Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the effects of porcine factor VIII (P-FVIII; Hyate:C) and other coagulation products employed in the management of patients with hemophilia A, on platelet activation in vitro. Exposure of normal resting platelets to P-FVIII resulted in platelet activation, as manifested by increased expression of the platelet surface activation markers CD62, CD63, and activated-GPIIbIIIa, and by activation-induced modulation of expression of normal platelet membrane glycoproteins CD41, CD42, and CD36. In contrast, platelet activation was not observed after exposure of the platelets to human FVIII, FEIBA, recombinant FVIIa, or cryosupernatant plasma. As with thrombin, exposure of platelets to P-FVIII resulted in the generation of platelet microparticles, an effect not seen not with the other products. In contrast to the characteristic reduction in expression in the number of CD42 molecules detected on thrombin-activated platelets, P-FVIII-stimulated platelets showed a small increase in CD42 expression. In contrast to thrombin, P-FVIII did not cause platelet dense granule release. The results indicate that therapeutic P-FVIII activates platelets, likely in ways that are different from the platelet activation seen with thrombin. The observed platelet activation and microparticle generation may provide a "hypercoagulable" mechanism for hemostasis with P-FVIII therapy separate from, and additional to, that due to increased circulating FVIII levels.
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PMID:Platelet activation induced by porcine factor VIII (Hyate:C). 949 69

Although platelet activation may play a role in coronary artery spasm, platelets activated following coronary vasospasm have not been clinically detected. We performed flow cytometric analysis of activation-dependent granular proteins, CD62P (P-selectin), CD63, PAC-1 (activated glycoprotein [GP] IIb/IIIa) and thrombospondin on the platelet plasma membrane in patients who exhibited acetylcholine-induced coronary vasospasm and compared findings with those in control patients without vasospasm. We simultaneously investigated the plasma levels of thrombin anti-thrombin III complex (TAT), plasmin alpha2-plasmin inhibitor complex (PIC), and thrombomodulin. In patients with vasospasm, the expression of CD62P, CD63 and PAC-1 on the platelet membrane surface increased in coronary sinus blood samples following coronary vasospasm, although the expression in aortic samples did not change. The TAT level also increased in the coronary sinus after vasospasm. Platelets might be activated by coronary vasospasm within the coronary circulation. The platelet activation process may be modulated by thrombin generation.
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PMID:Detection of platelets activated during acetylcholine-induced coronary vasospasm. 960 37

Moderate consumption of alcoholic beverages is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to inhibition by ethanol of platelet responses to some aggregating agents. Although ethanol is known to inhibit thrombin-induced secretion of platelet dense granule contents, the effect of ethanol on secretion of alpha-granule and lysosomal contents has not been studied. Using suspensions of washed platelets, and a range of thrombin concentrations (up to 0.1 U/ml), we examined the effect of 87 mM ethanol on secretion of [14C]serotonin from prelabelled platelets as a measure of secretion of dense granule contents. Secretion of alpha-granule and lysosomal contents was examined by flow cytometric measurement of the surface expression of CD62P (P-selectin) and CD63, respectively. Secretion of the lysosomal enzyme, beta-N-acetylglucosaminidase was also quantified. Results were expressed as % of maximum response induced by 1 U/ml thrombin. Ethanol inhibited the thrombin-induced secretion of both dense and alpha-granule contents (P <0.001, 2-way ANOVA), and of lysosomal contents (P <0.005 for CD63 expression and P <0.001 for beta-N-acetylglucosaminidase secretion). When platelets were pretreated with aspirin, thrombin-induced secretion of storage granule and lysosomal contents was slightly inhibited, but secretion was inhibited by ethanol to the same extent as the untreated platelets, indicating that this inhibition was independent of thromboxane A2. Surface expression of CD63 occurred at lower thrombin concentrations than those required for secretion of beta-N-acetylglucosaminidase, possibly due to the presence of some CD63 on granule membranes. Although the role of lysosomal contents in thrombus formation is not established, some constituents of storage granules are known to augment thrombus formation; ethanol's inhibition of their secretion by stimulated platelets may contribute to its beneficial effect on thromboembolism.
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PMID:Ethanol inhibits thrombin-induced secretion of the contents of human platelet dense and alpha-granules and lysosomes. 979 88

The relationship between platelet aging and early markers of membrane activation have not been defined clearly. Activation markers expressed during prolonged storage are similar if not identical to those that appear after exposure to thrombin. Using flow cytometry, we investigated platelet membrane expression of CD62P, CD63, and annexin V binding (i.e., loss of membrane asymmetry) in platelets stored for up to 11 days under standard blood banking conditions. We compared five apheresis platelets to two random donor platelet concentrates, and to one pooled platelet preparation from six single platelet concentrates before and after exposure to thrombin. CD62P, CD63 expression, and annexin V binding increased during storage albeit with different kinetics. The differential increments observed between resting and thrombin (1 unit/ml) activated platelets showed an inverse correlation to storage time for CD62P, CD63, and annexin V binding, which was identical to published survival curves. A difference between apheresis platelets and platelet concentrates was observed only on day 1. Our data indicate that the in vitro platelet reserve activity to thrombin activation mirrors that of radiolabeled platelet survival in vivo and that platelet cross-sectional residual life span can explain their diminished capacity to respond to thrombin as a function of viability.
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PMID:Platelet membrane early activation markers during prolonged storage. 1006 70

We compared several responses in thrombin-stimulated and collagen (type I)-stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase, Arg-Gly-Asp-Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal-transducing responses, exposure of CD62 (P-selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and beta-N-acetyl-glycosaminidase were studied as final activation responses. Clear differences between thrombin-stimulated and collagen-stimulated platelets were observed. First, practically all protein-tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen-induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen-stimulated platelets seems to be due to removal of the G-protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP.
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PMID:Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets. 1009 87

Spontaneous EDTA-independent cold platelet agglutination is a rare phenomenon that produces pseudothrombocytopenia when blood samples are analyzed in automated cell counters. We report a case of platelet cold agglutinins and an analysis by flow cytometry. A 49 year old woman presented with abnormal vaginal bleed secondary to uterine fibroids. Platelet clumping was observed in blood samples taken in EDTA-, heparin- and citrate-containing tubes. In flow cytometric tests, patient serum agglutinated 16% of normal platelets at 22 degrees C, and 7% of platelets after incubation at 37 degrees C; in contrast, 3% and < 1% of platelets were agglutinated at 22 and 37 degrees C, respectively, after incubation with normal serum. Minimal agglutination (< 10%) was observed with patient serum at a titre of 1:5 or at temperatures > 30 degrees C. After incubation at 4 degrees C, IgM antibody and C3 were increased on the patient's platelets; no significant amount of IgM or C3 was detected on normal platelets. The specificity of the platelet cold agglutinin was determined by competitive inhibition by monoclonal anti-CD41(GPIIbIIIa). Before the addition of monoclonal antibody, patient's serum agglutinated 16% of normal platelets at 22 degrees C; after addition of anti-CD41 only 2% of the platelets were agglutinated. This blocking effect was not observed with anti-CD42. The patient's platelets functioned normally as determined by CD62 and CD63 expression in response to thrombin, normal platelet aggregation in response to collagen, ADP, and ristocetin, and a normal template bleeding time. In summary, platelet agglutination by a platelet cold agglutinin was quantitated by flow cytometry, the responsible antibody was characterized as a low titre IgM with minimal activity > 30 degrees C, and competitive binding studies supported the GPIIbIIIa complex as the binding site for the antibody. Since the antibody did not affect platelet function, we believe that these patients will not suffer complications from their platelet cold agglutinin, but it could pose a problem under circumstances such as cardiac surgery with hypothermia.
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PMID:Platelet cold agglutinins: a flow cytometric analysis. 1035 Nov 32

Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.
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PMID:Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant. 1035 85

Platelet activation occurs during the collection, processing and storage of platelet concentrates. The effect of the platelet activation on the functional state of stored platelets remains however undefined. We employed flow cytometric analysis to evaluate the extent of platelet activation and the physiological response to thrombin stimulation of platelets stored for up to five days under routine blood bank conditions. Platelet surface expression of the activation markers CD62 and CD63 was examined, along with modulation of platelet membrane glycoproteins (GP) Ib and IIbIIIa. Platelet dense granule content was determined using a mepacrine uptake assay and the extent of platelet microparticle generation was quantified. Thirteen random-donor platelet concentrates prepared under routine conditions by a platelet-rich-plasma protocol were examined. Platelets were found to be activated following preparation on day 1. Although a gradual increase was seen with increasing storage time, this was not statistically significant for CD62 or CD63 expression, GPIIbIIIa or GPIb modulation or dense granule release; the generation of platelet microparticles did, however, increase with increasing storage time. The characteristic increase in surface expression of CD62, CD63 and GPIIbIIIa and decrease in GPIb and dense granule content in response to thrombin stimulation was observed with all concentrates, but these measures of platelet functional reserve showed decreasing platelet function with increasing storage time. The results indicate that platelets are activated by day 1, likely as a consequence of manipulation during collection and processing, but are not further progressively activated with increasing storage time; they do, however, become relatively hypofunctional with increasing storage.
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PMID:Flow cytometric analysis of platelet function in stored platelet concentrates. 1053 16

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.
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PMID:Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. 1057 93

We have investigated hemostatic parameters including platelet activation in 56 pediatric patients with or without cyanosis undergoing cardiopulmonary bypass (CPB) and cardiac surgery to repair congenital defects. Patients were participants in a study assessing the effects of tranexamic acid on surgery-related blood loss. Parameters monitored included blood loss, prothrombin F1.2, thrombin-antithrombin complexes, t-PA, PAI-1, plasminogen, fibrin D-dimer, and plasma factor XIII. Additionally, flow cytometry monitored platelet degranulation (P-selectin or CD63), as well as surface-bound fibrinogen, von Willebrand factor and factor XIIIa. Cyanotic patients had evidence of supranormal coagulation activation as both fibrin D-dimer and PAI-1 levels were elevated prior to surgery. While the extent of expression of P-selectin or CD63 was not informative, platelet-associated factor XIIIa was elevated in cyanotic patients at baseline. In both patient groups, CPB altered platelet activation state and coagulation status irrespective of the use of tranexamic acid.
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PMID:Hemostatic parameters and platelet activation marker expression in cyanotic and acyanotic pediatric patients undergoing cardiac surgery in the presence of tranexamic acid. 1066 55


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