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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of two protein C gamma-carboxyglutamic acid domain mutations in familial thrombosis, protein CVermont (Bovill, E. G., Tomczak, J. A., Grant, B., Bhushan, F., Pillemer, E., Rainville, I.R., and Long, G. L. (1992) Blood 79, 1456-1465), was investigated. Two single mutations (Glu20-->Ala and Val34-->Met) and the naturally occurring double mutation were created by site-directed mutagenesis and were expressed in human kidney 293 cells. Purified recombinant protein C with the mutation glutamate to alanine at position 20 is defective in the assays of activated partial thromboplastin time, factor Va inactivation, and fibrinolysis. Mutation from valine to methionine at position 34 has only a minor effect. Activation of Glu20 mutants by thrombin-thrombomodulin was not enhanced by phospholipid vesicles and showed a different calcium dependence compared with the wild type, suggesting that Gla20 is important in the interaction of the protein C Gla domain with a phospholipid-mediated site on the thrombomodulin molecule. Glu20-substituted protein C is not inhibited by calcium ion in its interaction with the calcium-dependent monoclonal antibody H-11, suggesting that this mutation has lost the calcium-induced, lipid-independent conformational transition of the protein C Gla domain. These data indicate that the loss of Gla20 causes the major familial dysfunction of protein C to associate with phospholipid as well as to undergo Ca(2+)-dependent, lipid-independent conformational changes and are consistent with the importance of Gla20 in both external and internal Ca2+ binding based upon the x-ray-derived structure of the homologous Gla domain in bovine prothrombin.
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PMID:Molecular mechanism for familial protein C deficiency and thrombosis in protein CVermont (Glu20-->Ala and Val34-->Met). 796 68

Three Streptoverticillium anticoagulants, SAC I, II, and III, which strongly inhibit human intrinsic blood coagulation, were each isolated in a homogeneous form from a culture fluid of Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852. SAC I, II, and III are simple proteins with molecular weights of around 12,000, and with isoelectric points of 9.7, 9.7, and 9.9, respectively. Their amino acid compositions are similar and each SAC possesses two disulfide bonds. The COOH-terminal residue of each of these proteins is phenylalanine. Together with the similarity of their protein chemical properties, the results of NH2-terminal amino acid sequence analysis of these SAC proteins strongly suggested that the deletion of Ser-Leu and Ser-Leu-Tyr from the NH2-terminus of SAC I (Ser-Leu-Tyr-Ala-Pro-...) results in the generation of SAC II and III, respectively. The amount of each SAC necessary to double the partial thromboplastin time was around 5 micrograms/ml. SAC I inhibited activated human factor XII and human plasma kallikrein. It also inhibited, but to a lesser extent, activated factor X. The inhibition constants (Ki) of SAC I toward activated factor XII and plasma kallikrein were 5.3 x 10(-8) and 7.2 x 10(-9) M, respectively. The SACs also inhibited some microbial serine proteases such as subtilisin Carlsberg and, to a lesser extent, mammalian serine proteases including bovine trypsin and alpha-chymotrypsin. Of these three inhibitors, only SAC I inhibited metalloproteases such as thermolysin in addition to these serine proteases.
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PMID:Isolation and characterization of Streptoverticillium anticoagulant (SAC), a novel protein inhibitor of blood coagulation produced by Streptoverticillium cinnamoneum subsp. cinnamoneum. 808 92

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
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PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13

The biological response to 4 different heparins after successive subcutaneous administration once daily for 5 days at a dose used for primary prophylaxis of deep vein thrombosis was investigated in a randomized cross-over study in 12 volunteers. Three different preparations of low molecular weight heparins (LMWH) were administered, 10,000 U unfractionated heparin (UFH) was used as a control. The anticoagulant properties in terms of anti-Xa activities, as measured by a chromogenic substrate assay or Heptest, showed high interindividual variations with peak levels 2 to 4 h following injections. There was a significantly higher increase of anti-Xa activities 3 h after administration at day 5, when compared with day 1, for two LMWH's, suggesting an accumulation of the anticoagulatory effect. The anticoagulant activity, especially when measured by Heptest, was significantly influenced by the body weight. This could be observed for all LMWH's. For the assessment of anticoagulant activity in LMWH-treated individuals, the chromogenic substrate assay and Heptest revealed maximal correlation (r = 0.51), while in UFH-treated individuals, peak correlation (r = 0.75) was observed between the partial thromboplastin time and thrombin clotting time. The chromogenic substrate method was the most sensitive anti-Xa assay, showing also the smallest interindividual variation. No significant influence of heparins neither on platelet count and function nor on fibrinolysis were recognized. Enhanced lipolytic activities were not observed. There was an increase of alanine aminotransferases induced by UFH as well as LMWH's, which, however, was most pronounced after UFH.
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PMID:Comparison of low molecular weight heparins and unfractionated heparin after successive subcutaneous administration. A randomized controlled study in healthy volunteers. 839 45

C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C-terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N-terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site-directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (beta-glucuronidase release). Mutants that contained the single residue substitutions Ile-6-->Gly or Tyr-13-->Gly were reduced in potency to 4-30% compared with wild-type rC5a. Other single-site glycine substitutions at positions Leu-2, Ala-10, Lys-4, Lys-5, Glu-7, Glu-8, and Lys-14 showed little effect on C5a potency. The double mutant, Ile-6-->Gly/Tyr-13-->Gly, was reduced in potency to < 0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in alpha-helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N-terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.
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PMID:Site-specific mutations in the N-terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor. 840 Dec 25

Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of approximately 10% that of wild-type rFVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII-tissue factor interaction. 841 32

The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.
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PMID:Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa. 899 32

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.
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PMID:Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va. 900 85

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165-->Ala, Lys166-->Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158-->Arg, Ser160-->Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TF(R158G160). In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TF(A165A166) as the cofactor, but only 3.5-fold with TF(R165G160). The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TF(R158G160) and 7-8-fold with TF(A165A166). This suggests that the molecular recognition of VII in complex with TF by the enzymes TF-VIIa and factor Xa are similar. Generation of factor IXa by TF(R158G160)-VIIa was unaltered, but reduced 2-fold with TF(A165A166). In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TF-VIIa complex.
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PMID:Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. 903 67

One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.
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PMID:A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent. 912 26


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