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R Rawala-Sheikh

Publications and source records attributed to R Rawala-Sheikh.

10 recordsLinked to original sources
PubMed1994

High-affinity, specific factor IXa binding to platelets is mediated in part by residues 3-11.

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 microM) revealed a stoichiometry of approximately 550 sites per platelet with a Kd of approximately 0.65 nM compared with a Kd of approximately 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 at positions 9, 10, and 11 results 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 VII with factor IX residues at positions 33, 34, 35, 39, and 40 displayed abnormal factor IXa binding and a decreased Vmax and a normal Km 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.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1992

The role of the first growth factor domain of human factor IXa in binding to platelets and in factor X activation.

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the first epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X (Lin, S.-W., Smith, K. J., Welsch, D., and Stafford, D. W. (1990) J. Biol. Chem. 265, 144-150). The chimeric protein, factor IX(Xegf1), as well as the wild-type, factor IXwt, produced in embryo kidney cells, and factor IX isolated from human plasma were radiolabeled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXa(Xegf1) (566 sites/platelet, Kd = 0.69 nM) and factor IXawt (590 sites/platelet, Kd = 0.61 nM) in the presence of factor VIIIa (5 units/ml) and factor X (1.5 microM) compared to factor IXaN (558 sites/platelet, Kd = 0.67 nM). The concentration of factor IXaN, factor IXawt, and factor IXa(Xegf1) required for half-maximal rates of factor Xa formation were 0.63, 0.7, and 0.83 nM, indicating that the Kdapp for binding of factor IXa(Xegf1) to the factor X activating complex on activated platelets is normal. These studies suggest either that the EGF-1 domain of factor IXa is not involved in factor IXa binding to platelets or that the EGF-1 domain from factor X when inserted into factor IXa, suffices to promote normal factor IXa binding.

Autoradiography↗
PubMed1992

Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation.

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, 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.

1-Carboxyglutamic Acid↗
PubMed1990

Comparative platelet binding and kinetic studies with normal and variant factor IXa molecules.

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P. N. (1989) J. Biol. Chem. 264: 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, equilibrium binding studies and kinetic studies of factor X activation were carried out with normal factor IXa and with two variant proteins: factor IXaAlabama (FIXaAL; Asp47----Gly substitution) and factor IXaChapel Hill (FIXaCH; Arg145----His substitution). In the absence of factors VIIIa and X, there were 331 binding sites/platelet for FIXaCH (Kdapp = 2.8 nM), and 540 sites/platelet for FIXaAL (Kdapp = 3.2 nM), compared with 540 sites/platelet (Kdapp = 2.3 nM) for normal factor IXa. The addition of factors VIIIa and X, both at saturating concentrations, had no effect on the number of binding sites for either normal or variant factor IXa, resulted in a decrease in the Kd for normal factor IXa to 0.67 nM, resulted in a suboptimal decrease in Kd for FIXaAL (1.4 nM), and had no effect on the Kd for FIXaCH. Kinetic studies of factor X activation at variable factor IXa concentration confirmed these values of Kd in the presence of factors VIIIa and X. Determination of rates of factor X activation at variable substrate concentrations yielded normal values of catalytic efficiency (kcat/Km) for the variant proteins, thereby indicating that the abnormally low rates of factor X activation obtained were a consequence of the low affinity binding of FIXaAL and FIXaCH to thrombin-activated platelets in the presence of factors VIIIa and X. These studies suggest that the presence of Asp47 and the cleavage of factor IX at Arg145-Ala146 are important structural features required for specific, high affinity factor IXa binding to platelets in the presence of factors VIIIa and X.

Blood Platelets↗
PubMed1990

Kinetics of coagulation factor X activation by platelet-bound factor IXa.

Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd approximately 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S.S., Rawala-Sheikh, R., & Walsh, P.N. (1989) J. Biol. Chem. 264, 3244-3251]. To determine the functional consequences of factor IXa binding to platelets, a detailed kinetic analysis of the effects of platelets, phospholipids, and factor VIII on factor IXa catalyzed factor X activation was done. In the absence of platelets, phospholipids, or factor VIII, the Michaelis constant (Km = 81 microM) was greater than 500-fold higher than the factor X concentration in human plasma. Unactivated platelets and thrombin-activated factor VIII, alone or in combination, had no effect on the kinetic parameters, whereas thrombin-activated platelets caused a major decrease in Km (0.39 microM) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 microM) near that of factor X in plasma and to increase the kcat 24,000-fold to 1240 min-1. Sonicated mixed phosphatidylserine/phosphatidylcholine vesicles (25/75, mol/mol) had kinetic effects similar to those of activated platelets. When factor IXa binding to thrombin-activated platelets and rates of factor X activation were measured simultaneously at saturating concentrations of factor X and factor VIIIa, the kcat was independent of factor IXa concentration, and the mean kcat value was 2391 min-1. The increase in catalytic efficiency (kcat/Km) in the presence of thrombin-activated platelets and factor VIIIa was (17.4 x 10(6))-fold.

Factor IX↗
PubMed1989

Platelet receptor occupancy with factor IXa promotes factor X activation.

To investigate the activated platelet surface as a locus for factor X activation, the functional consequences of factor IXa binding to platelets were studied. The concentration of factor IXa required for half-maximal rates of factor X activation in the presence of factor VIIIa and thrombin-activated platelets was 0.53 nM, which is close to the Kd (0.56 nM) for factor IXa binding to platelets under identical conditions, determined from equilibrium binding studies. In direct comparative experiments, there was a close correspondence between equilibrium binding of factor IXa to thrombin-activated platelets in the presence of factor VIIIa and kinetic determinations of factor X activation rates. Analysis by polyacrylamide gel electrophoresis revealed that 125I-labeled factor IXa bound to platelets was structurally intact and did not form covalent complexes with platelet proteins. Factor IXa active site-inhibited by 5-dimethylaminonaphthalene-1-sulfonyl glutamyl-glycylarginyl chloromethyl ketone was shown to be a competitive inhibitor of factor IXa binding in the absence (Ki = 2.3 nM) and presence (Ki = 0.43 nM) of factor VIIIa and factor X and of factor X activation (Ki = 0.4 nM) by factor IXa in the presence of factor VIIIa, indicating that the generation of factor Xa is not required for factor IXa binding and that factor IXa bound to activated platelets in the presence of factor VIIIa is closely coupled with rates of factor X activation. We conclude that factor IXa bound tightly to a platelet receptor in the presence of factor VIIIa is the enzyme active in factor X activation.

Blood Platelets↗
PubMed1989

Rapid purification of factor IX, factor X and prothrombin by immunoaffinity and ion exchange chromatography.

This study describes a rapid purification of factor IX, factor X and prothrombin by immunoaffinity and ion exchange chromatography. Human factor IX was purified from plasma in 34% yield using barium sulfate adsorption and immunoaffinity purification. The specific clotting activity of purified factor IX was 269 units/mg, with an apparent molecular weight of 57,000 in the presence of sodium dodecyl sulfate on polyacrylamide gels. The immunodepleted, factor-IX deficient plasma was chromatographed on a dextran sulfate agarose column which resolved prothrombin and factor X in highly purified states and with approximately 50% yield. The specific activities of prothrombin and factor X obtained by this procedure were 24 units/mg and 147 units/mg respectively. Both proteins isolated by this method showed a single component on SDS gel electrophoresis and the molecular weights of intact prothrombin and factor X were 72,000 and 67,000 respectively.

Ammonium Sulfate↗
PubMed1989

Comparative interactions of factor IX and factor IXa with human platelets.

Both factor IX and factor IXa were bound to gel filtered platelets in the presence of CaCl2 (2-20 mM) and human alpha-thrombin (0.06-0.2 units/ml) with maximal binding occurring in 10-20 min at 37 degrees C, and rapid reversibility was observed when unlabeled ligands were added in 100-fold molar excess. Competition studies with various coagulation proteins revealed that neither factor XI nor high molecular weight kininogen, at 300-fold molar excess, could compete with 125I-labeled factor IXa for binding sites on thrombin-activated platelets, whereas prothrombin and factor X, in 450-fold molar excess, could displace approximately 15 and 35%, respectively, of bound factor IXa in the absence of added factor VIII. Analysis of saturation binding data in the presence of CaCl2 and thrombin without factors VIII and X indicated the presence of 306 (+/- 57) binding sites per platelet for factor IX (Kd(app) = 2.68 +/- 0.25 nM) and 515 (+/- 39) sites per platelet for factor IXa (Kd = 2.57 +/- 0.14 nM). In the presence of thrombin-activated factor VIII (1-5 units/ml) and factor X (0.15-1.5 microM), the number of sites for factor IX was 316 (+/- 50) with Kd = 2.44 (+/- 0.30) nM and for factor IXa 551 (+/- 48) sites per platelet (Kd = 0.56 +/- 0.05 nM). Studies of competition for bound factor IXa by excess unlabeled factor IX or factor IXa, and direct 125I-labeled factor IXa binding studies in the presence of large molar excesses of factor IX, confirmed the conclusion from these studies that factor IX and factor IXa share approximately 300 low-affinity binding sites per thrombin-activated platelet in the presence of Ca2+ and in the absence of factor VIII and factor X, with an additional 200-250 sites for factor IXa with Kd(app) similar to that for factor IX. The presence of factor VIII and factor X increases by 5-fold the affinity of receptors on thrombin-activated platelets for factor IXa that participate in factor X activation.

Binding, Competitive↗
PubMed1989

Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.

Blood Coagulation Tests↗