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)

Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.
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PMID:Association of the factor VIII light chain with von Willebrand factor. 313 49

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113. 314 Aug 94

Blood coagulation factor V, the labile factor, is an important cofactor in the activation of prothrombin. Approximately 10 years ago, the first purification procedures for undegraded factor V from bovine and human plasma were reported. This was the starting point for a new area in the research on factor V structure-function relationships. In parallel to this, the structure of the even more labile anti-hemophilic factor (factor VIII) has been elucidated and the two proteins are found to be very similar in structure and in function. In this mini-review, I will focus on work performed in our laboratory, which has led forward to the proposal of a new structural model for factor V. It is based on results obtained with several different techniques, including protein chemistry, DNA technology and high resolution electron microscopy. In plasma, factor V circulates as a single chain, high molecular weight protein. During coagulation a limited number of peptide bonds are cleaved in the factor V molecule by thrombin. This leads to a great increase in biological activity. The active Va species is composed of a noncovalent complex between the N- and C-terminal fragments, whereas the activation fragments correspond to the carbohydrate-rich central portion of the molecule. The activity of factor Va is regulated through the selective degradation of the N-terminal heavy chain fragment by activated protein C. Purified human and bovine factor V was examined by high resolution transmission electron microscopy. Factor V was found to be composed of four major domains, three similar sized globular structures (diameter approx. 80 A) are linked via thin spacers to a larger central domain (diameter approx. 140 A). Activation with thrombin results in a reorganization of the molecule. The thrombin cleavage sites are positioned in the spacers between the different domains and two of the peripheral domains combine to form the active Va species. The new factor V model suggests that a unique and dramatic molecular reorganization occurs during the activation of factor V by thrombin and indicates that the low biological activity of single chain factor V is due to the physical separation of the N- and C-terminal domains by the large central region. Full biological activity can only be expressed after limited proteolysis by thrombin, when the two initially separated domains are free to combine to form the active factor Va molecule.
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PMID:A new model for coagulation factor V suggesting a unique mechanism of activation. 324 83

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.
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PMID:Calcium binding to the epidermal growth factor homology region of bovine protein C. 325 33

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.
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PMID:Proteolysis of factor Va by factor Xa and activated protein C. 361 Nov 8

Using the thrombin-cut [68-30 kilodalton (kDa)] myosin subfragment 1 (S-1) whose heavy chain has been selectively split within the central 50-kDa region, at Lys-560, with concomitant specific alterations of the ATPase and actin binding properties [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry 25, 1134-1140; Chaussepied, P., Mornet, D., Barman, T., Travers, F., & Kassab, R. (1986) Biochemistry 25, 1141-1149], we have isolated and renatured the COOH-terminal 30-kDa fragment associated with the alkali light chains by the procedure recently described [Chaussepied, P., Mornet, D., Audemard, E., Kassab, R., Goodearl, J., Levine, B., & Trayer, I. P. (1986) Biochemistry 25, 4540-4547]. The 30-kDa peptide preparation was found to exhibit a crucial feature of the native S-1; namely, it interacts with F-actin in an adenosine 5'-triphosphate (ATP)-dependent manner. Studies by ultracentrifugation, turbidity measurements, and chemical cross-linking experiments showed that the acto-30-kDa peptide complex was dissociated almost completely by the gamma-phosphoryl group containing ligands ATP, 5'-adenylyl imidodiphosphate, and pyrophosphate, to a lesser extent by ADP, and not at all by AMP and inorganic phosphate. The maximal dissociating effect is operating with the thrombic 30-kDa entity, whereas the 22-kDa fragment produced by staphylococcal protease is only slightly dissociated. In contrast, the tryptic 20-kDa fragment binds irreversibly to actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of polyphosphate recognition sites communicating with actin sites on the skeletal myosin subfragment 1 heavy chain. 379 May 30

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin-cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.
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PMID:Proteolytic inactivation of blood coagulation factor IX by thrombin. 406 23

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.
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PMID:Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma. 620 93

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79-80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79-80,000 (or 71-72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.
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PMID:Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. 641 99

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.
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PMID:Amino acid sequence of human beta-factor XIIa. 660 55


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