence of
CKII antigen in APSF or the platelet membrane fraction was verified by
immunoblotting using a polyclonal antibody against the
-subunit of
human CKII (Fig. 3B). An approximate 10-fold increase in
factor Va heavy chain phosphorylation was observed when using a Triton
X-100 extract of platelet membranes as a kinase source when compared
with the phosphorylation of the heavy chain by APSF alone (Fig.
3A, lanes 1 and 2). Further,
substantial kinase antigen was detected in the platelet membrane
fraction (Fig. 3B, lanes 2 and 3).
Thus, the low stoichiometry of factor Va phosphorylation previously
observed when using APSF alone as a source of kinase (21) is most
likely due to the fact that following activation of platelets with
-thrombin most of the kinase remains associated with the platelet
membranes, and APSF contains CKII activity associated with platelet
microvesicles (42).
|
Purification and Partial Characterization of Platelet CKII
The strategy to purify the platelet CKII was based upon a
combination of several methodologies used to purify CKII from various mammalian tissues (18, 43, 44). The difficulty in obtaining large
amounts of purified kinase is due to the fact that, unlike CKII
purified from other mammalian tissues, the platelet CKII is
membrane-associated and as a consequence extremely insoluble in the
absence of detergent. Washed platelets were activated with -thrombin
(1 unit/ml). Since the platelet CKII was found to be a
membrane-associated kinase, the platelet membranes were separated from
the soluble material by centrifugation. Activation by thrombin and
elimination of the supernatant was considered as a first purification step in the present study. The platelet pellet that was solubilized in
HBS++ containing Triton X-100 (0.5%) was found to contain
the majority of kinase activity (Fig. 4,
lane 1) and was used as the source of CKII. Further
purification of the kinase was carried out at 4 °C.
|
Since heparin is a potent inhibitor of the platelet CKII with respect to factor Va phosphorylation, the supernatant was loaded on a heparin-agarose column. Elution was performed with a NaCl gradient (0.15-1 M) followed by washing the column with 2 M NaCl. Fractions containing kinase activity, which eluted between 0.4-0.6 M NaCl, were pooled, dialyzed, and applied to the phosphocellulose column (Fig. 4, lane 4). Small amounts of kinase were detected in the flow-through of the column, most likely because of the column overload (Fig. 4, lane 2). No significant amounts of kinase were detected in the 2 M NaCl wash of the heparin-agarose column (Fig. 4, lane 3).
The phosphocellulose column was eluted with a NaCl gradient (0.15-0.5 M). No CKII antigen was observed in the flow-through of the
column (Fig. 4, lane 5). Fractions containing kinase antigen and activity were pooled (Fig. 4, lane 6), and following
dialysis they were applied to the Q-Sepharose column. The poor
immunostaining of the kinase in Fig. 4, lane 6, is due to
the fact that the kinase is eluted from the phosphocellulose column in
a large volume. No kinase antigen was detected the flow-through of the
Q-Sepharose as shown in Fig. 4, lane 7, whereas the
imunoreactivity of the eluate from the ion exchange column is depicted
in Fig. 4, lane 8. However, since the kinase was still
eluted in a large volume from the Q-Sepharose, the protein was
concentrated on a small heparin-agarose column. Fractions containing
kinase activity were pooled; dialyzed against HBS++, 0.5%
Triton X-100; and kept at 80 °C in frozen aliquots. The final CKII
preparation used for factor Va phosphorylation is shown in Fig. 4,
lane 9.
CKII was isolated from several mammalian tissues, and several studies
have demonstrated that the enzyme is a heterotetramer of
Mr 130,000 composed of two catalytic subunits
( and
', of Mr 36,000-44,000) associated
noncovalently to two
-subunits (Mr 26,000)
(27, 44). The holoenzyme could thus be a mixture of different
tetrameric forms. To evaluate the subunit composition of the platelet
CKII, the immunoreactivity of the platelet CKII kinase with respect to
the two isoforms of the
subunit was next assessed by using
polyclonal antibodies raised against specific epitopes of the
- and
'-subunits of human CKII (27). The
'-subunit is usually shorter
than the
-subunit (27) and is believed to be the product of a
different gene than the
-subunit (44). Fig.
5 demonstrates that the platelet CKII is
composed of both the
- and
'-subunit with
Mr 36,000 and 33,000, respectively as previously
shown (20). Platelet CKII preparation also contains a
Mr 26,000
-subunit (Fig. 5,
). These data
demonstrate that the platelet CKII is similar in subunit composition to
the other known CKII purified from other mammalian tissues and that at
least some of the holoenzyme exists as an
'
2
complex. It is also possible that there are two distinct populations of
CKII in platelets, one that is
/
and one that is
'
'/
.
|
CKII-mediated Phosphorylation of Human Factor V
Localization of Phosphate Incorporation--
Human factor V
incorporates radioactivity in the presence of
[-32P]ATP and the purified platelet CKII (Fig.
6, lane 1). Approximately 1.1 mol of 32PO4/mol of factor V were incorporated
in the factor V molecule in the presence of purified platelet-derived
CKII. Following activation by
-thrombin, most of the phosphate was
found to be incorporated in the heavy chain of human factor Va (Fig. 6,
lane 2). The fragments designated by a,
b, and c represent the Mr
220,000 precursor of the light chain of factor Va (a, amino
acid residues 710-2196), the Mr 150,000 activation peptide (b, amino acid residues 1019-1545), and
the Mr 71,000 activation peptide (c,
amino acid residues 710-1018). These fragments are highly glycosylated
and also contain some minor potential phosphorylation sites
(e.g. Ser804 that has acidic amino acids at
positions +1 and +5 and Ser1506 that has acidic amino acids
at positions +1 and +3) (6). The calculated stoichiometry for the
phosphorylation reaction of human factor V varied between 0.8 and 1.3 mol of 32PO4/mol of human factor V/Va,
indicating the presence of at least one phosphorylation site on the
human factor Va heavy chain. It is noteworthy that the extent of
phosphate incorporation into the heavy chain of factor Va varied from
one preparation to the other and was closely related to the amount of
the
-subunit present in the kinase preparation.
|
Significance of Phosphorylation-- We have previously demonstrated that upon phosphorylation, inactivation of the bovine cofactor by APC occurs faster than inactivation of its native counterpart (21). Acceleration of the inactivation process was observed to occur in the presence as well as in the absence of phospholipid vesicles (21). The data thus suggested that acceleration of the inactivation process may be the result of acceleration of the cleavage rate at the phospholipid-independent site (Arg506). The susceptibility of human factor Va, stoichiometrically phosphorylated by the platelet CKII (0.7-0.8 mol of 32PO4/mol of factor Va), to cleavage and inactivation by APC was next assessed and compared with the inactivation of the native cofactor (Fig. 7). Aliquots of the mixtures were also analyzed for immunoreactivity with a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the heavy chain of factor Va (Fig. 8) (34, 35). Fig. 7 shows that the phosphorylated cofactor (Fig. 7, filled circles) is inactivated approximately 3-fold faster than its native counterpart (Fig. 7, filled squares). Fig. 8 demonstrates that the heavy chain of native human factor Va persists for a longer time (Fig. 8B) as compared with the heavy chain of the phosphorylated cofactor (Fig. 8A). These data indicate an increase in the rate of cleavage at Arg506 of the phosphorylated cofactor. No increase in the rate of cleavage at Arg306 of the Mr 75,000 fragment was observed. The decrease in full-length factor Va heavy chain only results in a 3-fold decrease in activity, because the Mr 75,000 fragment intermediate still possesses cofactor activity (8) in the presence of the light chain of factor Va. The data demonstrate that phosphorylation of human factor Va increases its susceptibility to inactivation by APC because of an increase in the rate of cleavage at Arg506.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present data demonstrate that platelet CKII is associated with
the platelet membranes and stoichiometrically phosphorylates the heavy
chain of factor Va at Ser692. Phosphorylation of factor Va
increases its rate of inactivation by APC because of the increased rate
of cleavage at Arg506 (Fig.
9). The data suggest that factor Va
phosphorylation on the surface of platelets is critical for cofactor
activity regulation. It is noteworthy that cleavage at
Arg679 on the phosphorylated cofactor occurs slowly, as
demonstrated by the slow disappearance of the Mr
28,000/26,000 doublet representing the COOH-terminal portion of the
heavy chain of factor Va (Fig. 6). In contrast, cleavage at the COOH
terminus of the heavy chain occurs quickly when using isolated bovine
factor Va heavy chain (21). Platelet factor Va is more resistant to
inactivation by APC than its plasma counterpart, retaining
approximately 20-30% cofactor activity on the surface of activated
platelets (46). It has been also demonstrated that cleavage at
Arg679 during factor Va inactivation may account for the
loss of approximately 20% of cofactor activity (34). Collectively,
these data suggest that following activation of platelets and factor Va
binding, phosphorylation of the heavy chain of the cofactor by the
platelet CKII locally at the place of vascular injury may be
responsible, at least in part, for the increased resistance of factor
Va on the platelet surface. As a consequence, phosphorylation of factor Va by CKII may have a differential effect on the rate of cleavages at
Arg506 and Arg679 on the platelet surface,
accelerating the rate of the former while slowing the rate of the
latter, and is thus actively involved in the regulation of cofactor
activity. It has been also shown that platelet factor Va is
phosphorylated on the light chain by a platelet kinase that is similar
to protein kinase C and that the light chain of the platelet cofactor
is the major platelet phosphoprotein (22). Protein kinase C is
activated by diacylglycerol and tumor-promoting phorbol esters that
increase the affinity of the enzyme for Ca2+, resulting in
the activation of protein kinase C (47). Overall, the data suggest that
both kinases can phosphorylate factor V/Va locally at the place of
vascular injury, since it has been demonstrated that CKII is present on
the platelet membrane while the light chain kinase, which is most
likely one of four protein kinase C isoforms present in platelets (48),
is translocated upon -thrombin stimulation of platelets from the
cytosol to the plasma membrane and to the cytoskeletal components.
Since in vivo clotting occurs at the surface of platelet
membranes, it is almost certain that platelet and/or plasma factor V/Va
will come in contact with either of the kinases. Thus, in the presence
of high concentrations of ATP released locally by the dense granules
following activation of platelets, phosphorylation of factor V/Va will
occur. As a consequence, the protective role provided by platelets on
platelet factor Va inactivation (46) may be a consequence of
phosphorylation. However, the possibility of a protective effect of a
platelet receptor for factor Va on the platelet membrane should not be ignored.
|
The function and significance of the phosphorylation of various
proteins has begun to be partially elucidated. The location and
properties of many kinases have been defined the last few years.
Earlier data showed that platelets contain kinase activity (12), and
these findings were verified by the bulk of data published the last 20 years concerning protein phosphorylation. More recently, it has been
reported that platelets contain 14 yet unidentified serine/threonine
protein kinases (14). CKII is a widespread kinase in eukaryotic cells
(49-51). CKII is also localized in the nucleus and phosphorylates many
nuclear proteins. Although its physiological function remains
enigmatic, the fact that the enzyme is subjected to short-term
regulation by growth factors suggests that CKII may play a role in
intracellular signal transduction during the cell proliferation and
differentiation mechanisms. CKII is not regulated by a single cofactor
like cAMP, but its biological effects are differentially controlled in
each tissue. In addition, the large variety of proteins that are
substrates for CKII suggests that the latter plays a central role in
protein regulation. Recently it has been shown that CKII -subunit
can serve as an oncogene and that its dysregulated expression can transform lymphocytes in a two-step pathway with c-myc (52). The presence of CKII in human platelets was confirmed using immunogold electron microscopy with antibodies against CKII, and it has been suggested that CKII may be associated with platelet particles (20).
Controversial data exist as to whether CKII is constitutively expressed
on cells or is expressed following activation or stimulation by various
cytokines (51).
The present data clearly demonstrate that factor Va is phosphorylated on the heavy chain at a unique site (i.e. Ser692) by a platelet membrane-associated CKII. Since the present study is the first to show that the platelet CKII is membrane-associated and thus most likely has a transmembrane domain, it would be more appropriate to refer to the platelet CKII as a CKII-like activity. It is also possible that the platelet CKII is tightly associated to a membrane protein. It is noteworthy that platelet phosphorylation is actively involved in the factor Va-factor Xa interaction (53), since in the presence of kinase inhibitors the factor Va-factor Xa bimolecular interaction is differentially altered (53). It has also been shown that tissue factor pathway inhibitor (TFPI) is phosphorylated on Ser2 by CKII (54). It is noteworthy that upon platelet activation approximately 8% of TFPI found in plasma is secreted from platelets (55). Since plasma-isolated TFPI is already partially phosphorylated, it is conceivable that upon platelet activation and in the presence of the high amounts of ATP released by the platelet dense granules, platelet factor Va and platelet TFPI are readily phosphorylated. Thus, it is possible that factor Va and TFPI are two physiological substrates for the platelet CKII locally at the surface of platelets.
![]() |
ACKNOWLEDGEMENTS |
---|
I thank Dr. Ken Mann for supporting this project and for generously allowing use of his laboratory's facilities. I also thank Dr. Paula Tracy for constructive discussions, critical comments, and continuous support and enthusiasm concerning this project. I thank Shaw Henderson for excellent technical assistance and Drs. Richard Jenny and Paul Haley for providing some of the purified proteins used in this study as well as the platelet membranes. Finally, I thank Dr. David Litchfield for the polyclonal antibodies to CKII and Dr. William Church for peptide synthesis.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant-in-Aid 94017990 from the American Heart Association and by National Science Foundation Grant NSF/EPSCoR. A portion of this work was presented in an abstract form at the 69th Scientific Sessions of the American Heart Association, New Orleans, LA, November 1996 (56).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry,
Cleveland State University, Cleveland, OH 44115, Tel.: 216-687-2457; Fax: 216-687-9298; E-mail: m.kalafatis{at}csuohio.edu.
1 The abbreviations used are: APC, activated protein C; CKII, casein kinase II; PAGE, polyacrylamide gel electrophoresis; APSF, activated platelet soluble fraction; TFPI, tissue factor pathway inhibitor; PCPS vesicles, phospholipid vesicles composed of 75% phosphatidylcholine, 25% phosphatidylserine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|