Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.
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PMID:Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding. 163 19

Through immunohistochemical techniques, blood coagulation factors were identified in situ in fresh frozen sections of small cell carcinoma of the lung. Prothrombin/thrombin, factor VII, factor X, and antithrombin III were present in intercellular spaces and associated with tumor cells. Factor IX, factor XI, prekallikrein, and high molecular weight kininogen were identified as being associated with tumor cells but did not exist in intercellular spaces. Variable connective tissue staining but no tumor-related staining was observed for factor V, factor VIII-related antigen, factor XII, the B subunit of factor XIII, alpha 1-antitrypsin, alpha 2-macroglobulin, or alpha 2-antiplasmin. Neither consecutive tissue nor the tumor manifested platelet Ib and IIbIIIa surface glycoproteins. These divergent staining patterns suggested that the detected clotting factors had not merely diffused from permeabilized blood vessels, but were selectively localized in situ. While conditions may exist within tumor tissue that both retard and promote thrombin generation, we propose that interactions between the observed coagulation factors ultimately lead to local thrombin formation, which is responsible for the conspicuous fibrin deposits already described in small cell carcinoma of the lung. Thrombin formed locally might contribute to progression of this tumor. Inhibition of local thrombin formation by warfarin therapy could explain the beneficial effects of warfarin therapy in treating small cell carcinoma of the lung.
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PMID:Occurrence of blood coagulation factors in situ in small cell carcinoma of the lung. 282 13

Involvement of platelet membrane glycoproteins (GP) in interactions between platelets and tumor cells was studied by using two human tumor cell lines and two monoclonal antibodies against platelet membrane GP. HMV-I cells derived from vaginal melanoma induced platelet aggregation in heparinized plasma, which was not followed by coagulation. M7609 cells derived from colon adenocarcinoma also induced platelet aggregation in heparinized plasma, which, on the contrary, was followed by coagulation. Aggregating activities of the HMV-I cells were abolished by pretreatment with neuraminidase or trypsin, but M7609 activity was not labile to these enzymes. Aggregations induced by M7609 were inhibited by hirudin or MD805, while those by HMV-I were not. M7609 cells dose dependently shortened the recalcification time of normal as well as Factor IX-deficient plasmas, while they were not effective in shortening the time of Factor II- or Factor VII-deficient plasmas. The procoagulant activity of HMV-I cells was 1000 times less than M7609 on the basis of cell numbers. When human platelets were preincubated with monoclonal anti-GPIb or anti-GPIIb/IIIa complex antibodies, neither cell line could cause aggregations. These findings suggest that both GPIb and the GPIIb/IIIa complex on the platelet surface are involved in the thrombin-dependent and -independent platelet aggregations induced by tumor cells.
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PMID:Involvement of platelet membrane glycoprotein Ib and glycoprotein IIb/IIIa complex in thrombin-dependent and -independent platelet aggregations induced by tumor cells. 291 Apr 73

Blood coagulation studies carried out on 78 patients up to 30 h after they were bitten by Bothrops jararaca snakes demonstrated clotting defects in 37 patients which included afibrinogenemia, reduced levels of prothrombin, of factors V and VIII, thrombocytopenia and activation of the fibrinolytic system. Factor IX and X levels were within normal range for all patients. These in vitro data suggest that the disseminated intravascular clotting observed in vivo following envenomation may be triggered by the intravascular release of patient thrombin by snake venom enzymes.
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PMID:Alterations of the blood coagulation system after accidental human inoculation by Bothrops jararaca venom. 310 94

In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.
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PMID:Inhibition of prothrombin activation by factor X and factor IX Gla-peptides. 337 73

We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose-dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.
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PMID:The effect of platelets in the activation of human blood coagulation factor IX by factor XIa. 348 52

Previous studies have demonstrated a Factor IX and IXa binding site on the endothelial cell surface for which both the zymogen and enzyme compete with equal affinity. In this report, we demonstrate that the affinity of Factor IXa, but not Factor IX, for the cell surface is increased in the presence of both Factors VIII and X. When Factor Xa formation was studied in the presence of saturating concentrations of Factors VIII and X, the half-maximal rate was observed at a Factor IXa concentration of 151 +/- 12 pM. Active site-blocked Factor IXa, 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa, was a more effective inhibitor of Factor X activation (Ki = 124 pM) than was Factor IX (Ki = 3.0 nM). Radioligand binding studies carried out in the presence of Factors VIII and X confirmed the presence of a selective endothelial cell Factor IXa binding site with Kd = 127 +/- 27 pM. In contrast, when Factor IXa binding was studied in the absence of other coagulation factors, or in the presence of Factor VIII (thrombin-activated or unactivated) alone, this new high affinity site was not observed. Competitive binding studies indicated that Factor IXa was 12 times more effective as an inhibitor of Factor IX-endothelial cell binding in the presence of Factors VIII and X. Consistent with the increased affinity of Factor IXa binding in the presence of factors VIII and X, cell-associated Factor IXa coagulant activity decayed 7 times more slowly in the presence of these coagulation factors. These results demonstrate selective Factor IXa-endothelial cell binding in the presence of Factors VIII and X, suggesting this interaction could be a physiologic occurrence.
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PMID:The binding of factor IXa to cultured bovine aortic endothelial cells. Induction of a specific site in the presence of factors VIII and X. 392 79

Thrombin plays an essential role in the maintenance of haemostatic balance through several biochemical reactions. Its functions as well as the regulation of its activities seem to be decisive in controlling thrombus formation. The present paper endeavours to give a brief summary of the control of thrombin activity and the regulation of biological actions of thrombin. As to the control of the amount of the active enzyme, three possible ways are discussed, i.e. prothrombin synthesis, activation of prothrombin, and inactivation of thrombin by plasma proteinase inhibitors (antithrombin III, alpha-2-macroglobulin, alpha-1-proteinase inhibitor). Regarding the regulation of the various actions of thrombin, the most important biological targets of this enzyme are summarized, namely interaction with fibrinogen, activation of Factor XIII, Factor IX, Factor VIII, Factor V and prothrombin, and the binding to platelets, fibroblasts and endothelial cells. The limits of our present knowledge about the determination of the possible reaction routes catalyzed by thrombin are also considered. Finally, a tentative hypothesis is put forward to explain how the endogenous modulators like heparin determine the biological functions of thrombin.
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PMID:Thrombin and haemostasis: regulation of the biological functions of thrombin. (Facts and perspectives). 617 55

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.
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PMID: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. 620 16

A 17-year-old male with previously undiagnosed congenital Factor IX deficiency (13%) presented with gastrointestinal bleeding and a hepatic mass. Prolonged thrombin and Reptilase times, which partially corrected with CaCl2 and a discrepancy between thrombin-clottable and immunoreactive plasma fibrinogen, suggested a dysfibrinogenemia. Laparotomy disclosed metastatic hepatoma. Adequate hemostasis was obtained with clotting factor replacement, but wound healing was delayed. Patient fibrinogen purified with 2.1 M glycine migrated normally on immunoelectrophoresis and 7.5% polyacrylamide-SDS gel electrophoresis. However, fibrin monomers prepared from purified patient fibrinogen displayed impaired aggregation at high and low ionic strengths when compared with fibrin monomers from normal and control Factor IX deficient subjects. Aggregation of normal monomers was delayed when mixed 1:1 with patient monomers. Fibrinopeptide release was normal, and total sialic acid content was similar to that of normal and control fibrinogens. Chemotherapy, consisting of 5-FU given via intra-arterial hepatic infusion, was accompanied by significant transient clinical improvement which coincided with correction of thrombin clotting times and fibrin monomer aggregation. Reappearance of fibrinogen dysfunction occurred with clinical deterioration prior to death from metastatic hepatoma and sepsis. This case is the first to corroborate the postulated tumor marker role of dysfibrinogenemia in a patient with hepatoma by documenting a direct relationship with response to chemotherapy.
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PMID:Acquired dysfibrinogenemia in a hemophiliac with hepatoma: resolution of fibrinogen dysfunction following chemotherapy. 626 56


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