EC:3.4.21.69 - FACTA Search

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Query: EC:3.4.21.69 (APC)
16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study, including 33 consecutive patients was designed to assess the haemostatic alterations occurring during repair of thoracoabdominal aneurysms. The surgical procedure consisted in Dacron graft replacement of the diseased aorta, using neither cardiopulmonary bypass, nor any shunting technique, nor any heparin. Blood samples were drawn before anaesthesia, before and 30 min after unclamping, and on the first postoperative day. The measured parameters were: haematocrit, platelet count, bleeding, activated cephalin, thrombin and prothrombin times, and concentrations of fibrinogen, factors V, VII, X and II, anti-thrombin III, proteins C and S, fibrin degradation products, D-dimers, alpha 2-antiplasmin, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor, and serum protein. Eight patients developed severe multiple haemorrhages; 3 of them died during the procedure because of uncontrollable bleeding. Although the measured parameters were similar in the "bleeding" and control (n = 25) groups before surgery, there was, before unclamping in the first group, an important increase in activated cephalin and thrombin times, with a fall in concentrations of factor II and V, protein C, fibrinogen, and alpha 2-antiplasmin, and in platelet numbers. After unclamping, these changes worsened further, with an increase in prothrombin time and in fibrinogen levels (0,8 g.l-1), without any increase in fibrin degradation products. Abnormal bleeding started about 30 min after this in all the patients of the "bleeding" group. These changes, involving the fibrinolytic system as well as a fall in concentration of all the coagulation factors, can probably be partly explained by the clamping and unclamping of mesenteric vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Mechanisms and prediction of hemorrhagic complications during surgery of thoraco-abdominal aortic aneurysms]. 224 Jun 94

Vitamin K functions in animal cells as the cofactor of the enzyme vitamin K-dependent carboxylase which catalyzes the post-translational formation of gamma-carboxyglutamyl (Gla) residues in specific vitamin K-dependent proteins. These proteins include four blood coagulation factors (prothrombin and Factors VII, IX and X), other plasma proteins (protein C, protein S and protein Z), two proteins from bone (osteocalcin or bone Gla-protein and matrix Gla-protein), and other proteins from lung, kidney, spleen, testis, placenta and other tissues. In the proteins involved in blood coagulation the Gla residues are mandatory for the activation of the inactive proenzymes; this process occurs on phospholipid surfaces to which the proenzymes are bound via Gla residues and calcium ions. The energy needed in the carboxylation reaction is obtained from the oxidation of vitamin K hydroquinone to 2,3-epoxide of the vitamin. Specific enzymes, vitamin K epoxide reductase and vitamin K quinone reductases, catalyze consecutive reactions in which the vitamin K hydroquinone is regenerated, thus allowing continued use of the vitamin K molecule for the carboxylations. The oral anticoagulants, derivatives of 4-hydroxycoumarin and indan-1,3-dione, used as therapeutic agents in thromboembolic disease, are antagonists to vitamin K preventing the catalytic use of vitamin K in the carboxylations by irreversibly inhibiting vitamin K epoxide reductase.
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PMID:The metabolic functions and mechanism of action of vitamin K. 224 79

The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes. These results suggest meizothrombin generation is targeted toward anticoagulant function such as protein C activation, whereas alpha-thrombin generation is targeted toward procoagulant functions, such as fibrinogen clotting, factor V activation, and platelet aggregation.
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PMID:Multiple active forms of thrombin. IV. Relative activities of meizothrombins. 235 15

A family with inherited combined deficiency of factor V and von Willebrand factor (vWF) is reported. Hematological examination of 41 year-old female proband and her younger brother revealed prolonged prothrombin time and Kaolin partial thromboplastin time. The level of both factor V activity and factor V antigen markedly decreased, below 15% of normal. The decreased levels of factor VIII activity and vWF activity are also seen. Furthermore, abnormal mobilities were observed in crossed immunoelectrophoresis. The protein C, S antigens and activities, and protein C inhibitor activity were within normal. Four sons have received the 50% levels of factor V from their parents. One of them also showed the 50% of factor VIII and vWF activities. From above results, this family is thought to be a case of inherited deficiency of factor V and vWF, which are transmitted as an autosomal trait apparently.
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PMID:[A family of congenital combined deficiency of factor V and von Willebrand factor]. 236 42

To determine whether children treated with chronic peritoneal dialysis have a hypercoagulable state, various coagulation and fibrinolytic factor concentrations or activities were measured in 17 children undergoing chronic peritoneal dialysis. The patients had significantly increased activities of factors VII and VIII, and increased concentrations of von Willebrand factor (vWF), fibrinogen, factor XIIIA and factor XIIIS compared to reference values (P less than 0.001 in each case). The activated partial thromboplastin time was prolonged (P less than 0.001) and the thrombin clotting time was decreased (P less than 0.05) in these children. The prothrombin time and activities of factors XII, XI, IX, X, V and II were not significantly different from control values. Protein C concentrations were similar to normal, but antithrombin III concentrations were increased (P less than 0.05). Within the fibrinolytic pathway, decreased concentrations of plasminogen were found (P less than 0.001) and the concentrations of alpha-2-antiplasmin were increased (P less than 0.001). The plasma albumin concentration was below 33 g/l in 13 of the 17 children. The duration of treatment with peritoneal dialysis was directly correlated with vWF concentrations (P less than 0.001) and inversely correlated with factor VII concentrations (P less than 0.01). Of these patients 2 have since had clinical thrombotic episodes. The coagulation abnormalities found may have a role in the occurrence of thrombosis complicating renal transplantation.
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PMID:Coagulation abnormalities in chronic peritoneal dialysis. 239 81

Protein C, like the other vitamin K-dependent plasma proteins that participate in blood coagulation, except prothrombin, has at least one high affinity calcium-binding site that is independent of gamma-carboxyglutamic acid. Calcium binding to this site is required for activation of protein C by the thrombin-thrombomodulin complex. In an attempt to localize this calcium-binding site, we subjected protein C to limited tryptic digestion. A monoclonal antibody that recognizes a calcium-dependent epitope both in intact protein C, in gamma-carboxyglutamic acid-domainless protein C, and in activated protein C, was used to isolate a fragment from the tryptic digest. The fragment was derived from the light chain of protein C and consisted of the two domains that are homologous to the epidermal growth factor precursor. Half-maximal binding of the intact protein and of the isolated fragment by the antibody occurred at 100-200 microM Ca2+. The results suggest the presence of a Ca2+-binding site in the epidermal growth factor homology region of protein C.
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PMID:Calcium-dependent interaction between the epidermal growth factor precursor-like region of human protein C and a monoclonal antibody. 244 97

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding. 245 60

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.
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PMID:Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides. 247 15

The murine monoclonal antibody H-11 binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized by antibody H-11 contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions. By using a solid-phase immunoassay with 125I-labeled antibody and immobilized vitamin K-dependent protein, binding of the antibody to the vitamin K-dependent proteins was inhibited by increasing concentrations of calcium, manganese, and magnesium ion. The transition midpoints for antibody binding were in the millimolar concentration range and were different for each metal ion. In general, the transition midpoints were lowest for manganese ion, intermediate for calcium ion, and highest for magnesium ion. Antibody H-11 bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative. Binding of the antibody to the peptide was not inhibited by calcium ion. These data suggest that inhibition of antibody H-11 binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. This transition which is conserved in vitamin K-dependent proteins containing the H-11 antigenic site is likely due to a structural transition of the amino-terminal polypeptide possibly from a random (accessible) to ordered (inaccessible) structure.
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PMID:Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins. 247 38

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineated by immunoblotting and peptide studies and found to be present in the NH2-terminal 15 residues of protein C. Although residues corresponding to positions 10-13 of human protein C were necessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likely that Ca2+ occupation of a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NH2-terminal region of protein C which is favorable for antibody interaction, whereas Ca2+ binding to the low affinity site(s), known to be present in the gamma-carboxyglutamic acid domain, causes an unfavorable conformational change.
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PMID:Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains. 247 52

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