Identification and Partial Characterization of Factor Va Heavy Chain Kinase from Human Platelets*

Michael KalafatisDagger

From the Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 alpha -, alpha '-, and beta -subunits of human CKII demonstrated the coexistence of both alpha - and alpha '-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha 'beta 2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alpha alpha /beta beta and one that is alpha 'alpha '/beta beta . 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 beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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' alpha -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 alpha -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 [gamma -32P]ATP and a platelet kinase contained in the activated platelet soluble fraction (APSF) following activation of human platelets by alpha -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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. [gamma -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 alpha -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 alpha -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 alpha -, alpha '-, and beta -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 alpha -subunit of CKII was confirmed by using a commercially available rabbit polyclonal antibody raised against a synthetic peptide spanning region 70-91 of the alpha -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 [gamma -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 [gamma -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 (epsilon 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 alpha -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
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Abstract
Introduction
Procedures
Results
Discussion
References

Specificity of the Platelet Kinase

Previous data have demonstrated that while bovine factor Va heavy chain incorporates radioactivity in the presence of APSF and [gamma -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.


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Fig. 1.   Inhibition of the phosphorylation. Bovine factor Va was incubated with APSF and ATP as described under "Experimental Procedures." Following gel filtration, the protein components contained in the void volume of the column were subjected to an SDS-PAGE (5-15%) linear gradient followed by staining with Coomassie Blue (A). The gels were destained, dried, and subjected to autoradiography (B). Lanes 1 and 5, factor Va phosphorylated by APSF (control); lane 2, factor Va and APSF in the presence of heparin; lane 3, factor Va and APSF in the presence of peptide 73; lane 4, factor Va and APSF in the presence of peptide 67. The position of the molecular weight markers is indicated at the left.

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.


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Fig. 2.   Phosphorylation of the synthetic peptides. Incorporation of phosphate into both synthetic peptides was monitored as described under "Experimental Procedures" using P81 papers (36). Filled circles, phosphorylation of peptide 67; filled triangles, phosphorylation of peptide 73.

Localization of the Platelet Kinase

We have previously shown that bovine factor Va incorporates 32PO4 in the presence of APSF and [gamma -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 alpha -thrombin. To verify this hypothesis, factor Va heavy chain was phosphorylated by both APSF and the corresponding platelet membranes resulting from alpha -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 alpha -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 alpha -thrombin most of the kinase remains associated with the platelet membranes, and APSF contains CKII activity associated with platelet microvesicles (42).


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Fig. 3.   CKII is associated with the platelet membranes. Platelets were activated with alpha -thrombin (1 unit/ml). Following centrifugation, the pellet was raised in an equal volume of HBS++ containing 0.5% Triton X-100. Bovine factor Va heavy chain was phosphorylated by both fractions in the presence of [gamma -32P]ATP as described under "Experimental Procedures." Phosphorylated heavy chain was identified by autoradiography of an 8-18% SDS-PAGE. An aliquot of the phosphorylation mixture was analyzed for kinase antigen on a 5-15% SDS-PAGE followed by transfer to nitrocellulose and staining with an anti-alpha -subunit rabbit polyclonal antibody. A, identification of heavy chain phosphorylation; B, localization of CKII alpha -subunit. Lane 1, APSF and factor Va heavy chain; lane 2, membrane fraction from thrombin-activated platelets and factor Va heavy chain; lane 3, membrane fraction from thrombin-activated platelets alone, no heavy chain. The position of the heavy chain of bovine factor Va is indicated at the right of panel A, while the position of the alpha -subunit of CKII is shown at the right of panel B. Molecular weight markers are shown at the left of panels A and B.

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 alpha -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.


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Fig. 4.   Purification of platelet CKII. CKII from platelets was purified as described under "Experimental Procedures" using heparin-agarose, phosphocellulose, and Q-Sepharose. A sample of the polled fractions that was analyzed for CKII activity using either bovine factor Va heavy chain or human factor Va was also analyzed for CKII antigen. Following transfer to nitrocellulose the presence of the kinase in the pooled fractions was verified using a polyclonal antibody against the alpha  subunit of CKII. Lane 1, starting material; lane 2, heparin agarose flow-through; lane 3, high salt wash (2 M) of the column following gradient elution; lane 4, pooled fractions of the heparin-agarose column that were loaded on the phosphocellulose column; lane 5, phosphocellulose flow-through; lane 6, pooled fractions of the phosphocellulose column that were loaded on the Q-Sepharose column; lane 7, Q-Sepharose flow-through; lane 8, pooled fractions of the Q-Sepharose column that were loaded on a small heparin-agarose column to be concentrated; lane 9, final CKII preparation. The positions of the molecular weight markers are indicated at the left.

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 (alpha  and alpha ', of Mr 36,000-44,000) associated noncovalently to two beta -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 alpha  subunit was next assessed by using polyclonal antibodies raised against specific epitopes of the alpha - and alpha '-subunits of human CKII (27). The alpha '-subunit is usually shorter than the alpha -subunit (27) and is believed to be the product of a different gene than the alpha -subunit (44). Fig. 5 demonstrates that the platelet CKII is composed of both the alpha - and alpha '-subunit with Mr 36,000 and 33,000, respectively as previously shown (20). Platelet CKII preparation also contains a Mr 26,000 beta -subunit (Fig. 5, beta ). 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 alpha alpha 'beta 2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alpha alpha /beta beta and one that is alpha 'alpha '/beta beta .


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Fig. 5.   Subunit composition of platelet CKII. Partially purified platelet CKII was analyzed by SDS-PAGE followed by transfer to nitrocellulose and immunostaining with polyclonal antibodies raised against synthetic peptides mimicking the amino acid sequence of the alpha - or alpha '-subunit of human CKII (27) or with a polyclonal antibody raised against the beta -subunit of human CKII.

CKII-mediated Phosphorylation of Human Factor V

Localization of Phosphate Incorporation-- Human factor V incorporates radioactivity in the presence of [gamma -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 alpha -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 beta -subunit present in the kinase preparation.


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Fig. 6.   APC digestion of phosphorylated human factor Va. Human factor V was phosphorylated by purified platelet CKII and activated with alpha -thrombin. The stoichiometry of phosphorylation in the experiment shown was ~1.1 mol of phosphate/mol of factor Va. Following SDS-PAGE, radioactive fragments were visualized by autoradiography. Lane 1, phosphorylated human factor V; lane 2, factor V and alpha -thrombin (1 unit/ml); lanes 3-10, factor Va incubated for 1, 3, 5, 10, 20, 30, 45, and 60 min with APC. Molecular weight markers are shown at the left. a, the Mr 220,000 precursor of the light chain (residues 710-2196); b, the Mr 150,000 activation peptide (residues 1019-1545); c, the Mr 71,000 activation peptide (residues 710-1018) (6, 8).

Factor Va is inactivated by APC following three sequential cleavages of the heavy chain of the cofactor. Cleavage at Arg306 only occurs in the presence of a membrane surface and is responsible for the loss of approximately 80% of the cofactor activity (8, 9). Following incubation with APC and PCPS vesicles, aliquots of the mixture were analyzed by SDS-PAGE followed by autoradiography (Fig. 6, lanes 3-10). The data demonstrate that the radioactivity incorporated into the heavy chain of human factor Va disappears during the APC cleavage (Fig. 6, lanes 3-10) as a consequence of cleavage at Arg506. Concomitant with that disappearance, there is increase in the concentration of a radioactive Mr 28,000/26,000 doublet representing the COOH-terminal portion of the factor Va heavy chain (Fig. 6, lanes 3-8), which in turn is cleaved at Arg679. This latter cleavage is associated with the loss in radioactivity of the Mr 28,000/26,000 doublet (Fig. 6, lanes 9 and 10). These data are consistent with incorporation of radioactivity at the COOH-terminal portion of factor Va heavy chain. The most likely and unique candidate for phosphate incorporation within this region (amino acid region 680-709) is Ser692. These data are consistent with previous findings (21) and clearly demonstrate that factor Va heavy chain is a physiological substrate for platelet-mediated phosphorylation as is also the case for fibrinogen (45).

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.


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Fig. 7.   APC inactivation of phosphorylated and native human factor Va. Factor V was phosphorylated and activated with alpha -thrombin using similar conditions as described in Fig. 6. The rates of phosphorylated and native factor Va (200 nM) inactivation by APC (2 nM) in the presence of a membrane surface (PCPS vesicles, 50 µM) were measured by using a clotting assay in the presence of factor V deficient plasma. The percentage of initial cofactor activity of factor Va was plotted as a function of time following the addition of APC. The graph represents the average results found in four different experiments using three different CKII preparations. Filled circles depict inactivation of phosphorylated human factor Va, whereas filled squares show the inactivation of native human factor Va.


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Fig. 8.   Analysis of factor Va inactivation by APC. During one of the experiments shown in Fig. 7, aliquots were also analyzed by SDS-PAGE, followed by transfer to nitrocellulose and immunostaining with a monoclonal antibody that recognizes an epitope on the heavy chain of factor Va between amino acid residues 307 and 506 (34, 35). The thick arrow at the right of each panel identifies the heavy chain of factor Va. The smaller arrow (dotted line) represents the migration of the Mr 75,000 intermediate that derives from the heavy chain of factor Va following cleavage at Arg506. Lanes 1-8 represent membrane-bound human factor Va at 1, 3, 5, 10, 20, 30, 45, and 60 min following the addition of APC. Panel A represents the proteolysis of phosphorylated factor Va whereas panel B depicts the inactivation of native factor Va.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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.


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Fig. 9.   Schematic representation of human factor Va inactivation by APC. The heavy chain portion of the human cofactor (containing 709 amino acids) is composed of two A domains (A1-A2; A1 spanning region 1-303 and A2 spanning region 317-656) associated through a connecting region (amino acids 304-316) (6). The portion 657-709 at the COOH terminus of the heavy chain portion of factor Va contains a cluster of acidic amino acids and Ser692 that is phosphorylated by the platelet CKII (left). Phosphorylated and native plasma factor Va are inactivated by APC in the presence of a membrane surface following cleavages at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure and cleavage at Arg306 and Arg679, which are the inactivating cleavage sites. Phosphorylation of the cofactor increases the rate of cleavage at the lipid-independent site Arg506 (k1), as compared with cleavage at Arg506 of the native cofactor (k'1).

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 alpha -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.

Dagger 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.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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