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Biomedical subjects

J Stenflo

Publications and source records attributed to J Stenflo.

At least 109 records · Page 6Linked to original sources
PubMed1982

Amino acid sequence of the heavy chain of bovine protein C.

The amino acid sequence of the heavy chain of protein C from bovine plasma is reported together with supporting data. The heavy chain contains 260 amino acid residues and it has three carbohydrate side chains. Its structure was determined by sequenator degradation of fragments obtained by enzymatic and chemical cleavage. The sequence is homologous with those of the other serine proteases. On activation, an NH2-terminal tetradecapeptide is removed.

Amino Acid Sequence↗
PubMed1982

Thrombin-catalyzed activation of human coagulation factor V.

Human coagulation factor V was purified from freshly frozen plasma by a method that gave high yields of single chain factor V. The purified protein has an Mr = 330,000 and consists of a single polypeptide chain with the following NH2-terminal sequence: Ala-Gln-Leu-Gly-Gln-Phe-Tyr-Val. Limited proteolysis of factor V by thrombin led to formation of factor Va which has a cofactor activity 25- to 30-fold that of factor V. Two intermediates and four end products were formed in the course of activation. From the NH2 terminus of factor V the end products are termed (apparent molecular weights in parentheses), fragment D (105,000), E (71,000), C1 (150,000), and F1F2 (71-74,000). The carboxyl-terminal fragment F1F2 is a doublet presumably resulting from cleavage of one of two approximately equally sensitive bonds. The end products but not the intermediates resemble those obtained on thrombin activation of bovine factor V, indicating a different order of peptide bond cleavages. The thrombin-catalyzed activation of purified factor V resembles the activation of factor V in plasma as judged from the activation pattern of a 125I-labeled factor V tracer in coagulating plasma. Fragments D and F1F2 are held together by noncovalent interactions and constitute the biologically active factor V molecule as judged from reconstitution experiments utilizing the isolated fragments. The two fragments (E and C1), derived from the interior of the molecule, are activation peptides.

Enzyme Activation↗
PubMed1981

High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein.

Protein S, a recently described vitamin K-dependent plasma protein, is shown to exist in two forms in plasma--free protein and in complex with C4b-binding protein. C4b-binding protein is involved in the regulation of the rate of complement activation. A major proportion of C4b-binding protein in plasma is in complex with protein S. The complex is a major and previously unrecognized component of the group of plasma proteins that adsorbs to barium citrate. The complex dissociates in the presence of NaDodSO4, indicating that C4b-binding protein and protein S are held together by noncovalent bonds. Uncomplexed C4b-binding protein was purified from the supernatant after barium citrate adsorption. On NaDodSO4/polyacrylamide gels without reduction, it appeared to have a slightly faster migration rate than the C4b-binding protein dissociated from the complex with protein S. After reduction, the subunits of the two forms of C4b-binding protein appeared to have identical molecular weights. Furthermore, there is an equilibrium between free and bound protein S in plasma. The role of protein S in the complex is unknown.

Barium↗
PubMed1981

A new fluorogenic peptide substrate for vitamin K-dependent blood coagulation factor, bovine protein C.

Protein C is a precursor of plasma serine proteinases, and its active form inactivates specifically blood coagulation Factor V and Factor VIII. Since a specific and sensitive synthetic substrate for the activated protein C was not known, we studied its amidolytic activity toward 25 fluorogenic peptides of the type peptidyl-4-methylcoumaryl-7-amide (peptidyl MCA). The activated protein C, namely, bovine protein C activated by bovine alpha-thrombin, showed the highest activity toward Boc-Leu-Ser-Thr-Arg-MCA. The enzyme's Km and Kcat values for this substrate were calculated to be 3.3 x 10(-4) M and 8.4 s-1, respectively. Optimum conditions for measurement of activated protein C activity were studied with this substrate. Optimum pH was 8.5. For the maximum activity at pH 8.5, concentrations of 0.1 M NaCl and 1 mM CaCl2 had to be maintained in the reaction mixture. The fluorogenic peptide Boc-Leu-Ser-Thr-Arg-MCA was successfully applied to a simple and accurate assay of protein C during its purification.

Animals↗
PubMed1980

Conformational change of human prothrombin induced by calcium ions: an X-ray scattering study.

The scattered X-ray intensities from dilute solutions of prothrombin and Ca2+-prothrombin at 21 degrees C in 0.1 M Tris-HCl buffer of pH 7.4 indicate that the prothrombin molecule attains a more extended conformation when Ca2+ ions are bound. This is indicated from the distance distribution function: the largest distance within the prothrombin molecule increases from 130 to 150 A when Ca2+ ions are bound; the radius of gyration increases from 35.5 to 39.5 A. A comparison of the experimental scattering curves with theoretical scattering curves calculated for various triaxial bodies shows that the shape of the prothrombin molecule can be represented by two ellipsoids and that the effect of Ca2+ binding can be represented by a change in the angle between their major axes.

Calcium↗
PubMed1980

Inhibitory effect of activated protein C on activation of prothrombin by platelet-bound factor Xa.

Factor Xa binds to a receptor available on the platelet surface after the release reaction. The receptor consists of phospholipid and factor V. Factor Xa bound to the receptor catalyses the activation of prothrombin effectively. The effects of bovine protein C, a vitamin-K-dependent zymogen of a serine protease, on prothrombin activation by platelet-bound bovine factor Xa has been studied. Protein C was found to be activated (protein Ca) by thrombin formed in the prothrombin-platelet-factor Xa incubation. Protein Ca in contrast to the zymogen, protein C, or protein Ca inactivated with diisopropylphosphofluoridate inhibited prothrombin activation by factor Xa in the presence of platelets. protein Ca was found to destroy the receptor by proteolysis whereas direct binding of protein Ca to the receptor could not be demonstrated. The inhibition by protein Ca could be monitored as a parallel decrease in factor Xa binding and prothrombin activation. The receptor was protected by factor Xa from proteolysis by protein Ca. Protein Ca was also found to inhibit the interaction between prothrombin and the factor Xa platelet receptor. These results indicate that protein C after activation may have a role as a regulator of prothrombin activation in vivo.

Animals↗
PubMed1978

Binding of bovine coagulation factor Xa to platelets.

The binding of highly purified bovine coagulation factor Xa to washed bovine platelets was studied. 125I-labeled factor Xa underwent binding to a platelet receptor that became accessible only after induction of the platelet release reaction by thrombin or by the calcium ionophore A 23187. The zymogen factor X did not bind to platelets. The factor Xa binding was saturable, reversible, and correlated with the rate of thrombin formation. The number of factor Xa binding sites per platelet was 290--420 and the apparent association constant was estimated to be 2.8 x 109 to 1.0 x 1010 M-1. Diisoprophyl fluorophosphate-factor Xa bound to the same platelet receptor as factor Xa indicating that limited proteolysis of a receptor protein was not required for binding. The rate of factor Xa binding was rapid (2.1 x 10(6) to 2.9 x 10(6) M-1 s-1) and similar to that preveiously found for the rate of binding of polypeptide hormones to their receptors. Displacement of factor Xa from the platelet receptor by diisopropyl fluorophosphate-factor Xa effectively blocked thrombin formation. Low concentrations of factor Xa catalyze prothrombin activation more effectively in the presence of platelets than in the presence of phospholipid and factor V.

Animals↗
PubMed1978

Bovine protein C: amino acid sequence of the light chain.

The amino acid sequence of the light chain of bovine protein C was determined by sequenator analysis of the carboxymethylated light chain and fragments obtained by cyanogen bromide treatment, tryptic digestion after blocking of lysine residues, and cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole). The sequence was (in the standard one-letter code) A-N-S-F-L-X-X-L-R-P-G-N-V-X-R-X-C-S-X-X-V-C-X-F-X-X-A-R-X-I-F-Q-N-T-X-D-T-M-A-F-W-S-K-Y-S-D-G-D-Q-C-E-D-R-P-S-G-S-P-C-D-L-P-C-C-G-R-G-K-C-I-H-G-L-G-G-F-R-C-D-C-A-E-G-W-E-G-R-F-C-L-H-E-V-R-F-S-N-C-S-A-E-B-G-G-C-A-H-Y-C-M-E-E-E-G-R-R-H-C-S-C-A-P-G-Y-R-L-E-D-D-H-Q-L-C-V-S-K-V-T-F-P-C-G-R-L-G-K-R-M-E-K-K-R-K-T-L. The first eleven glutamic acid residues were carboxylated to gamma-carboxyglutamic acid (X). The NH2-terminal, vitamin K-dependent part showed an extensive homology to both prothrombin and factor X, whereas the rest of the chain showed a strong homology to factor X but little similarity to prothrombin.

Amidohydrolases↗
PubMed1976

A new vitamin K-dependent protein. A phospholipid-binding zymogen of a serine esterase.

Conclusive evidence is presented that a recently purified (Stenflo, J. (1976) J. Biol. Chem. 251, 355-363) vitamin K-dependent protein (arbitrarily referred to as Protein C) which is not related to prothrombin, Factors IX or X is also unrelated to Factor VII. It therefore appears to be a new, previously unrecognized vitamin K-dependent protein. In contrast to prothrombin, which binds to negatively charged phospholipid only in the presence of Ca2+ ions, Protein C, like the other vitamin K-dependent proteins, is a precursor of a serine esterase, presumably a protease, but it does not seem to be necessary for blood coagulation. Although the lipid-binding properties of Protein C may suggest that it is associated with membrane structures, its biological function remains unknown.

Blood Proteins↗