Biochemistry 1994, 33, 12048-12055
12048
High-Affinity, Specific Factor IXa Binding to Platelets Is Mediated in Part by Residues 3-1 1' Syed S. Ahmad,*,$Razia Rawala-Sheikh,$ Wing-Fai Cheung,ll Bradford A. Jameson,*,l Darrel W . Stafford,ll and Peter N. W a l s h ' J ~ ~ ~ # The Sol Sherry Thrombosis Research Center, Department of Biochemistry, and Department of Medicine, Temple University School of Medicine. Philadelphia, Pennsylvania 191 40, Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599, and Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 191 07 Received March 9, 1994; Revised Manuscript Received June 20, 1994"
To identify the amino acids in the Gla domain that mediate factor IXa binding to human platelets, we have used chimeric molecules and point mutations in the Gla domain of recombinant factor IX, based on molecular modeling using the coordinates of the Gla domain of bovine prothrombin, which reveals two surface structures whose sequences differ among factor IX, factor X , and factor VII. Binding to thrombin-activated platelets of factor IXa in the presence of factor VIIIa ( 2 units/mL) and factor X (1.5 pM) revealed a stoichiometry of -550 sites per platelet with a Kd of -0.65 nM compared with a Kd of -2.5 nM in the absence of factor VIIIa and factor X. In contrast, mutations of factor IX to factor X residues at positions 4 and 5 or a t positions 9, 10, and 11 resulted in decreases in the number of sites and affinity of factor IXa binding in the presence or absence of factor VIIIa and factor X. A chimera consisting of the Gla domain of factor VI1 with factor IX residues a t positions 33,34,35,39, and 40 displayed abnormal factor IXa binding and a decreased V,,, and a normal K , for factor X activation, and the replacement of amino acid residues 3-10 with those of factor IX restored normal binding and factor X activation kinetics to this chimeric protein. Our data indicate that the high-affinity, specific binding of factor IXa to activated platelets in the presence or absence of factor VIIIa and factor X is the major determinant of rates of factor X activation and that both factor IXa binding and factor X activation are mediated a t least in part by amino acids exposed on the surface of the Gla domain within positions 3-1 1, possibly by residues 4, 5, 9, 10, and 11. ABSTRACT:
The interaction between blood platelets and coagulation factors is essential for normal coagulation and hemostasis. In our previous studies we have examined the mechanism by which platelets can promote factor X activation by factor IXa (Ahmad et al., 1989a-q 1992a; Rawala-Sheikh et al., 1990). Recently, we began an analysis of the structural features of the factor IX molecule that are important for assembly of the factor X activating complex on the platelet surface (Ahmad et al., 1990,1992b;Rawala-Sheikh et al., 1992). These studies have shown that a major determinant of factor IXa binding to its normal human platelet receptor resides within its y-carboxyglutamic acid (Gla) domain (Rawala-Sheikh et al., 1992). In contrast, the first epidermal growth factor domain of factor IXa does not appear to be involved in factor IXa binding to platelets (Ahmad et al., 1992b). We have This study was supported by research grants from the National InstitutesofHealth (HL45486, HL25661, and HL46213 t0P.N.W. and HL38973 toD.W.S.), bythe W. W. SmithCharitableTrust(toP.".W.), and by The Council For Tobacco Research (Grant 3190 to S.S.A.). * To whom correspondence should be addressed at the Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad St., Philadelphia, PA 19140 (telephone 215-707-4375; Fax 215707-3005). The Sol Sherry Thrombosis Research Center, Temple University School of Medicine. Department of Biochemistry, Temple University School of Medicine. 11 Department of Biology, University of North Carolina. Jefferson Cancer Institute, Thomas Jefferson University. # Department of Medicine, Temple University School of Medicine. a Abstract published in Advance ACS Abstracts, September I , 1994. Abbreviations: EGF, epidermal growth factor; Gla, y-carboxyglutamic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NaDodS04, sodium dodecyl sulfate; PPACK, D-phenylalanylpropyllarginyl chloromethyl ketone.
*
0006-2960/94/0433- 12048$04.50/0
used both Gla-modified and Gla-domainless factor IXa molecules and concluded that factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX (Rawala-Sheikh et al., 1992). The purpose of the present studies was to identify specific amino acid residues within the Gla domain that mediate the binding of factor IXa to platelets. To accomplish this purpose, we have constructed a computer-generated model based upon the known structure of bovine prothrombin fragment 1 in order to identify amino acid residues exposed on the surface of the protein that might comprise a binding site for the platelet receptor. We reasoned that if such amino acid residues were altered to those present in highly homologous proteins such as factor VI1 or factor X, the capacity of the resulting mutated or chimeric protein to bind to platelets might be diminished. The assumption underlying this rationale is that the tertiary structure of the Gla domain is highly conserved in the vitamin K-dependent proteins, such as factor IX, factor X, factor VII, and prothrombin, and that surface residues unique to any particular protein mediate specific protein-protein interactions. Recent studies of bovine aortic endothelial cells using several factor IX-factor VI1 chimeras implicated specific residues within the Gla domain in factor IX binding (Cheung et al., 1991;Toomeyet al., 1992). Thesestudiesdemonstrated, using computer-generated models for the Gla domain of factor IX, that the endothelial cell binding determinant resides on a prominent surface within the first 11 amino acids of factor IX. Recent studies have ruled out any specific binding role for either the first EGF-like domain or the eight amino acid
0 1994 American Chemical Society
Specific Binding of Factor IXa to Platelets hydrophobic stack domain of factor IX in its interaction with its endothelial cell receptor (Cheung et al., 1992). In this paper we have used nine different recombinant factor IX (rFIX) molecules to show that the high-affinity, specific binding of factor IXa to activated platelets in the presence or absence of factor VIIIa and factor X is mediated at least in part by amino acids exposed on the surface of the Gla domain within positions 3-1 1, possibly by residues 4,5,9, 10, and 11.
EXPERIMENTAL PROCEDURES Materials. The chromogenic substrate, S2337 [Bz-Ile-Glu-
(y-piperidy1)Gly-Arg-p-nitroanilide] ,was purchased from AB Kabi Diagnostica (Stockholm, Sweden). p-Aminobenzamidine was obtained from Sigma Chemical Co. (St. Louis, MO). D-Phenylalanylprolylarginylchloromethyl ketone (PPACK) was purchased from Calbiochem-Behring Corp. (San Diego, CA). Carrier-free Na1251was obtained from Amersham Corp., Arlington Heights, IL. All other reagents and chemicals used were the same as previously reported (Ahmad et al., 1989a) and were obtained from Sigma Chemical Co. (St. Louis, MO), Aldrich Chemical Co. (Milwaukee, WI), and Calbiochem-Behring Corp. (San Diego, CA) and were of the highest grade commercially available. Proteins. Human coagulation proteins, including factor IX, factor IXa, factor VIII, factor X, and a-thrombin, were purified, assayed, and characterized as previously published (Ahmad et al., 1989a). The conditions used for activation of factor VI11 with human a-thrombin were identical to those previously published (Ahmad et al., 1989a). All proteins were >98% pure as determined by polyacrylamide slab gel electrophoresis in NaDodS04 (Laemmli, 1970). Protein concentrations were determined by the Bio-Rad dye binding assay according to instructions provided by the manufacturer (BioRad, Richmond, CA). Mutant proteins were prepared and characterized as previously described (Lin et al., 1990). The proteins, expressed in human embryo kidney cells, were purified from cell supernatants as reported previously (Lin et al., 1990; Cheung et al., 1991). Briefly, these proteins were purified on a monoclonal antibody affinity column (Smith et al., 1987) which binds to factor IX molecules that have undergone a normal conformational alteration in the presence of calcium ions. The purified chimeric factor IX molecules demonstrated normal fluorescence quenching (Cheung et al., 1991) in the presence of increasing concentrations of CaCl2 (data not shown). Analysis of Gla was performed at Merck Sharp and Dohme according to the procedure of Przysiecki et al. (1987). All the normal plasma-derived, the wild-type, and the chimeric factor IX molecules were radiolabeled with lZ5Iby the iodogen method as previously described (Ahmad et al., 1989a), and specific radioactivities of all proteins were in the range of (2.0-2.5) X lo6 cpmlpg. Activation of both normal and chimeric factor IX molecules by purified factor XIa was carried out as previously described (Ahmad et al., 1989a). We also utilized thep-aminobenzamidinefluorescenceassay to examine quantitatively the activation of normal, plasma-derived factor IX (IXN) and recombinant factor IX molecules as previously reported (Monroe et al., 1988; Lin et al., 1990). Following gel electrophoresis, autoradiograms of normal and chimeric factor IXa molecules were developed to provide structural characterization of *251-labeledproteins. Both recombinant proteins and factor IXa wild type (factor IXaWt)migrated under reducing conditions as two polypeptide chains of Mr 27 000 and 17 000 representing the heavy and light chains (Figure 1, lanes 2-1 1) and were indistinguishable from plasmaderived factor IXaN (Figure l , lane 2). Labeled factor IXN, which was a single band at M , 60 000 under reducing
Biochemistry, Vol. 33, No. 40, 1994
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conditions, is shown for comparison (Figure 1, lane 1). Proteinstainedgels of all unlabeled proteins used in this study showed entirely similar results (not shown), thus confirming the purity and chain composition of both plasma-derived and recombinant proteins. Binding Experiments. In a typical binding experiment, gel-filtered platelets [(3-4) X 108/mL] in calcium-free 4-(2hydroxyethy1)- 1-piperazineethanesulfonic acid (HEPES) Tyrode's buffer, pH 7.4, were incubated at 37 "C in a 1.5-mL Eppendorf plastic centrifuge tube with mixtures of unlabeled and radiolabeled factor IXa (0.1-20 nM), CaCl2 (5 mM), human a-thrombin (0.1 unit/mL) in the presence or absence of factor X (1.5 pM), and thrombin-activated factor VI11 (2 units/mL) as detailed previously (Ahmad et al., 1989a). Platelets were separated from unbound proteins as previously described (Ahmad et al., 1989a). The data were analyzed, and the number of binding sites and dissociation constants (&) were calculated from the mean of three independent determinations, each done in duplicate, as previously described (Ahmad et al., 1989a) using a Macintosh Quadra 900 computer (Apple Computer, Inc., Cupertino, CA) and the LIGAND program was modified by G. A. McPherson (Elsevier Science Publishers BV, The Netherlands, 1985). Measurement ofRates of FactorXa Formation. Activation of factor X by factor IXaN, factor IXaWt,or chimeric or mutant factor IXa molecules was performed at 37 "C in the presence of thrombin-stimulated gel-filtered platelets, factor VIIIa, and CaCl2 as described previously (Rawala-Sheikh et al., 1990). The details of experimental conditions and concentration of reactants are given in the Results section and in the figure legends. Calculation of Kinetic Constants. The kinetic constants for factor X activation by factor IXa were derived on the basis of a one enzymeone substrate model. The Michaelis constant (K,) and the maximum velocity (V,,,) were calculated from the mean f SEM of four to six independent experiments each performed in duplicate and analyzed by the LevenbergMarquardt method (Marquardt, 1963) utilizing Kaleida Graph (Synergy Software, PCS Inc., Reading, PA) run on a Macintosh Quadra 900 computer (Apple Computer, Inc., Cupertino, CA). Values of Kd were obtained from experiments in which rates of factor X activation were determined at different factor IXa concentrations as described previously (Ahmad et al., 1989a
