Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRgamma. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76(-/-)) or heterozygous (SLP-76(+/-)) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76(+/-) platelets, but not SLP-76(-/-) platelets (P <.01), and failed to stimulate annexin V binding to either SLP-76(+/-) or SLP-76(-/-) platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76(+/-) or SLP-76(-/-) platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76(+/-) and SLP-76(-/-) platelets (P <.001 versus unstimulated platelets). Similar results were obtained with platelets from FcRgamma null mice, for which collagen, but not convulxin, induced procoagulant activity (P <.01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76(+/-) platelets (P <.05), but not in SLP-76(-/-) platelets. SLP-76(-/-) platelets also exhibited less annexin V binding than SLP-76(+/-) platelets after costimulation with thrombin and convulxin (P <.05). These findings demonstrate that an intact GPVI/FcRgamma/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.
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PMID:Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen. 1235 93

In the present study we assessed the effect of platelet counts and rFVIIa on thrombin generation, platelet activation and clot formation after tissue factor pathway activation in human plasma aiming to investigate the mechanism by which rFVIIa induces haemostasis in patients with severe thrombocytopenia. Plasma samples with platelet counts from 5 x 10(9)/l to 150 x 10(9)/l were spiked with rFVIIa (1 micro g/ml) or buffer. Clotting was initiated in the presence of diluted thromboplastin. Thrombin generation was assessed using the Thrombogram-Thrombinoscope trade mark assay. The kinetics of platelet activation was assessed using flow cytometry to measure the expression the P-selectin on platelet membrane of washed platelets suspended in defibrinated homologous PPP. Thromboelastography was used to evaluate the effect of platelets and rFVIIa on the kinetics of clot formation and clot's firmness. In the presence of low platelet counts the endogenous thrombin potential (ETP) and the maximum concentration of generated thrombin (Cmax) were reduced by 60%-70%. The lag-time of thrombin generation and the time required to reach the Cmax (Tmax) were prolonged, the velocity of platelet activation was decreased and thrombus formation was delayed. Recombinant FVIIa accelerated thrombin generation and platelet activation but it did not significantly modify ETP or Cmax. Recombinant FVIIa enhanced platelet activation in a TF and thrombin dependent manner since its effect on the studied parameters was abolished when TF was omitted or when hirudin was added into the experimental system respectively. Recombinant FVIIa normalized the velocity of clot formation but it did not modify clot firmness, which depended mainly on platelets' count. In conclusion, in experimental conditions simulating severe thrombocytopenia rFVIIa in the presence of low amounts of TF, accelerates thrombin generation, without increasing the maximum amount of generated thrombin, thus leading in enhanced platelet activation and rapid clot formation.
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PMID:The role of platelets and recombinant factor VIIa on thrombin generation, platelet activation and clot formation. 1511 59

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.
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PMID:Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry. 1547 Dec 23

Haemorrhage is often responsible for the lethal course of acute myeloid leukaemia (AML). Previously, multiple platelet function defects were identified by flow cytometric analysis of platelet activation markers in AML. The role of flow cytometric analysis of platelet function in characterization of prognostic markers of haemorrhage in AML patients has not been well elucidated. The objective of this prospective study was to analyse platelet function in 50 AML patients at diagnosis and to compare results with clinical bleeding score, graded by common toxicity criteria. Platelet activation markers CD62P, CD42b, CD63 and PAC-1 were analysed following in vitro activation by thrombin receptor activating peptide. The following plasma haemostasis parameters were measured: soluble P-selectin, activated partial thromboplastin time, thrombin time, prothrombin time, D-dimer, fibrinogen, and von Willebrand factor antigen. In a multivariate analysis, P-selectin (CD62P) <36 molecules of equivalent soluble fluorochrome x 10(3) (P < 0.0015) and platelet count <40 x 10(9)/l (P = 0.01) were significant predictors of haemorrhage at diagnosis. Haemorrhage at diagnosis predicted grade 3-4 haemorrhage in the first 28 d following diagnosis (P = 0.018). The presented results indicate that low P-selectin is a prognostic marker of haemorrhage in AML.
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PMID:Prediction of haemorrhage in the early stage of acute myeloid leukaemia by flow cytometric analysis of platelet function. 1568 63

Ischemia-reperfusion (I/R) injury is associated with activation of coagulation and inflammation. Interestingly, various anticoagulants have been shown to reduce both coagulation and inflammation in animal models of kidney I/R injury. Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in the coagulation cascade. The aim of this study was to investigate the effect of fondaparinux in a lethal murine model of kidney I/R injury. A murine model of kidney I/R was established. In this model, we measured activation of the coagulation cascade and induction of inflammation. Administration of fondaparinux to I/R-injured mice reduced fibrin deposition in the kidney, reduced serum creatinine levels and increased survival from 0 to 44% compared with saline-treated control mice. Fondaparinux also reduced interleukin-6 and macrophage inflammatory protein-2 expression and decreased neutrophil accumulation in the injured kidneys. Finally, we showed that fondaparinux reduced thioglycollate-induced recruitment of neutrophils into the peritoneum and inhibited the binding of U937 cells to P-selectin in vitro. Our data suggest that fondaparinux reduces kidney I/R injury primarily by inhibiting the recruitment of neutrophils.
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PMID:The synthetic pentasaccharide fondaparinux reduces coagulation, inflammation and neutrophil accumulation in kidney ischemia-reperfusion injury. 1574 44

Selectins are carbohydrate-binding cell adhesion molecules that play a major role in the initiation of inflammatory responses. Heparin can bind to P-selectin, and its anti-inflammatory property is mainly due to inhibition of P-selectin. However, the strong anticoagulant activity of heparin limits its clinical use. We prepared periodate-oxidized, borohydride-reduced heparin (RO-heparin) by chemical modification and tested its anticoagulant and anti-inflammatory activities. Activated partial thromboplastin time (aPTT) assays showed that, compared with heparin, RO-heparin had greatly reduced anticoagulant activity. Intravenous administration of this compound led to reduction in the peritoneal infiltration of neutrophils in a mouse acute inflammation model. In vitro cell adhesion experiments demonstrated that the effect of RO-heparin on inflammatory responses was mainly due to inhibiting the interaction of P-selectin with its ligands. These results indicate that RO-heparin may be a safer treatment for inflammation than heparin, especially when selectin is targeted.
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PMID:P-Selectin-mediated acute inflammation can be blocked by chemically modified heparin, RO-heparin. 1599 51

Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.
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PMID:Modulatory effects of Escherichia coli capsular-derived sulfaminoheparosans and heparins on tissue factor-mediated activation of platelets: flow cytometric analysis. 1695 84

Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion. This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown. To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anticoagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin. In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model. These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NAC-pentasaccharide is protective in kidney I/R injury.
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PMID:A non-anticoagulant synthetic pentasaccharide reduces inflammation in a murine model of kidney ischemia-reperfusion injury. 1713 76

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.
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PMID:Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis. 1719 Aug 26

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.
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PMID:A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. 1729 77


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