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H R Roberts

Publications and source records attributed to H R Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1986

p-Amidino esters as irreversible inhibitors of factors IXa and Xa and thrombin.

A number of inhibitors of thrombin and factor Xa have been described; however, only one inhibitor of factor IXa has been reported. This compound, dansyl-Glu-Gly-Arg chloromethyl ketone (DEGER), inhibits porcine factor IXa with a second-order rate constant of 2.2 X 10(4) M-1 min-1. We now describe the synthesis and characterization of three p-amidinophenyl esters that inhibit human factor IXa with second-order rate constants comparable to those observed with human and bovine factor Xa and alpha-thrombin. These rate constants of inhibition, moreover, are 2-30-fold greater than observed when DEGER is employed to inhibit porcine factor IXa. Additional advantages of these derivatives include their ease of synthesis and low degree of toxicity. The p-amidinophenyl ester of benzoic acid was employed to inhibit human factor IXa, and the plasma clearance of the protein was studied in mice. These experiments demonstrate for the first time that the endothelial binding previously reported with factor IXa is independent of the active site, a finding similar to the behavior observed with factor Xa and alpha-thrombin in this and previous reports.

Acylation↗
PubMed1986

Hemophilic patients with an inhibitor to factor VIII treated with high dose factor VIII concentrate. Results of a collaborative study for the evaluation of factor VIII inhibitor titer, recovery and half life of infused factor VIII.

The long term treatment of hemophilic patients with an inhibitor to Factor VIII has been difficult although some success with immunosuppressive agents has been reported. Eighteen hemophilic patients, mainly from Bonn in Germany, but also from other countries, have completed a high dose Factor VIII treatment in an attempt to reduce their inhibitor titer and induce "immune tolerance" to Factor VIII. Plasma samples from the 18 patients collected before and after infusion of 50 units Factor VIII/kg body weight were sent to five laboratories to evaluate inhibitor titer, Factor VIII recovery and half life. This collaborative study demonstrated a close correlation from one laboratory to another concerning inhibitor titration and Factor VIII recovery. The conclusion of the study is that all patients treated with this protocol showed an undetectable or low inhibitor titer against Factor VIII indicating that they can be efficiently treated with Factor VIII.

Adolescent↗
PubMed1986

Granulocyte lysosomal cationic protein alters fibrin assembly: a possible mechanism for granulocyte control of clot structure.

The effect of highly positively charged molecules on the assembly and structure of thrombin-induced fibrin gels was studied with turbidity techniques. Lysosomal cationic proteins (LCP) extracted from rabbit heterophils produced enhanced lateral association of fibrin fibers. A crude extract of LCP had little effect on kinetics but produced an increase in the final fiber mass/length ratios. A purified fraction, LCP-5, of the crude extract enhanced both the initial rate of increase and final turbidity of fibrin gels. Mass/length ratios obtained for fibrin fibers as a function of LCP-5 concentration varied from 4.3 X 10(12) in the absence of LCP-5 to 7.8 X 10(12) daltons/cm at 1 microgram LCP-5/ml. Similar studies were conducted with protamine as the perturbant of fibrin assembly. Mass/length ratios were increased from 5.7 X 10(12) daltons/cm with no protamine to 8.5 X 10(12) daltons/cm at 100 micrograms protamine per milliliter. High concentrations of both LCP and protamine precipitated fibrinogen. This phenomenon may imply a mechanism by which the blood cellular components may modulate clot structure through the release of highly charged molecules such as LCP-5.

Animals↗
PubMed1985

Comparison of lipid binding and kinetic properties of normal, variant, and gamma-carboxyglutamic acid modified human factor IX and factor IXa.

The abilities of normal and three abnormal factor IXa molecules to activate factor X and to bind to phospholipid membranes have been compared to define the contributions of protein-lipid interactions and factor IXa light chain-heavy chain interactions to the functioning of this protein. The abnormal proteins studied had altered amino acid residues in their light chains. The heavy-chain regions, containing the active site serine and histidine residues, were normal in the abnormal proteins on the basis of titration by antithrombin III. The binding constants (Kd) for normal (N), variant [Chapel Hill (CH) and Alabama (AL)], and gamma-carboxyglutamic acid (Gla) modified (MOD) factors IX and IXa to phosphatidylserine (PS)/phosphatidylcholine (PC) small, unilamellar vesicles (SUV) were measured by 90 degrees light scattering. The Kd values for factor IXN binding were quite sensitive to the PS content of the membrane but less sensitive to Ca2+ concentrations between 0.5 and 10 mM. The zymogen and activated forms of both normal and abnormal factor IX bound with similar affinities to PS/PC (30/70) SUV. In the cases of factor IXaN and factor IXaAL, but not factor IXaCH or factor IXaMOD, irreversible changes in scattering intensity suggested protein-induced vesicle fusion. Since the activation peptide is not released from factor IXaCH, the normal interaction of factor IXa with a membrane must require the release of the activation peptide and the presence of intact Gla residues. The rate of factor X activation by normal and abnormal factor IXa was obtained by using a chromogenic substrate for factor Xa in the presence of PS/PC (30/70) SUV and 5 mM Ca2+.

1-Carboxyglutamic Acid↗
PubMed1985

Activation of normal and abnormal human factor IX with trypsin.

Human factor IX is activated to factor IXa beta when factor XIa cleaves two peptide bonds, Arg 145-Ala 146 and Arg 180-Val 181, to release an activation peptide. In factor IX Chapel Hill (IXCH), isolated from a hemophilia B patient with a mild bleeding disorder, the arginine 145 residue has been replaced with a histidine. Thus factor IXCH is activated by factor XIa by cleaving only at the Arg 180-Val 181 bond, leaving the activation peptide attached, and resulting in an activated species, factor IXa alpha CH, that, like normal factor IXa alpha, is only 20% as active as factor IXa beta. It is reported that both factor IX and factor IXCH could be activated by trypsin to forms of factor IXa beta and factor IXa beta CH that had clotting activities identical to factor XIa-activated factor IX. Amino-terminal amino acid sequence analysis showed that trypsin cleaved factor IX at the same bonds as did factor XIa; factor IXCH was cleaved at the Arg 180-Val 181 bond, as normal, and was cleaved near the histidine 145, at the Lys 142-Leu 143 bond, releasing a slightly larger activation peptide than from normal factor IXa beta. Metal ions had no effect on the rate of activation of factor IX by trypsin; however, metal ions had a profound effect on the rate at which further incubation with trypsin inactivated factor IXa. Calcium and manganese protected factor IXa from inactivation by trypsin more effectively than magnesium, which was more effective than no metal ion. It is concluded that trypsin can activate normal factor IX and factor IXCH to fully active IXa beta forms.

Amino Acid Sequence↗
PubMed1985

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F., Bourne, C.D., Hoke, R.A., Koehler, K.A., and Hiskey, R.G. (1984) Anal. Biochem. 139, 82-90). The modified enzyme was evaluated for coagulant activity and calcium-dependent fluorescence quenching. [14C]Formaldehyde was employed to allow quantitation of the modification and to facilitate localization of the modified residues in the primary structure of factor IXa. In the presence of the [14C]formaldehyde/morpholine reagent, factor IXa rapidly lost coagulant activity, which corresponded to incorporation of radiolabel. Examination of the relationship between protein modification (radiolabel incorporation) and the loss of coagulant activity suggested that modification of 1 mol of Gla/mol of factor IXa results in complete loss of factor IXa coagulant activity. Primary structure analysis of the radioactivity labeled factor IXa suggested that modification of any one of 11 Gla residues was responsible for the loss of coagulant activity. In the presence of calcium, modified factor IXa exhibited a smaller Gla-dependent decrease in protein fluorescence than native factor IXa, but the Gla-independent fluorescence change was the same for both proteins. It therefore appears that the Gla domain of factor IXa must be completely intact for the enzyme to undergo a functionally important calcium-dependent conformational change necessary for coagulant activity.

1-Carboxyglutamic Acid↗
PubMed1985

Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX.

We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX.

Alleles↗
PubMed1985

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.

Blood Coagulation↗
PubMed1985

Heparin-enhanced inhibitors during reversible disseminated intravascular coagulation.

Intravenous injection of homologous lung or brain tissue thromboplastin in dogs under general anesthesia induced changes of conventional hemostasis variables consistent with acute DIC (prolongation of prothrombin times, thrombin times, APTT, drop of fibrinogen and a transient reduction of the platelet count). The animals reacted with accelerated respiration and pulse rates. After recovery from anesthesia they resumed their normal activity as before. Fibrinogen reached a minimum within 40 min after the DIC trigger dose had been injected. Dependent on the size of the latter up to 80% of clottable fibrinogen was consumed. No consumption of antithrombin III and heparin cofactor II could be demonstrated by functional assays based on thrombin inhibition by diluted plasma in the presence of heparin or dermatan sulfate. Prothrombin measured amidolytically by an Echis Carinatus venom assay remained practically unchanged. These findings are consistent with free thrombin concentrations in the nanomolar range sufficient to clot fibrinogen rapidly without visibly affecting the up to 1,000 fold higher concentrations of inhibitors and prothrombin. Heparin administered before tissue thromboplastin virtually suppressed the evolution of DIC but its protective effect was overcome by higher trigger doses. Heparin injected after the induction of DIC had no protective effect. The reversible DIC model in dogs may be a promising tool to study activated coagulation in vivo at practically constant inhibitor concentrations. One dog can be used for several acute experiments with homologous tissue thromboplastin, thus the number of animals and their costs may remain within reasonable limits.

Animals↗
PubMed1985

Structure and function of factor IX: defects in haemophilia B.

The genetics of haemophilia B and the structure-function relationships of factor IX interactions with cofactors and substrates have been reviewed. Emphasis has been placed on contributions to our understanding made by analysis of variants. Amino acid substitutions at or near the site of activation lead to inactive factor IX or to factor IX species with decreased clotting activity. Release of the activation peptide is necessary for optimal interaction of factor IX with its cofactors and substrates. Abnormalities in the calcium binding region, whether Gla independent or dependent, also decrease clotting activity. The defects in haemophilia Bm variants somehow affect factor VII-tissue factor interactions with factor X. Other mutations may affect the factor IX heavy chain, probably at or near the active site. Amino acid substitutions may cause conformational changes in factor IX that interfere with other interactions such as with antithrombin III and factor VIII. Recombinant DNA techniques have been employed to analyse normal and abnormal factor IX genes. DNA sequence analysis of factor IX cDNA clones revealed the primary structure of the mature protein and a predicted leader peptide. Knowledge of the primary sequence of factor IX allowed identification of the specific defect in the factor IX Chapel Hill variant. Analysis of normal factor IX genomic clones has determined that the 35 kb gene is composed of eight coding exons and seven intervening sequences. Sequence analysis of the CRM+ variants will identify mutations disrupting the normal interactions of factor IX. Southern analysis of CRM- variants has revealed gross factor IX gene deletions in some cases. Such deletions have been employed for carrier deletion in some families. Restriction fragment length polymorphisms in the factor IX gene have also proven useful for carrier identification. Manipulations of the cloned factor IX gene to make specific mutations in vitro and improvements in the technology for expression of deliberately modified genes will further elucidate the relationships between factor IX structure and function.

Amino Acid Sequence↗
PubMed1985

Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody.

An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil-T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF-binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF-binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those of the Ab occurring in inhibitor von Willebrand's disease in which vWF inhibitor and binding values were similar, with a strong anamnestic response. The findings indicate that the vWS Ab binds to an epitope on the molecular vWF in such a way that causes only limited inhibition of vWF/ristocetin function and no inhibition of vWF/botrocetin function, suggesting that these two functional domains are at separate sites.

Aged↗
PubMed1984

Inactivation of human blood coagulation factor X by chemical modification of gamma-carboxyglutamic acid residues.

The inactivation of human factor X by incubation with a reagent known to chemically modify gamma-carboxyglutamic acid to gamma-methylene glutamic acid was studied. Incubation of factor X at pH 5.0 with a preincubated formaldehyde/morpholine mixture (0.9 M/1.0 M) resulted in a progressive decrease in factor X coagulant activity. In the presence of calcium (20 mM) the rate of factor X inactivation was decreased -3-fold. By using [14]C-formaldehyde, modified-factor X (less than 5% residual activity) was found to contain 7 mols of [14]C per mol of protein. Modified-factor X was not activated by Russell's viper venom in the presence of calcium, suggesting that the loss of coagulant activity was related to the inability of modified-factor X to be activated.

1-Carboxyglutamic Acid↗
PubMed1984

Self-association of bovine prothrombin fragment 1 in the presence of metal ions. Use of a covalent cross-linking reagent to study the reaction.

The interaction of metal ions with prothrombin fragment 1 has been shown by other investigators to result in conformational change(s). Previous studies based on hydrodynamic measurements from other laboratories have suggested that prothrombin fragment 1 will, in addition to undergoing conformational change, self-associate in the presence of metal ions but there is a question regarding the specificity and role of metal ions in this process. The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin fragment 1 and has focused on factors influencing the covalent dimerization of bovine prothrombin fragment 1 during reaction with this reagent. Optimal dimerization of bovine prothrombin fragment 1 required the involvement of cations in two separable processes. The "first role" is probably a reflection of a slow conformational change in the bovine prothrombin fragment 1 representing isomerization of a proline-containing peptide bond (Marsh, H. C., Scott, M. E., Hiskey, R. G., and Koehler, K. A. (1979) Biochem. J. 183, 513-517). The "second role" most likely reflects ion bridging with another prothrombin fragment 1 molecule. The ion specificities of these two roles are clearly different. Ions stabilizing the proper conformation (first role) are Ca2+, Sr2+, Mg2+, Mn2+, with Ba2+ having less capability in filling this role. Ions which have activity in the second role (ion bridge formation) are Ca2+, Sr2+, and Ba2+. Mn2+ and Mg2+ cannot fulfill the requirements of this second role and actually inhibit the function of Ca2+.

Animals↗
PubMed1984

Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors.

The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.

Animals↗
PubMed1984

Overview of inhibitors to factor VIII and IX.

The studies related to inhibitors of factor VIII and IX have clearly shown that these inhibitors are antibodies whether they arise in hemophiliacs lacking one of the factors or in patients with diseases associated with altered immune functions. Some of the most interesting data relates to the etiology of these inhibitors. If inhibitors are more likely to arise in CRM- hemophilia patients and if inhibitor development is familial, is it possible to define beforehand which patients are likely to develop inhibitors? If a subpopulation of patients at risk for inhibitor development could be identified, how would one prevent the event from occurring? Should these patients receive immunosuppressive agents prior to infusion of the missing factor? What is the relationship between the actual gene defect and inhibitor development? Is the prevalence of inhibitors in hemophilia B less common and if so are severely affected hemophilia A patients more likely to exhibit gross gene defects? Are there relationships between gene defect and immune response genes? Other major questions involve therapy. Can one induce specific immune tolerance at acceptable cost using low doses of factor concentrates or would it be better to develop other ways of eradicating inhibitors after they appear? What will be the future use of concentrates designed to by-pass the inhibitor, and is bypassing activity due to factors that are complexed to lipids or other substances that protect them from inhibitors? This overview has raised more questions than answers, but perhaps that is the purpose of an overview.

Animals↗