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P N Walsh

Publications and source records attributed to P N Walsh.

At least 19 recordsLinked to original sources
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

Fine mapping of the high molecular weight kininogen binding site on blood coagulation factor XI through the use of rationally designed synthetic analogs.

Using immunological and chemical cleavage techniques, we have previously identified a domain contained within residues Phe56-Ser86 in the first tandem repeat (A1) of the heavy chain of factor XI which binds high Mr kininogen (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154). We have now chemically synthesized peptides from corresponding homologous regions in the second (A2), third (A3), and fourth (A4) tandem repeats of the heavy chain (A2: Asn145-Ala176; A3: Asn235-Arg266; and A4: Gly326-Lys357). These peptides had no effect on the binding of factor XI to high Mr kininogen. Because of a lack of detailed structural information for the A1 domain, a molecular model of this region was constructed. This hypothetical model made distinct and testable predictions regarding potential surfaces and concomitant secondary structure. Specifically, the resulting structure depicted two juxtaposed beta-stranded stem-loops that, in conjunction with biological information, constitute a candidate surface for contact with high Mr kininogen. The hypothetical A1 model was, consequently, used as a predictive template in the rational design of two synthetic peptides (Val59-Arg70 and Asn72-Lys83). When both these peptides were added together and the binding of factor XI to high Mr kininogen was examined, a synergistic inhibitory effect was observed compared with each peptide added individually. Our data are consistent with the notion that the sequence of amino acids from Val59-Lys83 of the heavy chain of factor XI contains two antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of high Mr kininogen.

Amino Acid Sequence↗
PubMed1992

A low molecular weight platelet inhibitor of factor XIa: purification, characterization, and possible role in blood coagulation.

A low molecular weight platelet inhibitor of factor XIa (PIXI) has been purified 250-fold from releasates of washed and stimulated human platelets. Molecular weight estimates of 8400 and 8500 were determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, although a second band of Mr 5000 was present upon electrophoresis. The inhibitor does not appear to be one of the platelet-specific, heparin-binding proteins, since it neither bound to nor was affected by heparin. An amount of PIXI which inhibited by 50% factor XIa cleavage of the chromogenic substrate S2366 (Pyr-Glu-Pro-Arg-pNA-2H2O) only slightly inhibited (5-9%) factor XIIa, plasma kallikrein, plasmin, and activated protein C and did not inhibit factor Xa, thrombin, tPA, or trypsin, suggesting specificity for factor XIa. Kinetic analyses of the effect of PIXI on factor XIa activity demonstrated mixed-type, noncompetitive inhibition of S2366 cleavage and of factor IX activation with Ki's of 7 x 10(-8) and 3.8 x 10(-9) M, respectively. Immunoblot analysis showed that PIXI is not the inhibitory domain of protease nexin II, a potent inhibitor of factor XIa also secreted from platelets. Amino acid analysis showed that PIXI has no cysteine residues and, therefore, is not a Kunitz-type inhibitor. PIXI can prevent stable complex formation between alpha 1-protease inhibitor and factor XIa light chain as demonstrated by SDS-polyacrylamide gel electrophoresis. The inhibition by PIXI of factor XIa-catalyzed activation of factor IX and its capacity to prevent factor XIa inactivation by alpha 1-protease inhibitor, combined with the specificity of PIXI for factor XIa among serine proteases found in blood, suggest a role for PIXI in the regulation of intrinsic coagulation.

Amino Acid Sequence↗
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↗
PubMed1991

Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.

We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.

Amino Acid Sequence↗
PubMed1991

The 'Open Road' project: real jobs for people with mental handicap.

This article describes the key elements of a three-year innovative project underway in Dublin, Ireland, which is applying the principles of supported employment. It covers the development of more integrated day services for adults with mental handicap, programme participants and activities, and key issues to be resolved in maintaining effective programmes of this kind.

Adult↗
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↗
PubMed1990

Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86.

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.

Amino Acid Sequence↗
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

Inhibitors of 2-ketoglutarate-dependent dioxygenases block aspartyl beta-hydroxylation of recombinant human factor IX in several mammalian expression systems.

While a role has been ascribed to the gamma-carboxyglutamate (Gla) residues in vitamin K-dependent coagulation proteins and the enzyme catalyzing this posttranslational modification has been identified and partially characterized, both the functional significance of a second posttranslationally synthesized amino acid found in these proteins, beta-hydroxyaspartate (Hya), and the aspartyl beta-hydroxylating enzyme remain to be determined. We now report that inhibitors of 2-ketoglutarate-dependent dioxygenases, such as dipyridyl, o-phenanthroline, and pyridine 2,4-dicarboxylate, block hydroxylation of Asp64 in recombinant factor IX molecules produced in three different mammalian expression systems. This hydroxylation was not inhibited by the specific copper chelators 2,9-dimethylphenanthroline or D-penicillamine. The Gla levels in these proteins were unaffected by these compounds and demonstrate that carboxylation proceeds independently of hydroxylation. Using these Hya-deficient recombinant factor IX molecules we demonstrate that this residue does not play a significant role in factor IX binding to endothelial cells under equilibrium conditions. From additional binding studies we have concluded that the Gla domain of factor IX is a major cell binding domain of factor IX. Furthermore, in contrast to studies demonstrating a marked loss of one-stage clotting activity in recombinant factors IX following site-directed mutations of Asp64 to neutral or basic residues (Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., and Brownlee, G. J. (1988) EMBO J. 7, 2053-2061), we have not found a decrease of one-stage clotting activity with Hya-deficient factor IX. Hya-deficient proteins produced in this manner may prove to be more appropriate to elucidate the function of Hya than those produced by site-directed mutagenesis.

2,2'-Dipyridyl↗
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↗
PubMed1989

Functional domains in the heavy-chain region of factor XI: a high molecular weight kininogen-binding site and a substrate-binding site for factor IX.

To probe the molecular interactions of factor XI we have prepared two monoclonal antibodies (MoAbs; 5F7 and 3C1), each of which binds the heavy chain of reduced and alkylated factor XIa. Competitive solid phase radioimmunoassay (RIA) binding studies revealed that 5F7 and 3C1 are directed against different epitopes within factor XI. One antibody (5F7) blocked the surface-mediated proteolytic activation of factor XI and its binding to HMW kininogen, but had no effect on factor-XIa-catalyzed factor IX activation. The other antibody (3C1) is a competitive inhibitor of factor-IX activation by factor XIa, but blocked factor-XI binding to HMW kininogen only at 1,000-fold higher concentration than 5F7. Moreover, HMW kininogen had no effect on the kinetics of factor-XIa-catalyzed factor-IX activation. Furthermore, factor XI CNBr peptide fragments that bind to the 5F7 and 3C1 antibodies were isolated. The peptides that bound to the 5F7 antibody blocked the binding of HMW kininogen to factor XI but did not inhibit factor-XIa-catalyzed factor-IX activation. However, the peptides isolated by the 3C1 antibody inhibited factor-XIa-catalyzed factor-IX activation and had no effect on factor-XI binding to HMW kininogen. Our results indicate that distinct functional domains within the heavy chain region of factor XI are important for the binding of factor XI to HMW kininogen and for activation of factor IX by factor XIa.

Antibodies, Monoclonal↗