Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 mumol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of mu-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca2+. Ca(2+)-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.
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PMID:Participation of calpain in protein-tyrosine phosphorylation and dephosphorylation in human blood platelets. 774 63

Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta 3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.
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PMID:Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in human platelets. 787 93

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-beta (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-beta was found to be activated to a greater extent by brain G-protein beta gamma subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with mu-calpain of the intact PI-PLC-beta (150 kDa) caused a marked augmentation of its activation by beta gamma subunits. This enhanced PLC activation by beta gamma subunits was due to truncation by mu-calpain, producing a 100 kDa PI-PLC, but not by another protease, thrombin.
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PMID:Proteolytic modification of membrane-associated phospholipase C-beta by mu-calpain enhances its activation by G-protein beta gamma subunits in human platelets. 813 42

An antibody specific to the calpain cleavage site in talin, a cytoskeletal protein, was produced. This antibody selectively recognizes the C-terminal 200-kDa fragment generated when talin is digested by calpain and does not react at all with intact talin or the N-terminal 47-kDa fragment. To assess the involvement of calpain in the integrin-mediated signaling pathway, the effect of limited proteolysis of talin by calpain on platelet activation and aggregation was analyzed using this antibody. It was revealed that thrombin-stimulated platelet aggregation accompanies the autolytic activation of mu-calpain and the accumulation of the mu-calpain-generated 200-kDa fragment of talin. These changes were blocked by RGDS peptide which inhibits the binding of fibrinogen, an adhesive ligand, to the major integrin in platelets, alpha IIb beta 3, while RGES peptide, which has no fibrinogen-binding-inhibitory activity, had no effect. Membrane-permeable calpain inhibitors calpeptin and E-64d inhibited platelet aggregation, mu-calpain activation, and the limited proteolysis of talin. These results strongly suggest that calpain is involved in the integrin-mediated signal transduction pathway.
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PMID:Involvement of calpain in integrin-mediated signal transduction. 863 21

Phosphatidylserine exposure and microvesicle release give rise to procoagulant activity during platelet activation. We have previously shown that whereas the Ca2+ ionophore A23187 and 2,5-di-(t-butyl)-1, 4-benzohydroquinone, a Ca2+-ATPase inhibitor, induce phosphatidylserine exposure, only the former triggers microvesicle release. We now report that microvesicle formation with ionophore A23187 is specifically associated with mu-calpain activation, increased protein tyrosine phosphatase (PTP) activity and decreased tyrosine phosphorylation. The degree to which calpain and individual PTPs were activated in response to A23187 depended on the extent of bivalent cation chelation in the external medium. EGTA (2 mM) blocked or severely retarded their activation, and addition of extracellular Ca2+ in excess (2 mM) resulted in virtually immediate tyrosine dephosphorylation. Dephosphorylation was correlated with an increase in total PTP activity in platelet lysates. In platelets stimulated by a combination of thrombin and collagen, only the subpopulation undergoing microvesicle release and isolated by their binding to annexin-V-coated magnetic beads exhibited protein tyrosine dephosphorylation. Detection of PTP activity in an 'in-gel' assay showed the Ca2+-dependent appearance of active low-molecular-mass bands at 38, 36 and 27 kDa. Individual PTPs varied in their protease sensitivity to changes in intracellular Ca2+ levels. For example, PTP1B was a more sensitive substrate than SH2-domain-containing tyrosine phosphatase-1 for mu-calpain cleavage. Incubation of platelets with the PTP inhibitors, phenylarsine oxide and benzylphosphonic acid acetoxymethyl ester, led to increased tyrosine phosphorylation and the surface expression of aminophospholipids but little microvesicle formation. Furthermore, microvesicle release in response to ionophore A23187 was inhibited. We conclude that platelet microvesicle formation is associated with extensive protein tyrosine dephosphorylation.
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PMID:Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen. 967 17

Cardiac troponin T (cTnT) has been shown to be present in fragmented forms in human serum after acute myocardial infarction (AMI). While calpain-1 and caspase-3 have been identified as intracellular proteases able to cleave the N-terminus of cTnT, it is still unclear which proteases are responsible for the extensive and progressive cTnT fragmentation observed in serum of AMI-patients. In this pilot study we have investigated the possibility that human thrombin may be involved in this process. Purified human cTnT was spiked in unprocessed and deproteinated serum in the presence or absence of either purified human thrombin or PPACK thrombin inhibitor. After immunoprecipitation, SDS-PAGE and Western blotting we observed an increase in cTnT fragmentation when purified thrombin was added to deproteinated serum. Consequently, the addition of thrombin inhibitor to unprocessed serum resulted in a decrease of cTnT fragmentation. Our results suggest that multiple enzymes are involved in cTnT degradation, and that thrombin plays an important role.
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PMID:Cardiac troponin T degradation in serum is catalysed by human thrombin. 2781 55