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)

Selectins are Ca(2+)-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neutrophil but not monocyte activation inhibits P-selectin-mediated platelet adhesion. 753 48

Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells. To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin. P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine. We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin. With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance of P-selectin are very rapid. Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine. These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling.
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PMID:Histidine phosphorylation of P-selectin upon stimulation of human platelets: a novel pathway for activation-dependent signal transduction. 754 25

N,N,N-trimethylsphingosine (TMS), a stable synthetic sphingosine derivative, was investigated in a feline model of myocardial ischemia (90 min) and reperfusion (270 min) injury. TMS (60 micrograms/kg), administered intravenously 10 min before reperfusion, significantly attenuated myocardial necrosis (15 +/- 3 vs. 31 +/- 4% necrosis of area at risk, P < 0.01) and cardiac myeloperoxidase activities, a marker of neutrophil accumulation, compared with vehicle-treated cats. Endothelium-dependent relaxation to acetylcholine in ischemic-reperfused coronary artery rings treated with TMS was also significantly preserved compared with vehicle (73 +/- 4 vs. 34 +/- 4% vasorelaxation, P < 0.01). Polymorphonuclear neutrophil (PMN) adherence to coronary endothelium 270 min after reperfusion was markedly attenuated in the TMS group compared with vehicle-treated cats (37 +/- 5 vs. 76 +/- 5 PMN/mm2, P < 0.01). TMS also attenuated upregulation of P-selectin on coronary venular endothelium by immunohistochemistry. This was consistent with in vitro findings that TMS attenuates PMN adherence to thrombin-stimulated coronary endothelium and P-selectin upregulation on thrombin-stimulated cat platelets. A sphingolipid derivative, TMS at physiological concentrations exerts cardioprotective actions and preserves coronary endothelial function following myocardial ischemia and reperfusion in vivo. The effects appear to be mediated by the inhibition of PMN-endothelial interaction and subsequent accumulation into the ischemic myocardium. Thus TMS may be a useful agent in attenuating myocardial reperfusion injury.
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PMID:Myocardial and endothelial protection by TMS in ischemia-reperfusion injury. 754 41

Immunocytochemistry with gold-labeled antibodies was used to compare the effects of stimulation of human platelets with thrombin (1 U/ml) and the thrombin receptor activating peptide, SFLLRN (20 microM). After 3 min, redistribution of fibrinogen, von Willebrand factor, and P-selectin (GMP-140, CD62) was examined, the percentages of [14C]serotonin and beta-thromboglobulin released from pre-labeled platelets were measured, and the amount of thromboxane B2 formed was assayed. Upon stimulation with either thrombin or SFLLRN, the platelets had changed from their normal disc shape to spheroidal forms with short pseudopodia. Few alpha-granules remained, the open canalicular system was expanded (more so with SFLLRN) and contained most of the fibrinogen and von Willebrand factor, although small amounts were evident on the platelet surface. Most of the P-selectin was on the surface. Both thrombin and SFLLRN caused complete release of beta-thromboglobulin and 88.3 and 77.5% release of [14C]serotonin, respectively. However, formation of TXB2 caused by thrombin was 10 times greater than that caused by SFLLRN (969 +/- 173 vs 76 +/- 22 ng/10(9) platelets). Thus, the redistribution of platelet alpha-granule contents is similar with thrombin or SFLLRN stimulation and is unaffected by the extent of thromboxane formation.
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PMID:Effects of thrombin and the thrombin receptor activating peptide, SFLLRN, on redistribution of platelet alpha-granule contents are similar and independent of the extent of thromboxane formation. 755 92

Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because No has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME-induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti-P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or he nonblocking anti-P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression.
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PMID:Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets. Role of protein kinase C. 758 91

Inflammatory cell deposition in atherosclerotic blood vessels has been thought to relate to loss of endothelium-derived nitric oxide (NO). To examine whether cell deposition correlates temporally with the loss of NO activity, rat aortic rings were incubated with buffer, native LDL (n-LDL), oxidized LDL (ox-LDL), or the endothelium-derived relaxing factor synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) for 2 hours, and vascular contractile response to norepinephrine and relaxant response to acetylcholine, thrombin, and calcium ionophore A23,187 were examined. Thereafter, the rings were exposed to biotin-fluorescein isothiocyanate-labeled fluorescent or unlabeled leukocytes for 30 minutes. Cell adhesion was quantitated by fluorescent microscopy as well as by scanning electron microscopy. Incubation with n-LDL or ox-LDL did not affect either the contractile or the relaxant response of rings. However, leukocyte adhesion increased markedly in all ox-LDL-treated rings but not in those treated with n-LDL. Thus, leukocyte adhesion occurred independent of NO activity. In keeping with this concept, pretreatment of rings with the NO precursor L-arginine failed to influence leukocyte adhesion to rings incubated with ox-LDL. Treatment of rings with L-NAME also resulted in adhesion of a large number of leukocytes. Furthermore, all rings treated with ox-LDL or L-NAME demonstrated marked expression of P-selectin leukocyte adhesion molecules, determined by immunohistochemistry. Pretreatment of rings with the P-selectin blocking antibody PB1.3 markedly decreased deposition of leukocytes in rings exposed to ox-LDL.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Oxidized low-density lipoproteins facilitate leukocyte adhesion to aortic intima without affecting endothelium-dependent relaxation. Role of P-selectin. 758 92

The platelet-activating properties of plasma containing multispecific HLA antibodies were studied in plasma from several persons stimulated by platelet or red blood cell transfusion or by pregnancy. Antibodies were characterized by their lymphocytotoxic effects and by a monoclonal antibody antigen--capture enzyme-linked immunoassay. Plasma from each patient induced dose-dependent aggregation and release of adenosine triphosphate with antigen-positive platelet-rich plasma. Plasma from three patients induced normal aggregation and adenosine triphosphate release with platelet-rich plasma from donors who had taken platelet inhibiting medications (e.g. aspirin, piroxicam). In contrast, these platelets failed to release adenosine triphosphate when stimulated with 5 mumol/L adenosine diphosphate. Platelets were fully activated when saturated with HLA antibodies from one patient, although little or no stimulation was observed at 50% saturation suggesting that additional plasma cofactors and/or a threshold of bound antibody were required for activation. With a murine monoclonal antibody specific for P-selectin and antimurine immunoglobulin G labeled with iodine 125, plasma from each patient was found to induce P-selectin expression that was approximately 50% of that induced by 0.2 U/ml thrombin. Expression on platelets of P-selectin induced by plasma from one patient was independent of whether the donor had taken aspirin. Purified immunoglobulin G from this same patient stimulated platelet activation, as did the patient's serum, but no activation was observed with F(ab')2 fragments prepared from the immunoglobulin G. These studies demonstrate that HLA antibodies (1) mediate expression of P-selectin on the platelet surface, (2) are as potent as thrombin in activating platelets previously exposed to antiinflammatory agents, and (3) require an intact Fc domain to activate platelets.
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PMID:Characterization of platelet activation induced by HLA antibodies associated with alloimmune thrombocytopenia. 768 Mar 69

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.
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PMID:In vivo neutralization of P-selectin protects feline heart and endothelium in myocardial ischemia and reperfusion injury. 851 48

The use of cardiopulmonary bypass (CPB) during cardiac surgery is associated with a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time. However, the nature of the putative platelet function defect is controversial. In this study, blood was analyzed at 10 time points before, during, and after CPB. We used a whole-blood flow cytometric assay to study platelet surface glycoproteins in (1) peripheral blood, (2) peripheral blood activated in vitro by either phorbol myristate acetate, the thromboxane (TX)A2 analog U46619, or a combination of adenosine diphosphate and epinephrine, and (3) the blood emerging from a bleeding-time wound (shed blood). Activation-dependent changes were detected by monoclonal antibodies directed against the glycoprotein (GP)Ib-IX and GPIIb-IIIa complexes and P-selectin. In addition, we measured plasma glycocalicin (a proteolytic fragment of GPIb) and shed-blood TXB2 (a stable breakdown product of TXA2). In shed blood emerging from a bleeding-time wound, the usual time-dependent increase in platelet surface P-selectin was absent during CPB, but returned to normal within 2 hours. This abnormality paralleled both the CPB-induced prolongation of the bleeding time and a CPB-induced marked reduction in shed-blood TXB2 generation. In contrast, there was no loss of platelet reactivity to in vitro agonists during or after CPB. In peripheral blood, platelet surface P-selectin was negligible at every time point, demonstrating that CPB resulted in a minimal number of circulating degranulated platelets. CPB did not change the platelet surface expression of GPIb in peripheral blood, as determined by the platelet binding of a panel of monoclonal antibodies, ristocetin-induced binding of von Willebrand factor, and a lack of increase in plasma glycocalicin. CPB did not change the platelet surface expression of the GPIIb-IIIa complex in peripheral blood, as determined by the platelet binding of fibrinogen and a panel of monoclonal antibodies. In summary, CPB resulted in (1) markedly deficient platelet reactivity in response to an in vivo wound, (2) normal platelet reactivity in vitro, (3) no loss of the platelet surface GPIb-IX and GPIIb-IIIa complexes, and (4) a minimal number of circulating degranulated platelets. These data suggest that the "platelet function defect" of CPB is not a defect intrinsic to the platelet, but is an extrinsic defect such as an in vivo lack of availability of platelet agonists. The near universal use of heparin during CPB is likely to contribute substantially to this defect via its inhibition of thrombin, the preeminent platelet activator.
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PMID:The platelet function defect of cardiopulmonary bypass. 840 Feb 75

P-selectin, a receptor for neutrophils and monocytes, is an adhesion molecule on the surface of activated platelets that resides in the alpha granule membrane of unstimulated platelets. To determine whether phosphorylation of P-selectin might accompany platelet activation, P-selectin in resting and thrombin-stimulated platelets labeled with o-[32P]phosphate was immunoprecipitated with the monoclonal antibody AC1.2 directed against P-selectin. SDS-gel electrophoresis of the immunoprecipitates indicated about 10-20-fold higher levels of 32P incorporated into P-selectin from thrombin-activated platelets than in resting platelets, although both sets of platelets contained equivalent amounts of P-selectin. The lower limits of the molar ratio of phosphate to P-selectin in activated platelets is about 0.52 +/- 0.08. Other platelet agonists, including the thrombin receptor peptide (SFLLR), epinephrine, ADP, and collagen, similarly stimulated phosphorylation of P-selectin. The kinetics of P-selectin phosphorylation following thrombin stimulation was rapid, with maximum phosphorylation observed at 15-30 s. Phosphoamino acid analysis of the phosphorylated P-selectin revealed the rapid synthesis of phosphoserine, phosphothreonine, and phosphotyrosine, but 80-90% of the phosphotyrosine and phosphothreonine disappeared within 5 min of platelet activation while the maximal level of phosphoserine remained stable. The rapid phosphorylation and selective dephosphorylation of specific amino acids in P-selectin following platelet activation may be important for P-selectin function and signal transduction within platelets.
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PMID:Rapid phosphorylation and selective dephosphorylation of P-selectin accompanies platelet activation. 768 99


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