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)

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca(2+)-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and alpha-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by gamma-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (approximately 200% increase with 10(-6) MPMA, approximately 400% increase with 10(-8) M alpha-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or alpha-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented alpha-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayer, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.
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PMID:Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. 152 36

Interaction of fibrinogen with its receptors (glycoprotein IIb/IIIa complex) on platelet membranes leads to platelet aggregation. By means of gel filtration, CM-Sephadex C-50, and reverse-phase HPLC, an antiplatelet peptide, gabonin, was purified from the venom of Bitis gabonica. The purified protein migrates as a 21,100-Da polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and as a 11,000-Da peptide in the presence of beta-mercaptoethanol, indicating that gabonin is a disulfide-linked dimer. It is a polypeptide consisting of about 84 amino acid residues, rich in Asp, Pro, and half-cystine. Gabonin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, U46619, or thrombin in preparations of platelet-rich plasma and platelet suspension (IC50 = 340-1600 nM). It also blocked platelet aggregation of whole blood. However, it apparently did not affect the initial shape change and only slightly reduced ATP release caused by aggregation agonists. Gabonin did not inhibit the rise of cytosolic calcium in Quin-2-loaded platelets stimulated by thrombin. In addition, gabonin dose-dependently inhibited fibrinogen-induced aggregation of elastase-treated platelets. In conclusion, gabonin inhibits platelet aggregation mainly through the blockade of fibrinogen binding toward fibrinogen receptors of the activated platelets.
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PMID:An antiplatelet peptide, gabonin, from Bitis gabonica snake venom. 152 22

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.
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PMID:The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells. 153 May 95

The hamster gene encoding the 78-kDa glucose-regulated protein (Grp78) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. After induction with isopropyl beta-D-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affinity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca(2+)-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated less than 50% by CaCl2, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca2+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and dimers. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.
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PMID:Interactions of liver Grp78 and Escherichia coli recombinant Grp78 with ATP: multiple species and disaggregation. 153 51

The action of many agonists results in rapid production of sn-1,2-diacylglycerol (DAG). Using platelets as a model system, we previously identified a delayed phase of DAG accumulation that is temporally associated with secondary aggregation and secretion. In the present study, we examined the quantitative relationship between this delayed DAG accumulation and platelet aggregation and secretion. To quantitate the low levels of DAG in platelets, we used the sensitive DAG kinase assay and simultaneously compared DAG levels with aggregation and ATP secretion. In platelets stimulated by gamma-thrombin or collagen, there was a dose response between concentration of agonist and DAG accumulation. Significantly, a dose response was observed between DAG accumulation and extent of aggregation and secretion in platelets stimulated by either agonist. A concentration of either gamma-thrombin or collagen that caused secondary aggregation and secretion was associated with DAG accumulation above 0.2 pmol of DAG/nmol of phospholipid. Subthreshold concentrations of gamma-thrombin or collagen resulted in DAG levels less than 0.2 pmol/nmol of phospholipid. Thus, these data suggest that a to occur. Moreover, secretion was blocked when DAG production was blocked with aspirin or when protein kinase C was inhibited with sphingosine. We conclude that endogenously formed DAG plays a critical role in regulating secondary aggregation and secretion and, therefore, represents an important target for future antiplatelet agents.
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PMID:Quantitative analysis of diacylglycerol second messengers in human platelets: correlation with aggregation and secretion. 153 14

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

The role of residual donor white cells (WBCs) in producing the storage lesion of platelets used for transfusion was studied. The effect of prestorage WBC reduction on in vitro and in vivo measurements of the quality of stored platelet concentrates (PCs) was examined by using a newly developed WBC-reduction filter capable of preparing PCs with a mean residual WBC concentration of less than 1 per microL. For in vitro studies, a triplet study design was used, in which WBC-reduced PCs were matched to standard PCs and to WBC-enriched PCs obtained from the same donor at the same phlebotomy. Twelve donors were studied. Prestorage WBC reduction resulted in a higher pH and pO2 and a lower pCO2 than in standard PCs. In accord with previous in vitro studies, a significant rise in plasma glycocalicin and lactate dehydrogenase was measured during storage, but the levels were not significantly different in WBC-reduced PCs and standard PCs. Platelet aggregation and ATP release in response to graded doses of thrombin was similar in WBC-reduced and standard PCs. In vivo recovery and survival studies were comparable in WBC-reduced and standard PCs. Although the residual donor WBC content of PCs has a significant impact on storage pH, pO2, and pCO2, prestorage WBC reduction does not affect platelet structure, function, or viability as assessed by in vitro or in vivo measurements.
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PMID:The effect of prestorage white cell reduction on the function and viability of stored platelet concentrates. 158 38

There exist no data yet about platelet storage in teflon bags. Therefore, a paired study has been performed to evaluate their suitability for the 5 days storage of platelet concentrates (PC) from the cell separator Fresenius AS-104. The PC's of the AS-104 were stored in teflon as well as in polyolefin bags each (PL-732). In addition, PC's of the Baxter CS-3000 were stored in PL-732. PH, cell counts, platelet morphology, aggregation, adherence and ADP/ATP concentration, plasma beta TG, glucose, lactate and thrombin-antithrombin III complexes were investigated at day 0, 3 and 5 of storage. The results show that the teflon bags are quite suitable for 5 day storage of the PC's. But the functional superiority of fresh PC's from the AS-104 seemed to be reduced by storage in teflon bags compared to storage in PL-732. The unfavorable size and geometry of the teflon bags used could have been the cause. Therefore, size and geometry were consequently changed in the meantime.
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PMID:Teflon bags for 5-day storage of platelet concentrates (PC) from cell separator. 162 21

Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2.
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PMID:Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. 163 Nov 1

Platelet antibodies identified in the plasma of three multiply transfused patients and a woman who had delivered a baby with neonatal alloimmune thrombocytopenia were investigated for their platelet activating properties. Three patients possessed multispecific HLA antibodies reactive with 90 to 100% of the cells on a lymphocytotoxic panel. These antibodies were also detected using the MAIPA assay and MAb w6/32, which recognizes an epitope common to all HLA class I molecules. In addition to HLA antibodies, three of the patients possessed platelet-specific antibodies that were identified by the MAIPA assay as anti-HPA-1a and anti-HPA-3a (one patient) and anti-HPA-1b (two patients). Each of the HLA antibodies when reacted with platelets expressing the corresponding HLA antigens, potently induced aggregation and release of ATP from dense granules. In contrast, the HPA-1b antibodies induced platelet agglutination, but failed to trigger ATP release. However, platelets coated with these latter antibodies were now refractory to subsequent stimulation by ADP. Similarly, when HLA antibodies were reacted with platelets to produce suboptimal activation, the platelets could now be stimulated only poorly or not at all by either epinephrine or thrombin. This was also true for anti-HPA-1b, which, although not inducing aggregation or ATP release by itself, was capable of almost completely blocking thrombin-induced platelet activation. The thrombin-inhibiting activity of these antibodies could partially be reversed by pretreating antibody-coated platelets with epinephrine immediately followed by stimulation with thrombin. These findings suggest that transfused platelets may either be activated or inhibited by reaction with various platelet antibodies. Therefore it is conceivable that the presence of platelet reactive antibodies in multiply transfused recipients may contribute to the increased thrombotic and hemorrhagic symptoms often observed among these patients.
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PMID:Human platelet activating antibodies. 163 66


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