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)

P-selectin, also known as GMP-140, PADGEM or CD62, is expressed on the surface of thrombin-activated platelets and endothelial cells (EC). It is a member of the selectin family of adhesion molecules that regulate leucocyte interactions with the blood vessel wall. In this study we have found that peptides derived from both the lectin (residues 19-34 and 51-61) and epidermal growth factor (EGF)-like (residues 127-139) domains inhibit the adhesion of peripheral blood mononuclear cells (PBMC), elutriated monocytes and a monocytic cell line (U937) to thrombin-activated EC. This inhibition occurred in a concentration-dependent manner and the peptide most active at the lowest concentrations was the one derived from the EGF-like motif (127-139). The scrambled forms of these peptides, identical in amino acid composition to the authentic peptides but with altered sequences, were not inhibitory. Thrombin-activated platelets supported adhesion of U937 cells and this adhesion was dramatically inhibited by the two peptides derived from the lectin-like domain (residues 19-34 and 51-61). All three peptides, when conjugated to BSA and coated on plastic plates, mediated U937 cell adhesion. This study shows, for the first time, that two sites on P-selectin, the lectin and EGF-like domains, are involved in the adhesion of monocytes to thrombin-activated EC.
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PMID:Two sites on P-selectin (the lectin and epidermal growth factor-like domains) are involved in the adhesion of monocytes to thrombin-activated endothelial cells. 752 45

The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.
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PMID:Thrombin binding to platelets and their activation in plasma. 752 34

The lectin-like domain of P-selectin, an adhesive receptor (also known as PADGEM, GMP-140 or CD62) is implicated in platelet or endothelial cell interactions with leukocytes. The aim of this study was to characterize the lectin-like domain of rat P-selectin by the use of synthetic peptides. The lectin and EGF-like domains of rat P-selectin were cloned in our laboratory and shown to present very strong homologies to its human counterpart. Peptides corresponding with the lectin-like domain of P-selectin were tested for their ability to inhibit thrombin-activated platelets rosetting to neutrophils. Peptides 23-30 (A) and 76-90 (C), but not peptide 51-61 (B), inhibited thrombin activated rat platelets interactions with rat neutrophils (A = 33%, C = 46%, P < 0.05). Using a combination of peptides (A+B = 35%, P = 0.008 and A+C = 62%, P < 0.001), we observe different degrees of inhibition of platelets binding to neutrophils. The IC50 of peptides A+C was 0.11 mM. LYP-20, an anti-human P-selectin monoclonal antibody, was also observed to inhibit thrombin-activated rat platelets binding to rat neutrophils in a very significant manner (57% of inhibition, P < 0.001). Moreover, heparin inhibited thrombin-stimulated platelet/neutrophils rosetting (36% of inhibition, P < 0.01). These results show the importance of two sites (23-30 and 76-90) on the lectin-like domain of P-selectin in mediating platelet-neutrophil interactions in rats. Such peptides may be potent in vivo inhibitors of cell-cell interactions involving P-selectin.
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PMID:Two sites (23-30, 76-90) on rat P-selectin mediate thrombin activated platelet-neutrophil interactions. 753 Jan 56

The natural history of acute myocardial infarction has been dramatically changed by the advent of thrombolytic treatment, with a 30% mortality reduction, a better recovery of ventricular function, and a better quality of life. This treatment notwithstanding, failure or delay in achieving reperfusion, along with reocclusion and bleeding, still worry clinicians and challenge researchers to improve thrombolytic regimens and concomitant antithrombotic treatments. Platelet activation, at least in part because of thrombolytic treatment itself, plays a pivotal role in the pathogenesis of resistance to lysis and rethrombosis. The aim of this study was to compare in vitro the effects on platelets of therapeutic concentrations of streptokinase (SK) and recombinant type plasminogen activator (rt-PA). The effects of plasmin and thrombin were also studied as a reference. Fluorescence flow cytometry was used to evaluate (1) fibrinogen binding and (2) surface expression of GMP-140, a sensitive marker of platelet release reaction. Platelet function was further studied by measuring the release of carbon 14-labeled serotonin, beta-thromboglobulin, plasminogen activator inhibitor-1 (PAI-1) and the generation of thromboxane (TxB2). We found that 10 nmol/L SK and 14 nmol/L rt-PA increased fibrinogen binding to platelets by 12 +/- 2 and 10 +/- 4 times, respectively (p = not significant). At the same concentrations, SK, but not rt-PA, also induced the platelet release reaction: surface expression of GMP-140 was increased by 6 +/- 1.5 times by SK and 1.3 +/- 0.2 times by rt-PA (p < 0.05). TxB2 production was not modified by plasmin and plasminogen activators. Our data showed that plasmin and SK stimulate fibrinogen receptor expression and platelet degranulation. Conversely, rt-PA, at concentrations up to 14 nmol/L, only promotes fibrinogen binding. These results, coupled with the less-pronounced lytic state induced by rt-PA, could explain the higher incidence of reocclusion accompanying rt-PA therapy in comparison with SK that occurs unless effective "adjunctive" antithrombotic treatment is used. Neither of the thrombolytic agents activates the arachidonate pathway. Thus aspirin is probably not an ideal agent to be used in conjuction with thrombolytic agents. We speculate that newer approaches, particularly RGD peptides and antibodies against GP llb/llla, might produce better results.
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PMID:Streptokinase and rt-PA activate platelets by a different way: implications on the rethrombosis rate after their administration in myocardial infarction. 753 Dec 12

It is now well established that monocytes adhere to endothelial cells activated by oxidized low-density lipoproteins (LDL). However, the adhesive receptors on endothelial cells involved in binding monocytes, following an insult by oxidized LDL, remains to be elucidated. In this study we have looked at the effect of native or oxidized LDL on the expression of P-selectin. Native LDL (N-LDL) was oxidized by incubation with either endothelial cells (EC-LDL) or copper (Cu-LDL), or in culture medium as a control (C-LDL). Expression of P-selectin was assayed with an anti-P-selectin (CD62) monoclonal antibody (LYP20). Results show that EC-LDL and Cu-LDL, but not N-LDL or C-LDL, induce the expression of P-selectin by human umbilical-vein endothelial cells (HUVECs). Induction of P-selectin by low concentrations (20 micrograms/ml) of LDL is directly related to the state of oxidation of the LDL particles. In addition, high concentrations (100 micrograms/ml) of N-LDL also activate HUVECs by inducing P-selectin expression. This expression was sustained for a period of over 1 h on LDL-activated endothelial cells, in contrast with thrombin- or histamine-activated endothelial cells, whose P-selectin levels fall within 15-20 min after induction. E-selectin, in contrast with P-selectin, could not be induced by endothelial cells treated with low or high concentrations of oxidized LDL. Results in this study show that P-selectin expressed by oxidized-LDL-treated endothelial cells are involved in mediating the adhesion of a monocytic cell line (U937) or monocytes in peripheral-blood mononuclear cells. An anti-P-selectin monoclonal antibody (LYP20) inhibited the binding of U937 cells and monocytes. These results strongly suggest that P-selectin is involved in the early stages of atherogenesis.
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PMID:Oxidized low-density lipoprotein induces the expression of P-selectin (GMP140/PADGEM/CD62) on human endothelial cells. 753 99

Platelets are activated by substances from the subendothelial matrix in endothelial lesions or by factors in the plasma coagulation cascade. Conversely, activated platelets are potent activators of this cascade. Only activated platelets express the adhesion molecules Gp53, GMP140 and thrombospondin on the plasma membrane. The postmortem activation status of platelets, therefore, can be determined immunoelectron microscopically by immunogold labeling of antibodies against these glycoproteins. Our studies revealed that the vast majority of these antigens were located within the granules postmortem, hence the platelets had not been activated. Thrombin-induced activation of platelets in vitro was only possible in the early postmortem interval, as demonstrated by labeling of the adhesion molecules on the plasma membrane. Later, such activation was no longer possible even though thrombin-induced fibrin formation gave the appearance of "coagulated blood". In forensic medicine, these findings can possibly be applied to distinguish intravital clotting from the postmortem coagulation phenomena and intravital hematomas from postmortem hematomas.
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PMID:The postmortem activation status of platelets. 753 69

Long-term exposure of platelets to prostacyclin or iloprost (100nM, 3hr) results in receptor desensitization measured as decrease in 3H-iloprost binding sites by 47 +/- 14%. Desensitized platelets respond with an increased adhesion to endothelial cells. The mechanism of increased adhesiveness was studied by measuring the expression of the adhesion molecule CD62p (p-selectin; GMP140) on washed human platelets by flowcytometry. In thrombin stimulated platelets CD62p expression was dose-dependently reduced by iloprost. In receptor desensitized platelets IC50 for iloprost inhibition of thrombin-induced CD62p expression increased from 0.48 +/- 0.10 to 2.4 +/- 0.7 nM.
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PMID:Diminished inhibition of adhesion molecule expression in prostacyclin receptor desensitized human platelets. 753 85

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

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.
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PMID:Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine, and dipyridamole. 909 1

Previous studies have suggested that qualitative changes in platelet bound fibrinogen modulate platelet aggregation. The present study used confocal scanning laser microscopy to further evaluate post-ligand binding events over a 60-minute time course. When fluorescein isothiocyanate (FITC)-streptavidin was added to ADP-stimulated platelets 1 minute after biotinylated fibrinogen binding at 22 degrees C, bound fibrinogen was found in variously sized patches on the cell surface. When streptavidin was added 60 minutes later, bound fibrinogen had been cleared from the platelet surface and was observed in clusters penetrating into platelets to various extents. ADP-activated platelets did not stain with a monoclonal antibody against CD62 suggesting that platelets were not permeabilized during the experiment and had not released alpha-granules. Additional studies using either biotinylated fibrinogen that had been prelabeled with FITC-streptavidin or FITC-labeled fibrinogen revealed similar patterns of platelet-associated fibrinogen clearance and redistribution. Pretreatment of platelets with cytochalasin D prevented this redistribution. Dual labeling experiments using biotinylated fibrinogen and FITC-streptavidin as well as a monoclonal anti-GPIIIa antibody labeled with rhodamine-conjugated anti-mouse IgG demonstrated the co-localization of fibrinogen and GPIIIa. Similar observations were made with fibrinogen bound to thrombin-stimulated platelets. In contrast, fibronectin bound to thrombin-activated platelets retained a predominantly surface membrane distribution under identical experimental conditions. Since surface-cleared fibrinogen was accessible to exogenous FITC-streptavidin under conditions that did not lead to platelet permeabilization, the data suggest fibrinogen deposition in compartments that are accessible to the extracellular milieu. This is consistent with the ability of exogenous plasmin to completely remove cleared fibrinogen pools without detectable fibrinogen reexpression on the platelet surface or alpha-granule secretion. The data provide morphological evidence for the selective, GPIIb-IIIa mediated, actin-dependent clearance of bound fibrinogen from the activated platelet surface, suggesting a mechanism for preventing and limiting thrombus development.
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PMID:Bound fibrinogen distribution on stimulated platelets. Examination by confocal scanning laser microscopy. 767 79


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