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Biomedical subjects

W Kisiel

Publications and source records attributed to W Kisiel.

At least 163 records · Page 9Linked to original sources
PubMed1984

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin.

Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.

Animals↗
PubMed1983

The occurrence of beta-hydroxyaspartic acid in the vitamin K-dependent blood coagulation zymogens.

Previous work has shown that two vitamin K-dependent plasma zymogens, factor X and protein C, each contain one residue of erythro-beta-hydroxyaspartic acid. In the present study, prothrombin, factor VII and factor IX were subjected to amino acid analyses for beta-hydroxyaspartic acid. Factor IX and factor VII each contain one residue of erythro-beta-hydroxyaspartic acid. Edman sequence analyses revealed that this residue occurs at position 64 in human and bovine factor IX. Inasmuch as the nucleotide sequence codes for aspartic acid at this position, it appears highly likely that beta-hydroxyaspartic acid is formed in these proteins by a post-translational hydroxylation of aspartic acid. In contrast, neither human nor bovine prothrombin contain beta-hydroxyaspartic acid.

Amino Acid Sequence↗
PubMed1983

Complete amino acid sequence of the light chain of human blood coagulation factor X: evidence for identification of residue 63 as beta-hydroxyaspartic acid.

The complete amino acid sequence of the light chain of human blood coagulation factor X has been determined by automated Edman degradation of peptides isolated from chemical and enzymatic digests of the carboxymethylated light chain. The protein consists of 139 amino acid residues, which include 11 residues of gamma-carboxyglutamic acid. The first 100 residues of the human factor X light chain exhibit approximately 80% homology when compared to the amino-terminal sequence of bovine factor X light chain. This homology decreases to approximately 50% in the remaining 39 residues of the carboxyl-terminal region of the protein. Proton nuclear magnetic resonance spectroscopy and mass spectrometry analyses of isolated residue 63 identified this residue as L-erythro-beta-hydroxyaspartic acid, a hitherto unrecognized amino acid in proteins. Evidence is also presented for the presence of this residue in the corresponding regions of the light chains of bovine factor X and bovine protein C. The biological function of beta-hydroxyaspartic acid in these proteins is unknown.

Amino Acid Sequence↗
PubMed1983

Use of human factor VIIa in the treatment of two hemophilia A patients with high-titer inhibitors.

Two patients with hemophilia A complicated with high-titer alloantibodies have been treated by repeated infusions of microgram quantities of pure human Factor VIIa. Patient 1 was presented with a gastrocnemius muscle bleeding that involved the knee joint. Upon treatment with Factor VIIa the circumference of the muscle decreased and joint mobility increased substantially. Patient 2 was given Factor VIIa concurrent with tranexamic acid in association with the extraction of two primary molars. No significant gingival bleeding occurred after Factor VIIa and tranexamic acid treatment. Furthermore, no deleterious side effects or increase of the alloantibody level were observed in either patient throughout the Factor VIIa infusion. These results, although limited and preliminary in nature, suggest that trace quantities of Factor VIIa can act as a Factor VIII bypassing activity and restore hemostasis in these patients.

Adolescent↗
PubMed1983

A technique for specific removal of factor IX alloantibodies from human plasma: partial characterization of the alloantibodies.

A method for specific removal of large amounts of factor IX:C alloantibodies by a resin to which highly purified factor IX was linked (factor IX CH-Sepharose) is described. Factor IX was isolated from human plasma by a three-step procedure, including barium citrate adsorption and elution, DEAE-Sepharose CL-6B chromatography, and dextran sulfate agarose chromatography. Approximately 100 mg factor IX was obtained from 60 liters of plasma. The preparation was about 95% pure as judged by SDS-PAA gel electrophoresis. Its specific coagulant activity was 160 U/mg (IX) and its factor IX clotting antigen (IX:Ag) 500-600 U/mg. Essentially quantitative coupling of the factor IX preparation to activated CH-Sepharose 4B was obtained (4 mg factor IX/ml gel; 2300-3000 U/IX:Ag/ml). This resin bound 1500-2000 U factor IX inhibitor/ml gel and could be re-used at least 5 times without any loss in binding capacity. The binding capacity was dependent on the flow rate. No signs of activation of the coagulation, fibrinolytic, or complement system were observed in vitro. Using this factor IX resin, factor IX alloantibodies were isolated and found to consist of two portions, one minor bound to the resin only in the presence of Ca2+ and another major portion Ca2+ independent. The specific inhibitory activity/milligram IgG of the Ca2+-dependent alloantibodies was about 5 times higher in the presence of Ca2+. It is concluded that 25 ml of the factor IX resin described can remove about 40,000 factor IX inhibitor units (comparable to 120,000 Bethesda U) in one run, provided the flow rate does not exceed 20 ml/hr. By using such a technique for removal of antibodies it seems feasible to convert hemophilia-B patients complicated with inhibitors against factor IX into ordinary hemophilia-B patients for treatment at an emergency or in association with major surgery.

Calcium↗
PubMed1983

Enzymological aspects of blood coagulation.

Recent studies on the mechanism of initiation and regulation of blood coagulation are reviewed. In the intrinsic blood coagulation pathway, factor XII, prekallikrein (or factor XI) and high molecular weight kininogen from a complex on an anionic surface, such as exposed subendothelium at the site of vascular trauma. In complex, zymogen factor XII activates prekallikrein (or factor XI) by limited proteolysis to initiate the coagulation cascade. A similar initiating mechanism may be operative in the extrinsic pathway, where zymogen factor VII, complexed with a lipoprotein (tissue factor) and calcium ions, converts factor X to factor Xa. Factor Xa converts prothrombin to thrombin which converts soluble fibrinogen to an insoluble fibrin network which physically arrests the flow of blood from the damaged vasculature. In addition, thrombin converts protein C to activated protein C. Activated protein C functions as a negative regulator in the coagulation process by degrading factor VIIIa and factor Va.

Blood Coagulation↗
PubMed1982

Studies of the capacity of factor Xa to protect factor Va from inactivation by activated protein C.

Blood coagulation Factor Xa and activated protein C are both serine proteases derived from circulating, vitamin K-dependent precursors. They express, respectively, procoagulant and anticoagulant properties through Ca2+ and phospholipid-dependent interactions with coagulation Factor Va. The present studies were undertaken to determine whether Factor Xa and activated protein C interact independently or competitively with Factor Va. The interactions were assessed by examining the activated protein C-catalyzed inactivation of Factor Va in the absence and presence of Factor Xa at various concentrations. The results indicated that the two proteins compete for Factor Va and that Factor Xa protects Factor Va from inactivation by activated protein C. The ability of Factor Xa to protect Factor Va was observed either in the absence of a Factor Xa substrate or under conditions in which the complex of Factor Va and Factor Xa was engaged in the conversion of substrate (prethrombin 1) to thrombin.

Enzyme Activation↗
PubMed1982

Evidence of normal functional levels of activated protein C inhibitor in combined Factor V/VIII deficiency disease.

Human activated protein C (APC) is a plasma serine protease that possesses amidolytic and anticoagulant activity. The rate at which the amidolytic and anticoagulant activity of APC was neutralized in normal plasma was essentially identical to that observed in plasma obtained from four individuals with combined Factor V/VIII deficiency disease. Incubation of radioiodinated APC with either normal human plasma or the combined Factor V/VIII-deficient plasmas resulted in the formation of a stable complex (Mr = 96,000) of the enzyme and a plasma protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pretreatment of the radiolabeled APC with diisopropyl fluorophosphate prevented the formation of the enzyme-protein complex. On the basis of its ability to form a complex with radiolabeled APC, the APC-binding protein was purified to homogeneity from normal human plasma by ammonium sulfate fractionation, heparin-agarose chromatography, and QAE-Sephadex A-50 chromatography. The APC-binding protein (Mr = 54,000) is a glycoprotein, and possesses an amino-terminal sequence of Gly-Arg-Thr-Cys-Pro-Lys-Pro-Asp. The amino-terminal sequence of the APC-binding protein exhibited considerable homology with bovine colostrum inhibitor and pancreatic trypsin inhibitor, but no apparent sequence homology with the plasma serine protease inhibitors. Affinity-purified antibody against APC-binding protein immunoprecipitated a complex of radiolabeled APC and native APC-binding protein from normal human plasma. Complex formation was virtually eliminated in plasma immunodepleted of the APC-binding protein. Quantitative electroimmunoassay indicated essentially equal levels of APC-binding protein antigen in normal plasma compared with plasma from four patients with combined Factor V/VIII deficiency disease.

Blood Proteins↗
PubMed1981

Interaction of calcium with bovine plasma protein C.

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.

Animals↗
PubMed1981

Studies on the structures of the carbohydrate moiety of human prothrombin.

Human prothrombin contains three asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and Nab3H4 reduction. All of the oligosaccharides contain N-acetylneuraminic acid. The neutral oligosaccharides obtained from all acidic oligosaccharides by sialidase digestion are identical. By the combination of sequential exoglycosidase digestion and methylation analysis, the structures of the asparagine-linked sugar chains of human prothrombin were confirmed to be as follows: (sequence in text).

Carbohydrate Sequence↗
PubMed1979

Molecular properties of the Factor V-activating enzyme from Russell's viper venom.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.

Amino Acid Sequence↗
PubMed1979

The carbohydrate of bovine prothrombin. Occurrence of Gal beta 1 leads to 3GlcNAc grouping in asparagine-linked sugar chains.

Bovine prothrombin contains three asparagine-linked sugar chains in 1 molecule. The sugar chains were quantitatively released from the polypeptide backbone by hydrazinolysis. All of the oligosaccharides thus obtained contain N-acetylneuraminic acid. Sialidase treatment of these acidic oligosaccharides released three isomeric oligosaccharides, N-1, N-2 and N-3. N-3 was a typical complex type asparagine-linked sugar chain widely found in other glycoprotein, while N-1 and N-2 were unique, because they contain Gal beta 1 leads to 3GlcNAc grouping in the outer chain moiety. By comparing the data of methylation analysis of the acidic oligosaccharides before and after sialidase treatment, the structures of the sugar chains of bovine prothrombin were confirmed as a mixture of NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn and their partially desialized forms.

Acetylglucosamine↗
PubMed1979

Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin.

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.

Blood Coagulation↗
PubMed1978

Interaction of vitamin K dependent proteins with membranes.

The membrane-binding characteristics of six vitamin K dependent plasma proteins, which have homologous amino acid sequences, were compared. All of these proteins display calcium-dependent membrane binding and the identified equilibria for protein-membrane binding are qualitatively the same for all proteins. Quantitative characteristics of these protein-membrane interactions allow organization into distinct subgroups. Protein C and factor VII form a subgroup which has extemely low affinity for bilayer membranes; prothrombin, factor X, and protein S form the tightest complexes with membranes and factor IX displays intermediate affinity. In the presence of manganese (which substitutes for calcium in a cation-dependent protein transition), calcium titration of protein-membrane binding shows the same calcium dependence for all proteins except prothrombin which requires lower calcium. These protein-membrane binding characteristics agree very well with the relatedness of these proteins based on their partial amino-terminal sequences.

Amino Acid Sequence↗