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 head of the myosin molecule (i.e., subfragment 1 with a heavy chain of 95 kDa) is usually obtained by chymotryptic cleavage in the presence of a divalent cation chelator. In the present work, we used another specific proteolytic enzyme, thrombin, to produce a limited cut within the myosin molecule, resulting in a new species of N-terminal fragment. Treatment of skeletal muscle myosin yielded a 97-kDa split heavy chain associated with intact light chains, corresponding to a single cut. The ATPase activities of this new S-1 derivative were slightly affected by the breakdown. It recognized actin in an ATP-dependent manner, as expected, with an affinity 2-5 times higher than that of the usual chymotryptic S-1 preparation but with a very different electron microscopic pattern. Functional differences are noted, and we involve them more precisely in relation to possible structural aspects of the additional C-terminal segment extending the usual S-1 heavy chain from 95 to 97 kDa.
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PMID:New subfragment 1 of skeletal muscle myosin obtained by thrombin cleavage. 213 79

Evidence for the participation of the 1-7 and 18-28 N-terminal sequences of actin at different steps of actin-myosin interaction process is well documented in the literature. Cross-linking of the rigor complex between filamentous actin and skeletal-muscle myosin subfragment 1 was accomplished by the carboxy-group-directed zero-length protein cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide. After chaotropic depolymerization and thrombin digestion, which cleaves only actin, the covalent complex with Mr 100,000 was characterized by PAGE. The linkage was identified as being between myosin subfragment 1 (S-1) heavy chain and actin-(1-28)-peptide. The purified complex retained in toto its ability to combine reversibly with fresh filamentous actin, but showed a decrease in the Vmax. of actin-dependent Mg2(+)-ATPase. By using e.l.i.s.a., S-1 was observed to bind to coated monomeric actin or its 1-226 N-terminal peptide. This interaction strongly interfered with the binding of antibodies directed against the 95-113 actin sequence. Moreover, S-1 was able to bind with coated purified actin-(40-113)-peptide. Finally, antibodies directed against the 18-28 and 95-113 actin sequence, which strongly interfered with S1 binding, were unable to compete with each other. These results suggest that two topologically independent regions are involved in the actin-myosin interface: one located in the conserved 18-28 sequence and the other near residues 95-113, including the variable residue at position 89. Other experiments support the 'multisite interface model', where the two actin sites could modulate each other during S-1 interaction.
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PMID:Characterization of an actin-myosin head interface in the 40-113 region of actin using specific antibodies as probes. 214 51

Thrombomodulin (TM), a major anticoagulant protein at the vessel wall, serves as a potent cofactor for the activation of Protein C by thrombin. Previous work has indicated that (rabbit) TM is a proteoglycan that contains a single polysaccharide chain, tentatively identified as a sulphated galactosaminoglycan, and furthermore suggested that this component may be functionally related to additional anticoagulant activities expressed by the TM molecule [Bourin, Ohlin, Lane, Stenflo & Lindahl (1988) J. Biol. Chem. 263, 8044-8052]. Results of the present study establish that (enzymic) removal of the polysaccharide chain abolishes the inhibitory effect of TM on thrombin-induced fibrinogen clotting as well as the promoting effect of TM on the inactivation of thrombin by antithrombin, but does not affect the ability of TM to serve as a cofactor in the activation of Protein C. Studies of yet another biological activity of rabbit TM, namely the ability to prevent the activation of Factor V by thrombin [Esmon, Esmon & Harris (1982) J. Biol. Chem. 257, 7944-7947], confirmed that TM markedly delays the conversion of the native 330 kDa Factor V precursor into polypeptide intermediates, and further into the 96 kDa heavy chain and 71-74 kDa light-chain components of activated Factor Va. In contrast, the activation kinetics of a similar sample of Factor V incubated with thrombin in the presence of chondroitinase ABC-digested TM did not differ from that observed in the absence of TM. It is concluded that the inhibitory effect of TM on Factor V activation also depends on the presence of the polysaccharide component on the TM molecule.
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PMID:Functional role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Effects on inactivation of thrombin by antithrombin, cleavage of fibrinogen by thrombin and thrombin-catalysed activation of factor V. 216 42

The validity of the hypothesis that Factor Xa activates Factor V in heparinized plasma was examined by establishing the temporal relationships between Factor V proteolysis and prothrombin consumption in plasma. Factor V was cleaved into Factor Va heavy chain (approx. 110 kDa) and an intermediate (approx. 230 kDa) 30 s after CaCl2 was added to contact-activated plasma (CAP). The larger fragment was converted into Factor V activation peptide (approx. 150 kDa) and Factor Va light chain (approx. 80 kDa) 15 s later. Heparin (approx. 0.05 microM) delayed Factor V proteolysis in CAP by at least 30 s. On supplementing CAP with 1 nM-Factor Xa or 1 nM-thrombin, Factor V was activated 15 s later. Heparin prolonged by 15 s and 45 s the time required to demonstrate Factor V activation in CAP supplemented with Factor Xa and thrombin respectively. Factor V was activated 20 s after tissue factor and CaCl2 were added to plasma, both in the absence and in the presence of approx. 0.05 microM-heparin. In contrast, hirudin and D-Phe-Pro-Arg-CH2Cl (two thrombin inhibitors more effective than heparin) delayed Factor V activation in this plasma by at least 30 s. The fragments of Factor V obtained in heparinized CAP suggest thrombin escapes inhibition and contributes to Factor V activation in that plasma. Production of Factor Va heavy chain and the 230 kDa Factor V fragment invariably preceded efficient prothrombin activation. These observations suggest that heparin, hirudin and D-Phe-Pro-Arg-CH2Cl delay Factor V activation by inhibiting thrombin. The availability of Factor Xa markedly moderates the ability of heparin to inhibit Factor V activation.
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PMID:Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl. 226 68

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin-activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme-linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
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PMID:Membrane expression of platelet calpain. 231 Aug 27

Coagulation factor V (fV) is a single-chain glycoprotein (Mr 330,000; domain structure A1-A2-B-A3-C1-C2) that is activated to factor Va (fVa; Mr 174,000) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2; Mr 94,000) and a light chain (A3-C1-C2; Mr 74,000). We analyzed the ultrastructure of scanning transmission electron microscope images of bovine and human fV, bovine fVa, and its constituent light chains and heavy chains. Factor V molecules had irregularly globular (10-12 nm) to oblong (8-14 nm) core structures which commonly displayed a peripheral satellite appendage of variable morphology attached to the core by a narrow stalk. Scanning transmission electron microscope mass analyses indicated that monomolecular bovine fV molecules had a mass of 322 +/- 45 kDa and human fV, 315 +/- 31 kDa. Factor Va molecules were irregular, globular (8-12 nm) structures that resembled the fV core structure, lacked the satellite appendage representing B domainal structures, and had a mass of 180 +/- 22 kDa. Our findings permit us to propose a structural model of fV suggesting the relative orientation of its closely associated light chain and heavy chain core components and indicating that these constituents remain associated in the transition from fV to fVa.
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PMID:Structural model of factors V and Va based on scanning transmission electron microscope images and mass analysis. 234 7

The reaction of alpha 2-macroglobulin (alpha 2M) with the two-chain enzyme plasma kallikrein results in covalent bond formation between the catalytic subunit and the inhibitor. We have recently published a model of alpha 2M which suggests that this phenomenon may be a general mechanism when multisubunit proteinases are inactivated by alpha 2M. In order to test this hypothesis, we studied the reactions of factor Xa, plasmin, streptokinase-plasmin and alpha-thrombin with alpha 2M. In the case of factor Xa the catalytic heavy chain demonstrated greater than 99% covalent incorporation while over 97% of the light chain failed to crosslink to the inhibitor. Preferential binding of the catalytic light chains of plasmin (70% covalent incorporation) and plasmin in complex with streptokinase (79% covalent incorporation) was also observed. Finally, 82% covalent incorporation of the catalytic heavy chain of alpha-thrombin was found. These studies demonstrate that in the case of multisubunit proteinases, the chain containing the active site demonstrates preferential binding as predicted by the model supporting placement of the site of covalent binding close to the "bait region" of alpha 2M.
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PMID:Specificity of alpha 2-macroglobulin covalent cross-linking for the active domain of proteinases. 243 19

A 5-Gla prothrombin, adsorbable onto barium oxalate and containing an admixture of one- and two-chain molecules, has been purified from the plasma of steers on a dicoumarol regimen for three years. Double-chain molecules were not detected in any of our six other variants containing zero to nine Gla residues. In this double-chain variant, the peptide bond between Arg52-Asn appears to be missing, as the preparation showed amino-terminal alanine and aspartic acid, and released both double- and single-chain F1 upon digestion with thrombin. The molecular masses of the F1 and its heavy chain were 24,000 and 19,000 daltons, respectively. The double-chain variant was similar to its single-chain counterpart [Malhotra, O.P. (1979) Thromb. Res. 14, 439-448] in that it (a) yielded thrombin equivalent to 30% of normal in three hr, (b) moved in the alpha 1 region in the absence of calcium ions and between the alpha 1 and alpha 2 macroglobulins in the presence of calcium ions, (c) contained five gamma-carboxyglutamyl residues (Gla), and (d) showed comparable antigenic activity with normal prothrombin.
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PMID:Purification and characterization of dicoumarol-induced prothrombins. Evidence of 5-Gla variant with two (or more) polypeptide chains. 244 45

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.
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PMID:The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody. 244 82

We have used immunoblotting of purified factor VIII (FVIII) to determine whether or not changes in FVIII chain specificity occur during the course of an inhibitor. Serial plasma samples from 15 inhibitor patients (13 hemophilic and two spontaneous) were analyzed. Nine of the 15 antibodies, all with epitopes on the 44-kilodalton (Kd) thrombin fragment of the 92-Kd FVIII heavy chain and/or the 72-Kd thrombin fragment of the 80-Kd FVIII light chain, showed no change in FVIII chain specificity. However, six of the inhibitors analyzed showed changes in FVIII fragment specificity. Four inhibitors (three hemophilic and one spontaneous) reactive with 72-Kd thrombin fragment also became reactive with the 44-Kd thrombin fragment after an anamnestic response to FVIII infusion. Another inhibitor with epitopes on both the 54-Kd and 44-Kd thrombin fragments lost most of its reactivity with the 44-Kd fragment but retained its reactivity with the 54-Kd fragment following a FVIII infusion. The inhibitor later regained its 44-Kd-fragment reactivity but lost its 54-Kd-fragment reactivity following treatment with FEIBA, FVIII inhibitor bypassing activity. The last inhibitor studied had an antibody to either the 44-Kd fragment or to both the 44-Kd and 72-Kd fragments during anamnestic responses to FVIII. These data indicate that a FVIII inhibitor patient can potentially produce antibody to multiple areas on the FVIII molecule and that this must be taken into account in the design of specific therapeutic products.
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PMID:Immunoblot analysis shows changes in factor VIII inhibitor chain specificity in factor VIII inhibitor patients over time. 245 82


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