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)

Stimulated platelets secrete a variety of physiologically active substances that affect many neutrophil functions. We have examined the capacity of platelets to modulate superoxide anion generation by neutrophils. The amounts of superoxide anion produced by neutrophils in the presence of platelets were markedly enhanced when platelet-neutrophil coincubations were stimulated with agents that simultaneously activate both cell types, as the calcium ionophore A23187 and sodium arachidonate. This effect was dependent upon the number of platelets added to the incubation media and was not affected by inhibitors of arachidonic acid pathway or by preincubation of platelets with an antibody anti-P-selectin. The hypothesis of an involvement of purine nucleotides released by platelets during aggregation on the observed effect of enhancement of superoxide anion generation by neutrophils was then tested. Experimental evidence indicates that platelets release, during A23187-induced aggregation, amounts of ATP that are of the same order (5-10 microM) of those demonstrated to enhance superoxide anion generation by neutrophils. In addition, platelet lysates mimicked the effect of intact platelets in enhancing superoxide anion generation by A23187 stimulated neutrophils. Interestingly, at variance with the results obtained with intact platelets and platelet lysates, supernatants of thrombin-stimulated platelets did not increase O2.- by neutrophils. The enhancing effect of these supernatants was, however, restored when platelets were preincubated with an antibody anti P-selectin. These data indicate that platelets, through the release of purine nucleotides, enhance superoxide generation by neutrophils, thus increasing the cytotoxic potential of these cells.
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PMID:Platelet-neutrophil interaction and superoxide anion generation: involvement of purine nucleotides. 872 Aug 96

In an in vitro study the effect of various thrombin inhibitors (argatroban, efegatran, DuP 714, recombinant hirudin and PEG-hirudin) on platelet activation in whole blood was investigated. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregation of platelets. Blood was anticoagulated with either argatroban, efegatran, DuP 714, hirudin or PEG-hirudin at final concentrations of 10 micrograms/ml. Blood samples were then incubated at 37 degrees C either with saline, r-tissue factor, arachidonic acid, adenosine diphosphate or collagen. At definite times (1, 2.5, 5, 10 min) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed a platelet activation after all agonists used. All thrombin inhibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after activation with the other agonists an increased percent CD-62 expression was found with a maximum after 2.5 to 5 min. The results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inhibited by these agents. The observations further suggest that while thrombin inhibitors may control serine proteases, these agents do not inhibit the activation of platelets mediated by other agonists.
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PMID:Flow cytometric evaluation of the effect of various thrombin inhibitors on platelet activation in whole blood. 873 29

Platelet degranulation occurs when platelets are activated. Alpha degranulation releases P-selectin whereas lysosomal degranulation releases GP53. A correlation between these two markers might therefore be expected. We studied the correlation between P-selectin and GP53 in 50 patients with myeloproliferative disorders (MPD), 35 normal controls and 105 disease controls (patients with inflammatory bowel disease [IBD, n = 52], rheumatoid arthritis [RA, n = 26] and coronary artery disease [CAD, n = 27]) by flow cytometry before and after stimulation with thrombin ex vivo. There was no significant correlation between percentage expression of P-selectin and GP53 in unstimulated samples in normal individuals; r = 0.13, P = 0.3, n = 34. Mild thrombin stimulation (10 mU/ml) led to both alpha and lysosomal degranulation with a strong correlation (r = 0.62, P < 0.001, n = 35). Disease controls (IBD, RA and CAD) showed similar trends. In patients with MPD, in contrast, a strong correlation between the expression of these platelet activation markers was demonstrable in unstimulated samples (r = 0.37, P = 0.007, n = 50). P-selection and GP53 expression in stimulated samples also correlated well. The data support the existence of different control pathways for the steady state expression of P-selection and GP53. Heterogeneous steady state responses of P-selectin and GP53 may be physiological and loss of this heterogeneity may be a hitherto unreported and pathologically important feature of MPD. This lack of correlation appears to be specific to MPD and is not simply a function of increased in vivo platelet activation.
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PMID:Correlation of GP53 and P-selectin expression in myeloproliferative disorders and normal controls. 873 10

It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.
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PMID:Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets. 873 21

A reduction in the ability of GPIb to bind specific MoAbs or ligands (vWF) has been reported in platelets exposed to thrombin in suspension. We have analyzed modifications in the presence of glycoproteins (GPs) on platelets activated under flow conditions in a system which allows limited thrombin and fibrin generation. Normal blood anticoagulated with low molecular weight heparin (LMWH, Dalteparin 20 IU/ml) was recirculated for up to 10 min at 800 s-1 through annular chambers containing denuded arterial segments. Aliquots of blood were removed from the reservoir at 0, 1, 5 and 10 min and immediately mixed with paraformaldehyde. Membrane glycoproteins: GPIb (CD42b), GPIIb-IIIa (CD41a), GPIV (CD36); and activation dependent antigens: P-selectin (CD62P) and lysosomal glycoprortein (CD63), were detected in whole blood by dual color flow cytometry. Circulation of through the perfusion system resulted in platelet activated as demonstrated by the increased percentage of platelets positive for antigens CD62P and CD63. A gradual increase in the binding of MoAbs directed against GPIb, GPIIb-IIIa, and GPIV epitopes was noted during the entire perfusion period. Observed differences in mean fluorescence intensities at all the observation times were statistically significant (P < 0.001). Our results obtained on platelets in an experimental thrombosis system indicate that GPIb, GPIIb-IIIa and GPIV remain on the surface of activated platelets and actually increase their expression. Alterations detected at the level of GPIb in platelets activated by thrombin in suspension may not take place under in vivo situations.
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PMID:Redistribution of membrane glycoproteins in platelets activated under flow conditions. 873 22

Cardiopulmonary bypass circuits cause morbidity during cardiac operations. Plasma proteins and cellular components are stimulated by contact with the cardiopulmonary bypass circuit and can cause bleeding and postperfusion syndrome. This is especially true in patients undergoing reoperative cardiac procedures, which carries a higher risk of postoperative bleeding and prolonged ventilation compared with primary cardiac surgical procedures. Recently, cardiopulmonary bypass circuit surfaces have been coated with antithrombotic agents to improve their biocompatibility. This study evaluated the effect of a heparin-coated cardiopulmonary bypass system (Duraflo II, Baxter Bentley Healthcare Systems, Irvine, Calif.) on thrombin formation, platelet stimulation, and leukocyte activation in patients undergoing reoperative coronary artery bypass grafting or valve operation. Fifty patients were selected and randomly assigned to a standard noncoated control system (n = 26) or the Duraflo heparin-coated system (n = 24). Similar heparin doses were used in both groups (3 mg/kg). The heparin-coated group used a completely heparin-coated bypass circuit including the cardiotomy reservoir; arterial filters were heparin-coated in both groups. Samples were obtained before cardiopulmonary bypass, 30 minutes into cardiopulmonary bypass, 5 minutes after crossclamp removal, and 5 minutes after protamine administration. Thrombin formation (thrombin-antithrombin III by enzyme-linked immunosorbent assays) and platelet activation (beta-thromboglobulin by enzyme-linked immunosorbent assays; P-selectin expression by flow cytometry) were assayed. Leukocyte activation was determined by quantitative and qualitative analysis of arterial filters by scanning electron microscopy in six patients from each group. In both circuits, thrombin values increased markedly 30 minutes into cardiopulmonary bypass compared with baseline values (p < 0.001) (heparin-coated, 7 +/- 5 to 96 +/- 115 ng/ml; noncoated, 10 +/- 9 to 115 +/- 125 ng/ml). Platelet activation as measured by beta-thromboglobulin (heparin-coated, 104 +/- 100 to 284 +/- 166 IU/ml; noncoated, 81 +/- 74 to 288 +/- 277 IU/ml) and P-selectin expression (heparin-coated, 1.5% +/- 1.5% to 6.4% +/- 6.1%; noncoated, 1.4% +/- 1.1% to 6.2% +/- 4.3%) also significantly increased 30 minutes into cardiopulmonary bypass compared with baseline values (p < 0.001). Platelet activation and thrombin generation did not differ between the two circuits at any time. Granulocyte activation and platelet deposition did not differ between the two circuits when arterial filters were evaluated. Both groups had similar heparin and protamine administration, blood transfusions, postoperative alveolar-arterial oxygen gradient, time to extubation, length of intensive care unit stay, and overall morbidity and mortality. Clinical outcome and blood loss did not differ between the groups. We conclude that heparin-coated cardiopulmonary bypass circuits did not improve biochemical or clinical markers of biocompatibility in a reoperative patient population.
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PMID:Biocompatibility of heparin-coated extracorporeal bypass circuits: a randomized, masked clinical trial. 875 16

Platelet activation in addition to blood coagulation abnormality is regarded as a primary factor of thrombosis by atherosclerotic obstruction. Therefore, the platelet activation on atherosclerosis is considered to correlate with shear stress generated between blood cells and endothelial cells in blood flow. P-selectin on the surface of the platelet membrane is measured by flowcytometry as a marker of platelet activation. Herein, we examined the phenomena of platelet activation and adherent platelets with leukocytes (ad-P.L) on stored platelet concentration (PC) and chronic rheumatoid arthritis (RA), moreover, of the adherent platelets or polymorphonuclear cells (PMN) with endothelial cells (EC) in shear stress by using an apparatus we devised. The rate of platelet activation and ad-P.L increased in PC with storage, and the sensitivity of platelets to thrombin decreased. The rate of platelet activation and ad-P.L increased in RA in vivo. Many adherent platelets with EC were found at a low shear rate on normal EC but at a high shear rate on denatured EC without any specific adherent property. Adherent PMN with EC had several hundred times more denatured EC than normal EC and the relationship with shear rate disappeared on denatured EC. The platelet activation and relationship between platelets or leukocytes and EC as to the cause of thrombosis are important subjects for future studies.
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PMID:[Adherent reaction among activated platelets, polymorphonuclear cells and vascular endothelial cells under thrombin stimulation or shear stress]. 881 62

Polymorphonuclear leukocytes (PMN) are directly involved in development of ischemic myocardial injury. Adhesion of PMN to endothelial cells is an initial step that triggers a sequential process leading to acute inflammatory responses. Interaction between P-selectin and its oligosaccharide ligand, sialyl Lewis x (sLex), plays an important role in the early stage of the adhesion. To examine the role of P-selectin in various animal disease models especially in rats, we have cloned rat E- and P-selectin cDNAs and established monoclonal antibodies against these rat selectins. In this report, we describe the generation and characterization of anti-rat P-selectin antibodies (ARPs). These antibodies detect cell surface P-selectin on thrombin-stimulated rat platelets. More importantly, intravenous administration of ARP2-4 reduced infarction developed after 30 min of ischemia followed by 24 h of reperfusion in a rat myocardial injury model. In addition, similar protective effect was also observed by administration of a sLex-oligosaccharide. These results indicate that cell adhesion mediated via P-selectin is involved in the development of ischemia and reperfusion injury in rat heart.
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PMID:Reduction of rat myocardial ischemia and reperfusion injury by sialyl Lewis x oligosaccharide and anti-rat P-selectin antibodies. 884 11

In patients with mitral valve prolapse (MVP) a high incidence of valvular abnormalities with a history of previous cerebrovascular disease has been reported and an embolic mechanism has been proposed. Aim of this study is the study of platelet and coagulation activation in patients with MVP. Fifty-four patients affected by MVP (mean age 46 +/- 15 yrs, 22 males, 32 females) and 50 control subjects, age- and sex-matched, were tested for platelet activation [P-selectin and GpIIb-IIIa platelet surface expression at rest and after stimuli by flow cytometric analysis, Beta-Thromboglobulin (TG) and Platelet Factor 4 (PF4) plasma levels by ELISA, platelet-rich-plasma (PRP) and whole blood spontaneous platelet aggregation (SPA)] and for activation of blood coagulation (Prothrombin activation fragment F1+2 plasma levels by ELISA). P-selectin, GpIIb-IIIa expression, Beta-TG, PF4 and SPA were found similar in MVP patients and in controls. However, in patients with severe mitral regurgitation (MR) the percentage of activated platelets which express P-selectin after stimuli was slightly but significantly (p < 0.05) lower in comparison to MVP patients without or with mild to moderate MR and to controls. Moreover, in patients with severe MR F1+2 levels (median 1.6 nmol/L, range 0.6-2.6 nmol/L) were significantly higher (p < 0.001) than both in controls (median 0.95 nmol/L, range 0.2-1.4 nmol/L) and in patients without or with mild to moderate MR (median 1.0 nmol/L, range 0.4-2.3 nmol/L). Our findings suggest that MVP is not responsible per se for blood clotting activation, but in patients with severe mitral insufficiency an increase in thrombin generation can occur. These alterations in hemostatic system may represent a mechanism by which MR increases the risk of thromboembolic events in patients with MVP.
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PMID:Platelet and blood clotting activation in patients with mitral valve prolapse. 887 Jan 74

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.
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PMID:In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. 887 31


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