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

---

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

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.
...
PMID:Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. 46 91

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.
...
PMID:Improved procedures for the purification of selected vitamin K-dependent proteins. 78 72

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation. 131 45

Protein C is a plasma, vitamin K-dependent zymogen of a serine protease that can inhibit blood coagulation. Protein C is regulated by a series of reactions known as the protein C pathway. The importance of this pathway is seen in the occurrence of thrombosis in individuals with deficiencies in elements of the pathway like protein C and protein S. Work on several steps in this pathway has revealed that mechanisms involved in activation of protein C and the expression of its anticoagulant activity have features that allow for the expression of the anticoagulant activity away from sites in which procoagulant reactions occur, but not systemically. Thrombin, the principal procoagulant enzyme at the site of an injury, is converted to an anticoagulant enzyme at distant sites through its interaction with the endothelial cell protein thrombomodulin. Structural and functional studies have revealed the importance of several domain structures in the modulation of thrombin activity. Structural features of both activated protein C and its substrates (coagulation factors V and VIII) are such that they require the localization of enzyme and substrate on the surface of phosphatidyl serine containing membranes for optimum activity.
...
PMID:Regulation of blood coagulation by the protein C system. 131 8

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.
...
PMID:The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives. 133 92

1. Guinea-pig blood clots rapidly and the clots retract in glass tubes. The prothrombin time is long and the activated partial thromboplastin time short compared to human. The Russel viper venom time is similar to human. 2. Factors VII and X assay at levels far below and factors V, VIII and XII assay far above human levels. Other coagulation factors (fibrinogen, II, IX, XI, Fletcher and Fitzgerald) assay within or close to the human range. 3. The thromboplastin generation test results for guinea-pigs and humans are similar. 4. Platelets are numerous and small. They aggregate with ADP, arachidonic acid and pig plasma, variably with ristocetin and poorly with bovine collagen or thrombin. On electron microscopy, platelets appear small with many dark granules (dense bodies). There is an open canicular system. Glycogen particles are sparse. Microtubules are occasionally seen, mitochondria are rare and alpha-granules are not readily distinguished from dark granules. 5. Ristocetin cofactor is very low, assaying at < 16% of human (< 0.16 U/ml). 6. Leukocyte counts are variable (6300-17,000 per microliters) and differential counts show neutrophils slightly lower and lymphocytes slightly higher than average human counts. 7. Guinea-pig erythrocyte parameters fall within human ranges. 8. Protein electrophoresis shows total protein and albumin to be slightly lower than human. 9. Antithrombin III, Protein C and alpha 2-antiplasmin assay within the human range and plasminogen at very low levels. 10. Bleeding times are consistently about 4 min.
...
PMID:Comparative hematology: studies on guinea-pigs (Cavia porcellus). 135 40

Human protein C is a vitamin K-dependent plasma glycoprotein that circulates as an inactive zymogen. At the endothelial cell surface, thrombin in complex with the integral membrane protein thrombomodulin converts protein C to its active form by specific cleavage of an activation peptide. The activated form of protein C has potent anticoagulant activity as a feedback regulator of thrombin generation (reviewed in refs 4-6), and also has profibrinolytic, anti-ischaemic and anti-inflammatory properties. Protein C is effective in the treatment of model and human thrombotic diseases but, except when it has been used to treat genetic or acquired deficiencies and microvascular thrombosis, it is administered as the activated enzyme, which has a short biological half-life. We have altered two putative inhibitory acidic residues near the thrombin cleavage site, which results in a 30-fold increase in substrate utilization by alpha-thrombin. We combined these changes with a genetically altered glycoform to generate a zymogen protein C with a 60-fold increased cleavage rate by free alpha-thrombin, independent of its cofactor thrombomodulin. We show that this 'proform' of protein C, unlike the natural circulating zymogen, can be activated by thrombin generated in clotting human plasma, resulting in an inhibition of further clot formation. Our data therefore show that we have engineered a site-activated agent, which only has anticoagulant activity when significant amounts of thrombin are being generated.
...
PMID:Enhancing protein C interaction with thrombin results in a clot-activated anticoagulant. 143 7

Protein C is a natural anticoagulant glycoprotein which prevents intravascular clot formation. Protein C functions as an anticoagulant when converted to an active serine protease (activated protein C). Activated protein C is formed at the site of the endothelial injury in response to blood clotting and helps limit the size of blood clots. We tested the hypothesis that by temporarily blocking the activation of intrinsic protein C, we could reduce subsequent surgical blood loss from a microvascular surgical wound. The formation of activated protein C was blocked systemically by intravenous administration of a monoclonal antibody (HPC4) which binds to circulating protein C and prevents its conversion to activated protein C. Domestic pigs were blindly pretreated with intravenous HPC4 or saline then underwent partial-thickness skin graft harvesting to create a reproducible microvascular wound. Blood loss was measured from each wound and the hemostatic effect of protein C blockade was compared to intravenous saline alone as well as to topical thrombin or thromboplastin. We found that blocking the activation of protein C significantly (P = 0.005) reduces surgical blood loss in this model by 27% compared to saline control animals. Intravenous HPC4 performed equally as well as topical thrombin or tissue thromboplastin. In addition, topical thrombin acted synergistically with HPC4 to reduce blood loss an additional 44% (P = 0.01) as compared to intravenous HPC4 or topical thromboplastin alone. Autopsies performed 1 week after HPC4 treatment showed no evidence of systemic thrombosis resulting from the protein C blockade.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Blockade of protein C activation reduces microvascular surgical blood loss. 152 31

Widespread intravascular coagulation is common in patients with sepsis. Coagulation abnormalities may result from exposure to endotoxin, from tumor necrosis factor alpha or interleukin 1 release, or from the actions of a more specific mediator, such as vascular permeability factor. The result is marked activation of the contact and coagulation systems; simultaneously, there is decreased fibrinolysis and depressed levels of the inhibitors of the contact and coagulation systems. Multiple agents are being studied to correct these abnormalities. Antithrombin III holds promise because it inhibits a number of factors important in contact and coagulation activation, not just thrombin. Plasminogen activators may prove helpful in increasing fibrinolysis during sepsis; because they have been associated with rebound thrombin generation, however, plasminogen activators may be most effective if used in conjunction with hirudin or a synthetic hirudin analogue. Bradykinin may offset hypotension in sepsis. Protein C may inhibit thrombin formation and also complex with plasminogen activator inhibitor 1, thereby promoting fibrinolysis. Other agents that may prove effective include alpha 1-antitrypsin Pittsburgh, C1-esterase inhibitor, monoclonal antibodies to contact factors, soybean trypsin inhibitors, thrombomodulin, prostaglandin I2, and aprotinin. There are no data to support the use of heparin or fibronectin, except in limited circumstances.
...
PMID:Modulators of coagulation. A critical appraisal of their role in sepsis. 162 18

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.
...
PMID:Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. 167 22


1 2 3 4 5 6 7 8 9 10 Next >>

---