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)

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.
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PMID:Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2. 1171 32

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-alpha (TNF-alpha) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-alpha production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.
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PMID:Participation of protease-activated receptor-1 in thrombin-induced microglial activation. 1184 73

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.
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PMID:Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation. 1185 34

1. We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)-activating peptides (PAR-APs) [SFLLRNPNDKYEPF-amide (TRAP), TFLLR-amide (PAR1AP) and AYPGKF-amide (PAR4AP)]. 2. PAR agonist-induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence. 3. Aggregation inhibitors, including prostacyclin (PGI(2)), nitric oxide-releasing agent (S-nitroso-glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist-induced aggregation to pharmacological inhibition. 4. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. 5. The aggregatory potencies of PAR-APs were not modified by the aminopeptidase inhibitor, amastatin. 6. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. 7. Both PGI(2) and GSNO reduced PAR agonist-induced aggregation and diminished GPIIb/IIIa up-regulation. 8. PAR agonist-induced aggregation was aspirin-insensitive indicating a minor role for TXA(2). 9. In contrast, phenanthroline and apyrase significantly enhanced the anti-aggregatory effects of aspirin against thrombin-, PAR1AP- and TRAP-induced aggregation suggesting the involvement of ADP- and MMP-2-dependent pathways. 10. PAR4AP-induced aggregation (but not PAR1AP-induced aggregation) was entirely ADP-dependent (abolished by apyrase) and resistant to phenanthroline (MMP-2-independent). 11. Thus, the mechanisms of PAR1 and 4-induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors.
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PMID:Mechanisms of action of proteinase-activated receptor agonists on human platelets. 1187 18

Protease-activated receptor (PAR)-mediated vascular relaxations have been compared in coronary arteries of different diameters isolated from both humans and pigs. Thrombin, trypsin, and the PAR1-activating peptide, TFLLR, all caused concentration-dependent relaxation of both large (epicardial; approximately 2 mm internal diameter) and small (intramyocardial; approximately 200 microm internal diameter) human coronary arteries. EC(50) values for thrombin (0.006 u ml(-1) in epicardial, 1.69 u ml(-1) in intramyocardial) and trypsin (0.02 u ml(-1) in epicardial, 1.05 u ml(-1) in intramyocardial) were significantly (P<0.01) greater in intramyocardial arteries. By contrast, EC(50) values for TFLLR were not different between epicardial (0.35 microM) and intramyocardial (0.43 microM) arteries. In porcine coronary arteries, EC(50) values for relaxations to thrombin (0.03 u ml(-1) in epicardial 0.17 u ml(-1) in intramyocardial) were also significantly (P<0.01) greater in the smaller arteries. EC(50) values for both TFLLR and the PAR2-activating peptide, SLIGKV, were not different between the two different-sized pig coronary arteries. PAR1-immunoreactivity was localized to the endothelium of human epicardial and intramyocardial arteries and both PAR1- and PAR2-immunoreactivity was observed in endothelial cells of equivalent porcine arteries. These findings indicate that enzymatic activation of endothelial cell PARs in human (PAR1) and porcine (PAR1 and PAR2) coronary arteries is markedly reduced in intramyocardial arteries when compared with epicardial arteries, suggesting increased regulation of PAR-mediated vascular responses in resistance-type arteries.
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PMID:Enzymatic activation of endothelial protease-activated receptors is dependent on artery diameter in human and porcine isolated coronary arteries. 1205 27

In the present study, the antiplatelet effect and its mechanism of a new synthetic compound YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] were examined. YD-3 inhibited the aggregation of washed human platelets caused by protease-activated receptor (PAR) 4 agonist peptide GYPGKF (IC50 = 0.13 +/- 0.02 microM), but had no or little effect on that by thrombin, PAR1 agonist peptide SFLLRN, collagen or U46619. YD-3 produced a parallel, rightward shift of the concentration-response curve for GYPGKF without decreasing of the maximum platelet aggregation, indicating a competitive antagonism. In contrast to human platelets, both thrombin- and GYPGKF-induced mouse platelet shape change and aggregation were completely inhibited by YD-3. YD-3 also selectively prevented GYPGKF-induced intracellular Ca2+ mobilization in human platelets. Furthermore, in the PAR1-desensitized human platelets, thrombin induced a relatively slow rise and decay of calcium mobilization that was significantly inhibited by YD-3. In addition, the synergistic effect of SFLLRN and GYPGKF on platelet activation was prevented by YD-3. YD-3 also inhibits both fMLP-stimulated neutrophil- and purified cathepsin G-induced platelet aggregation, which has been demonstrated to be PAR4-dependent. Taken together, our results suggest that YD-3 selectively inhibits PAR4-dependent platelet activation through blockade of PAR4. To the best of our knowledge, it is the first non-peptide PAR4 antagonist.
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PMID:Selective inhibition of protease-activated receptor 4-dependent platelet activation by YD-3. 1208 82

Protease-activated receptor 4 (PAR4) is cleaved by thrombin at the R47-G48 peptide bond. Unlike PAR1, PAR4 does not contain a sequence readily predicted to interact with thrombin anion binding exosite-I. HPLC kinetic results on hydrolysis of PAR4 peptides (38-51 and 38-62) reveal that extending the sequence from the active site toward the exosite does not promote further binding interactions with thrombin. One-dimensional-proton line-broadening NMR indicates that the amino acids occupying the P(4)-P(1) positions of PAR4 (38-47), 44PAPR(47), come into direct contact with the thrombin surface. Less contact arises from the Leu43 at the P(5) position. Two-dimensional total correlation spectroscopy and two-dimensional transferred nuclear Overhauser effect spectroscropy studies on this complex reveal that Leu43 is flexible and can exhibit two conformational states. The binding mode observed for PAR4 peptides is similar to that of PAR1 peptides. PAR4 takes advantage of a distinctive sequence to optimize its interactions with the thrombin active site surface.
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PMID:Protease-activated receptor 4-like peptides bind to thrombin through an optimized interaction with the enzyme active site surface. 1213 67

Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease alpha-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric by PAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by alpha-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5'-diphosphate, uniquely assemble an IQGAP2/arp2/3-actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.
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PMID:IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization. 1251 16

Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.
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PMID:Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells. 1253 82

Investigations determined the mechanism(s) by which Arg-Pro-Pro-Gly-Phe (RPPGF) inhibits thrombin-induced platelet activation. High concentrations of RPPGF inhibit thrombin-induced coagulant activity. RPPGF binds to the active site of thrombin by forming a parallel beta-strand with Ser214-Gly216 and interacts with His57, Asp189, and Ser195 of the catalytic triad. RPPGF competitively inhibits alpha-thrombin from hydrolyzing Sar-Pro-Arg-paranitroanilide with a Ki = 1.75 +/- 0.03 mM. Other mechanisms were sought to explain why RPPGF inhibits thrombin activation of platelets at concentrations below that which inhibits its active site. Soluble RPPGF blocks biotinylated NATLDPRSFLLR of the thrombin cleavage site on protease-activated receptor (PAR)1 from binding to the peptide RPPGC (IC50 = 20 microM). The soluble recombinant extracellular domain of PAR1 (rPAR1EC) blocks biotinylated RPPGF binding to rPAR1EC (IC50 = 50 microM) bound to microtiter plates, but rPAR1EC deletion mutants missing the sequence LDPR or PRSF do not. RPPGF and related forms prevent the thrombin-like enzyme thrombocytin from proteolyzing rPAR1EC at concentrations that do not block thrombocytin's active site. These studies indicate that RPPGF is a bifunctional inhibitor of thrombin: it binds to PAR1 to prevent thrombin cleavage at Arg41 and interacts with the active site of alpha-thrombin.
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PMID:Mechanisms of Arg-Pro-Pro-Gly-Phe inhibition of thrombin. 1259 31


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