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)

CD31/platelet endothelial cell adhesion molecule 1 (PECAM-1) is expressed on platelets, endothelial cells, myeloid cells, monocytes, and certain lymphocyte subsets. It has been shown that CD31 is involved in the homophilic and heterophilic cellular adhesion and leukocyte transendothelial migration, but little is known about the role of CD31 in platelet functions. Previously we have shown that monoclonal antibody (MAb) AAP2 produced in our laboratory bound to a 110-kD platelet antigen and gave an enhanced binding to activated platelet membrane. In this study we demonstrated that platelet lysate depleted of the antigen through adsorption by an AAP2-solidified affinity column was bound by MAbs against CD62 and CD42 but not by MAb 5.6E against CD31 or AAP2 on the immunoblot. Rabbit antibodies against CD31 completely inhibited the binding of AAP2 to platelets in the flow cytometry analysis. This indicates that AAP2 is specifically against CD31. 125I-labeled AAP2 bound to resting platelets with 5587 +/- 1765 sites/platelet and a Kd of 1 nmol/L and to thrombin-activated platelets with 17,625 +/- 4865 sites/platelet and a Kd of 0.24 nmol/L. Addition of 10 micrograms/mL AAP2 inhibited the aggregation induced by 4 mumol/L ADP by 78.6%, 6 mumol/L epinephrine by 79.4%, 1 microgram/mL collagen by 78.7%, and 0.25 U/mL thrombin by 29%. The platelet aggregation was completely restored when higher concentration of agonists were used. MAb 5.6E did not have any effect on platelet aggregation. These results suggest that AAP2 binds to a special epitope of CD31 on platelets and that CD31 is involved in platelet aggregation.
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PMID:Binding properties and inhibition of platelet aggregation by a monoclonal antibody to CD31 (PECAM-1). 940 5

Evidence is increasing that platelets can initiate and propagate inflammatory processes by interacting with leucocytes and the vascular endothelium. Platelets have been shown to bind to neutrophils, existing as platelet/neutrophil complexes (PNC) within the circulation. We describe a simple flow cytometric method for assessing and investigating platelet interactions with neutrophils in small volumes of whole blood. Twenty-five percent (sd 6%) of circulating neutrophils from healthy adults were associated with platelets. Formation of these platelet-neutrophil complexes was CD62P (P-selectin) and divalent cation dependent. Platelet activation (with ADP or thrombin) caused a rapid and sustained rise in %PNC which differed from the pattern of free platelet activation as assessed by CD62P expression. F-met-leu-phe induced neutrophil activation but did not increase the percentage PNC. Platelet activation also caused increased neutrophil CD11b/CD18 expression which was most marked on neutrophils complexed with platelets. This straightforward technique is simple, reproducible, and allows assessment of platelet-neutrophil interactions and activation of neutrophils. It may also provide a method for estimating platelet activation in whole blood.
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PMID:Investigation of platelet-neutrophil interactions in whole blood by flow cytometry. 946 29

The platelet-sized particle formation in the human megakaryoblastic leukaemia cell line MEG-01 and its subline MEG-01s was examined. MEG-01 and MEG-01s cells spontaneously released platelet-sized particles into the culture medium, in which the cells occasionally extended cytoplasmic processes similar to those of megakaryocyte proplatelets. Scanning electron microscopic images showed cytoplasmic processes elongated from blebs on the MEG-01 and MEG-01s cell surface and were constricted between segments of platelet size. Immunofluorescence staining with anti-tubulin antibody showed that the cytoplasmic processes contained microtubules that were organized into a ring, which is a characteristic of circulating platelets. Some platelet-sized particles, probably released by ruptures at the sites of the process constriction, were metabolically active in an MTT assay (about 50%). Some particles also expressed the platelet-specific glycoproteins GPIIb, IIIa and GMP-140. Rarely, in response to thrombin, particles underwent a shape change from spherical to a shape with irregular membrane protrusions and fine filopodia, and aggregating with one another. The particles also had increased GMP-140 (P-selectin) expression with the addition of thrombin. These results show the usefulness of the MEG-01 and MEG-01s cell lines for the study of thrombopoiesis.
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PMID:Platelet-like particle formation in the human megakaryoblastic leukaemia cell lines, MEG-01 and MEG-01s. 948 40

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

Although thrombopoietin has been shown to promote megakaryocyte (MK) proliferation and maturation, the exact mechanism and site of platelet formation are not well defined. Studies have shown that MKs may transmigrate through bone marrow endothelial cells (BMEC), and release platelets within the sinusoidal space or lung capillaries. In search for chemotactic factor(s) that may mediate transmigration of MKs, we have discovered that mature polyploid MKs express the G protein-coupled chemokine receptor CXCR4 (Fusin, LESTR). Therefore, we explored the possibility that stromal cell-derived factor 1 (SDF-1), the ligand for CXCR4, may also induce transendothelial migration of mature MKs. SDF-1, but not other CXC or CC chemokines, was able to mediate MK migration (ED50 = 125 pmol/liter). The MK chemotaxis induced by SDF-1 was inhibited by the CXCR4-specific mAb (12G5) and by pertussis toxin, demonstrating that signaling via the G protein-coupled receptor CXCR4 was necessary for migration. SDF-1 also induced MKs to migrate through confluent monolayers of BMEC by increasing the affinity of MKs for BMEC. Activation of BMEC with interleukin 1beta resulted in a threefold increase in the migration of MKs in response to SDF-1. Neutralizing mAb to the endothelial-specific adhesion molecule E-selectin blocked the migration of MKs by 50%, suggesting that cellular interaction of MKs with BMEC is critical for the migration of MKs. Light microscopy and ploidy determination of transmigrated MKs demonstrated predominance of polyploid MKs. Virtually all platelets generated in the lower chamber also expressed CXCR4. Platelets formed in the lower chamber were functional and expressed P-selectin (CD62P) in response to thrombin stimulation. Electron microscopy of the cells that transmigrated through the BMEC monolayers in response to SDF-1 demonstrated the presence of intact polyploid MKs as well as MKs in the process of platelet formation. These results suggest that SDF-1 is a potent chemotactic factor for mature MKs. Expression of CXCR4 may be the critical cellular signal for transmigration of MKs and platelet formation.
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PMID:Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation. 968 31

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.
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PMID:The F2-isoprostane 8-epiprostaglandin F2alpha increases platelet adhesion and reduces the antiadhesive and antiaggregatory effects of NO. 971 31

A reconstituted high density lipoprotein (rHDL) prepared for clinical use was tested for its influence on platelet activity modulated by various stimuli. In a first series of in vitro experiments, rHDL was added to blood in a concentration series, and platelet rich plasma (PRP) was isolated. Platelets were stimulated with arachidonic acid, collagen, epinephrine or ADP, and platelet aggregation was assessed. rHDL mediated a dose dependent inhibition of the platelet activity. With purified platelets rHDL inhibited the release reaction induced by collagen, but not by thrombin, as measured by CD62P (P-Selectin) expression on the plasma membrane. Ex vivo experiments were performed with PRP from volunteers, previously infused with 25 mg rHDL/kg body weight and 40 mg rHDL/kg body weight, respectively. Platelet activity in PRP was assessed before, and up to 30 h after the end of the rHDL infusion. A transient inhibition of the platelet aggregation induced by arachidonic acid and collagen was observed which was more pronounced in the group receiving 40 mg rHDL/kg body weight. In both groups of experiments, in vitro and ex vivo, the inhibition of the platelet activity was also dependent on the stimulus used.
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PMID:Reconstituted high density lipoprotein (rHDL) modulates platelet activity in vitro and ex vivo. 971 59

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

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.
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PMID:Kinetics of platelet P-selectin mobilization: concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO donor SNAP. 986 63


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