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H R Roberts

Publications and source records attributed to H R Roberts.

At least 91 records · Page 5Linked to original sources
PubMed1990

Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain.

Factor IX Alabama is a factor IX variant in which a glycine has been substituted for Asp47 in the first epidermal growth factor (EGF) domain. The structural defect in factor IX Alabama results in a molecule with 10% of normal coagulant activity. The interactions of immunoaffinity-purified factor IX Alabama with its activator, cofactors, and substrate have been investigated to determine the functional defect in the variant. Factor IX Alabama is activated by factor XIa/calcium at near normal rates. Calcium fluorescence-quenching experiments indicate that high affinity calcium binding in the first EGF domain is not altered in factor IX Alabama. The active site of factor IXa Alabama is fully competent to activate factor X in the absence of calcium when using polylysine as a surface to catalyze the reaction. Factor IXa Alabama has only 64% of normal factor IXa activity in the presence of 300 microM CaCl2 in the polylysine-catalyzed system although apparent high affinity calcium binding constants are similar. Factor IXa Alabama has 52-60% of normal activity in a calcium/phospholipid vesicle system. The addition of factor VIIIa to the phospholipid vesicle system decreases the relative rate of factor IXa Alabama to 18-19% of normal. Three-dimensional computer-aided models of the first EGF domain of normal factor IX and factor IX Alabama indicate no major structural alterations resulting from the glycine substitution for Asp47. The model of the first EGF domain of normal factor IX predicts a calcium-binding site involving Asp47, Asp49, Asp64, and Asp65. Our binding data, however, indicate that Asp47 is not necessary to form the high affinity binding site. We conclude that Asp47 in normal factor IX coordinates to the bound calcium, inducing a conformational change in the molecule essential for proper interaction with factor X and factor VIIIa.

Binding Sites↗
PubMed1990

Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B.

We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5' to the proposed translation start site, and 60 bp 3' to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.

Amino Acid Sequence↗
PubMed1990

Calcium ion binding to human and bovine factor X.

Human and bovine factor X contain 11 and 12 glutamyl residues, respectively, within the first 40 amino terminal residues that are post-translationally modified to gamma-carboxyglutamyl (Gla) residues. We have measured calcium ion binding to human factor X by equilibrium dialysis. This is the first examination of calcium ion binding to human factor X. We have also re-examined the equilibrium dialysis binding of calcium ions to bovine facor X in order to compare the two species. The data was analysed using a variety of models that allow for more than one class of binding site and for co-operativity among binding sites. Calcium ion binding to human factor X fits a model that had two classes of sites: one class with a single site that had an affinity of 0.1 mM and a second class with 19 equivalent, non-interacting sites with an average affinity of 3.5 mM. There was no evidence for co-operativity in calcium ion binding. Calcium ion binding to bovine factor X was best stimulated by a model that assumed one tight site, four co-operative sites, and 18 equivalent, non-interacting sites. To examine the co-operativity seen in calcium ion binding to bovine factor X, calcium ion binding to isolated Gla region (residues 1-44) and Gla-domainless factor X was measured by equilibrium dialysis. Calcium ion binding to Gla-domainless factor X was simulated by a model that had two classes of sites: one class with a single site that had an affinity of 0.25 mM, and a second class that had 15 sites with very low affinity sites (greater than 15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1989

Mechanism by which recombinant factor VIIa shortens the aPTT: activation of factor X in the absence of tissue factor.

Clinical trials have recently begun using high concentrations of activated recombinant factor VII (rFVIIa) for the treatment of hemophilic patients with inhibitors. Unexpectedly, the activated partial thromboplastin time (aPTT) was observed to be significantly shortened during infusion of the rFVIIa. To determine the mechanism for this shortening, the effect of rFVIIa on both the prothrombin time (PT) and the aPTT of normal and various factor-deficient plasmas was examined. rFVIIa shortened the PT of all plasmas tested except FX and FV deficient plasmas. rFVIIa also shortened the aPTT of all plasmas tested except FX and FV deficient plasmas. Since there is no added tissue factor (TF) in aPTT reagents, rFVIIa appeared to shorten the aPTT in the absence of TF. To investigate this possibility, the activity of rFVIIa in a purified system containing only FX, phospholipid vesicles (1:1 PS:PC), and calcium was examined. In this system, rFVIIa activated factor X in the absence of TF. If any component of the purified system was omitted, there was no detectable activation of FX. Thus it appears that calcium and phospholipids are required for the activation of FX by rFVIIa in the absence of TF. Increasing the concentration of rFVIIa increased the rate of FX activation, but the rate of activation was always much lower than that observed with even trace amounts of tissue factor. We conclude that high concentrations of rFVIIa, in the presence of calcium and phospholipid, can directly activate FX in the absence of TF and hence account for the shortening of the aPTT in inhibitor patients treated with rFVIIa.

Blood Coagulation Tests↗
PubMed1989

Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo.

Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

Amino Acid Sequence↗
PubMed1989

Molecular defect in factor IXHilo, a hemophilia Bm variant: Arg----Gln at the carboxyterminal cleavage site of the activation peptide.

A genomic DNA library and the enzymatic DNA amplification technique were used to isolate human factor IX coding sequences of a hemophilia Bm variant, factor IXHilo. A point mutation that resulted in the substitution of a glutamine (CAG) for an arginine (CGG) at amino acid 180 was found in exon VI of the factor IX gene (G----A at nucleotide 20519). This mutation alters the carboxy terminal cleavage site for the activation peptide at Arg180-Val181. The arginine residue at the activation peptide cleavage site is conserved in mouse, canine, bovine, and human factor IX, suggesting that the arginine at amino acid 180 is important for normal cleavage. Sequencing of all of the coding regions of factor IXHilo revealed no other mutations. We have also shown that the point mutation in exon VI creates a new Dde I restriction site, which, in combination with the enzymatic DNA amplification technique, provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first report of the molecular defect in a hemophilia Bm patient with a markedly prolonged ox brain prothrombin time.

Amino Acid Sequence↗
PubMed1989

Use of recombinant activated factor VII for treatment of a retropharyngeal hemorrhage in a hemophilic patient with a high titer inhibitor.

Treatment options during an acute hemorrhage for a hemophilic patient who has an inhibitor (antibody) to factor VIII (FVIII) are limited. If the inhibitor titer is high, even massive doses of FVIII are not sufficient to neutralize the antibody. Likewise, immunoadsorption techniques or plasmapheresis cannot remove enough antibody to permit treatment with FVIII. Allergic reactions and cross-reacting inhibitors complicate therapy with porcine FVIII. Prothrombin complex concentrates (PCC) may be effective but the mechanism is unclear. The theory that activated factor VII (FVIIa) is the active principle in PCC prompted our treatment of a patient with a recombinant FVIIa (rFVIIa) product (NOVO Industries). The patient presented with a large retropharyngeal hemorrhage and an initial inhibitor titer of 129 Bethesda units (BU). Despite Autoplex therapy (100U/kg), tracheal compression by the hematoma increased and asphyxiation was imminent. RFVIIa therapy (60 micrograms/kg) was substituted for Autoplex and nine doses were given without complication. The hemorrhage was controlled. By 18 hr breathing was normal and swallowing and speech were greatly improved. Clinically, the patient dramatically responded to rFVIIa. In addition, the purity, the lack of known infectious agents, and the ease of administration make rFVIIa a potentially attractive new therapy. Use of this product also promises to further our understanding of in vivo hemostasis.

Adult↗
PubMed1988

Use of p-aminobenzamidine to monitor activation of trypsin-like serine proteases.

The fluorescent compound p-aminobenzamidine was used to monitor activation of the trypsin-like serine proteases trypsin, thrombin, and blood coagulation factors IXa and Xa. p-Aminobenzamidine, when bound to the activated forms of these proteases but not the corresponding zymogens, displayed an increase in fluorescence. This fluorescence increase was coincident with activation as measured by synthetic substrate hydrolysis, physiological coagulation activity, and the appearance of activation products on gel electrophoresis. The activation of proteolytically modified factor X was also monitored. These results suggest that following p-aminobenzamidine fluorescence is a convenient procedure for monitoring activation of trypsin-like serine proteases.

Amidines↗
PubMed1988

Influence of divalent and monovalent cations on some active site properties of human factor Xa.

The effect of divalent and monovalent cations on the hydrolysis of BzIleGlu(OR)GlyArgpNA(S-2222) was compared to the rate of inactivation of factor Xa by dansyl-GluGlyArg-chloromethylketone(DERG-CK). At substrate concentrations below Km, an approximate four-fold increase in amidase activity was observed in the presence of manganese ions while a three-fold increase was observed with calcium ions. The presence of magnesium ions resulted in a two-fold increase in amidase activity. Similar increases in the rate of inactivation of factor Xa by DERG-CK were observed. Na+ ions had a marked enhancing effect of both factor Xa amidase activity and inactivation by DERG-CK. Kinetic parameters for the hydrolysis of S-2222 by factor Xa were obtained in the presence and absence of Ca++ and Na+. Vmax values increased in the presence of either Ca++ or Na+. Km values increased in the presence of Ca++ while there was a modest decrease in Km in the presence of Na+. It is suggested that the enhanced activity of factor Xa is a reflection of changes in the reactivity of active site residues.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Interaction of polylysine with bovine factor Xa: effect of divalent cations.

Poly-L-lysine has been demonstrated to partially replace biological cofactors in the activation of prothrombin by factor Xa. The present study was initiated to determine if poly-L-lysine has an effect on the enzymatic activity of factor Xa in the absence of prothrombin. At low ionic strength (50 mM Tris-Cl, pH 8.0, ambient temperature), poly-L-lysine inhibits amidase activity (S-2222) of bovine factor Xa with high affinity (Ki = 7 nM). The inhibition was readily reversed by 100 mM NaCl. The inhibition was also markedly reduced by the addition of 1.0 mM CaCl2 but not by MnCl2 or MgCl2. All three metal ions enhance amidase activity in the absence of poly-L-lysine. Poly-L-lysine also inhibits the amidase activity of factor Xa from which the gamma-carboxyglutamic acid domain has been removed by limited proteolysis with chymotrypsin (factor Xa-GD) but with somewhat lower avidity (Ki = 35 nM). As with native factor Xa, calcium ions reduce the observed inhibition while either manganese or magnesium ions are much less effective. The amidase activity of factor Xa-GD is enhanced with any one of the three divalent cations. These results provide additional support for the existence of a functionally significant binding site for calcium ions outside of the gamma-carboxyglutamic domain of factor Xa.

Amidohydrolases↗
PubMed1987

Spontaneous antithrombin in a patient with benign paraprotein.

A 66-year-old man with peptic ulcer disease developed a paraprotein that resulted in a spontaneously prolonged prothrombin time, activated partial thromboplastin time, and thrombin clotting time. Although the reptilase time was normal, the thrombin clotting time failed to correct with the addition of normal plasma, calcium, or protamine sulfate. The patient's purified fibrinogen was normal, but his serum contained an IgG that inhibited the clotting of normal plasma and purified fibrinogen in the presence of thrombin. In contrast to previously described paraproteins, this patient's IgG appeared to inhibit the activity of thrombin per se rather than to interfere with fibrinogen cleavage or fibrin polymerization. Although immunoprecipitation between thrombin and the paraprotein could not be demonstrated, the patient's purified IgG, in the presence of thrombin, decreased the thrombin activity on a chromogenic substrate. Further, increasing concentrations of thrombin overcame the inhibitory effect of the patient's paraprotein. Thus, the patient's paraprotein appeared to possess antithrombin activity.

Aged↗
PubMed1987

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Animals↗
PubMed1987

Factor IXAlabama: a point mutation in a clotting protein results in hemophilia B.

Factor IXAlabama is a variant factor IX molecule responsible for a clinically moderate form of hemophilia B. Twenty-five kilobases (kb) of the variant gene, including seven exons coding for the structural protein, were cloned and characterized. The restriction map and the arrangement of coding regions are identical to those of the normal gene. DNA sequence analysis of the coding regions revealed a single base-pair difference between the gene for factor IXAlabama and the normal factor IX gene. An adenine to guanine transition in the first nucleotide of exon d causes the substitution of a glycine codon (GGT) for the normal aspartic acid codon (GAT). This point mutation results in a single amino acid substitution at residue 47 of the zymogen and represents the genetic defect in factor IXAlabama.

Amino Acid Sequence↗