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Thrombin
Kinds of Thrombin Terms modified by Thrombin Selected AbstractsNeuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca2+ mobilization, and apoptosis ,DEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001Irina V. Smirnova Abstract Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca2+]i flux, receptor cleavage, and elevation of rest [Ca2+]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87,100, 2001 [source] Synthesis and Characterization of Thrombin Conjugated ,-Fe2O3 Magnetic Nanoparticles for HemostasisADVANCED ENGINEERING MATERIALS, Issue 12 2009Ofra Ziv Abstract Thrombin is the final protease produced in the clotting pathways. Thrombin has been used in the clinic more than six decades for topical hemostasis and wound management. In human plasma the half-life of thrombin is shorter than 15 seconds due to close control by inhibitors. In order to stabilize thrombin, this enzyme was conjugated covalently and physically to ,-Fe2O3 magnetic nanoparticles. The physical conjugation was accomplished through adsorption of thrombin to BSA coating on the nanoparticles. The coagulant activity of the covalently bound thrombin was significantly lower than that of the physically adsorbed thrombin. Leakage of the physically bound thrombin into PBS containing 4% HSA was negligible. The physical conjugation of thrombin onto the nanoparticles stabilized the thrombin against its major inhibitor antithrombin III and improved its storage stability. At optimal CaCl2 concentration, the clotting time by the bound thrombin is shorter than that of the free enzyme. This novel conjugated thrombin may be an efficient candidate for topical hemostasis and wound healing. [source] Aptamer-Conjugated Nanoparticles Efficiently Control the Activity of ThrombinADVANCED FUNCTIONAL MATERIALS, Issue 18 2010Yen-Chun Shiang Abstract Thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) for highly effective control of thrombin activity towards fibrinogen are demonstrated. While a 29-base long oligonucleotide (TBA29) has known no enzymatic inhibitory functions for thrombin-mediated coagulation, the ultrahigh anticoagulant potency of TBA29 -Au NPs can be demonstrated via the steric blocking effect, at two orders of magnitude higher than that of free TBA29. The surface aptamer density on the Au NPs is important in determining their enzymatic inhibition of thrombin and their stability in the presence of nuclease. The practicality of 100TBA29 -Au NPs (100 TBA29 molecules per Au NP) for controlling thrombin-mediated coagulation in plasma is found, and the 100TBA29 -Au NPs has an ultra binding affinity towards thrombin (Kd = 2.7 × 10,11M) due to their high ligand density. The anticoagulant activity of TBA29 -Au NPs is found to be suppressed by TBA29 complementary sequence (cTBA29) modified Au NPs (cTBA29 -Au NPs), with a suppression rate 4.6-fold higher than that of cTBA29. The easily prepared and low-cost TBA29 -Au NPs and cTBA29 -Au NPs show their potential in biomedical applications for treating various diseases related to blood clotting disorders. In principle, this study opens the possibility of regulation of molecule binding, protein recognizing, and enzyme activity by using aptamer-functionalized nanomaterials. [source] Thrombin potently enhances swelling-sensitive glutamate efflux from cultured astrocytesGLIA, Issue 9 2007Gerardo Ramos-Mandujano Abstract High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of 3H -glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5,5 U/mL) elicited small 3H -glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of 3H -glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca2+ (Ca2+i) concentration ([Ca2+]i). Preventing Ca2+i rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%,22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca2+ -sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC50 15.8 ,M), DCPIB (IC50 4.2 ,M). These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma. © 2007 Wiley-Liss, Inc. [source] Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: Involvement of phospholipase A2, cyclooxygenase, and lipoxygenaseGLIA, Issue 2 2004Kyung-ae Ji Abstract Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000. J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokine-induced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI-P97 and WHI-P154) had little effect on thrombin-induced CIS expression. In contrast, cytosolic phospholipase A2 (cPLA2), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA2, cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N-acetyl-cysteine [NAC] and trolox) reduced thrombin-induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE2 and LTB4 generated by COX and LO, mediate CIS expression. Since interferon-, (IFN-,)-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS-transfected cells compared to control vector-transfected cells, CIS could have anti-inflammatory activity. These data suggest that thrombin-stimulation of ROS and prostaglandin and leukotriene production via the cPLA2, COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses. © 2004 Wiley-Liss, Inc. [source] Thrombin induces cyclooxygenase-2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2- and p38 MAPK-dependent pathway in murine macrophagesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2009Huey-Ming Lo Abstract Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti-thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in macrophages. Thrombin-induced COX-2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)-binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX-2 expression by thrombin was functionally linked to release of PGE2 and PGI2 but not thromboxane A2 into macrophage culture medium. Thrombin-induced COX-2 expression and PGE2 production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase-activated receptor 1 (PAR1)-activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist-SCH79797 could attenuate thrombin-induced COX-2 expression and PGE2 release. Taken together, we provided evidence demonstrating that thrombin can induce COX-2 mRNA and protein expression and PGE2 production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK-dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions. J. Cell. Biochem. 108: 1143,1152, 2009. © 2009 Wiley-Liss, Inc. [source] Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neuronsJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Haripriya Vittal Rao Abstract Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated. [source] Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice cultureJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Hiroshi Katsuki Abstract Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent. [source] The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective?JOURNAL OF NEUROCHEMISTRY, Issue 1 2003Guohua Xi Abstract In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood,brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved. [source] Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Rosaria Meli Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1,10 U/mL) and PAR1-AP (1,100 µm) induced a significant concentration-dependent increase both of IFN-,- (250 U/mL) or TNF-,- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS,PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulogTM (60 µg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response. [source] Preconditioning with thrombin can be protective or worsen damage after endothelin-1-induced focal ischemia in ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2006Petra Henrich-Noack Abstract The serine protease thrombin has shown direct neuroprotective and neurotoxic effects on brain tissue in cerebral ischemia. Previous data suggested that thrombin-induced protection in vivo can be achieved by preconditioning rather than by acute treatment. In the current work, we used a model of mild ischemia to investigate the effects of preischemic intracerebral thrombin injection on neural damage. By intracerebral injection of endothelin-1 in freely moving animals, we achieved middle cerebral artery occlusion (MCAO), and 7 days postischemia we performed histological quantification of the infarct areas. Thrombin was injected as a preconditioning stimulus intracerebrally 7 days or 2 and 3 days before ischemia. For acute treatment, thrombin was injected 20 min before MCAO. Thrombin induced significant neuroprotection when given 7 days before endothelin-1-induced MCAO but was deleterious when given 2 and 3 days before the insult. The deleterious effect was not seen when thrombin was given acutely before ischemia. Our data demonstrate that preconditioning with thrombin can protect against damage or worsen ischemic damage. Its effect depended on the time interval between thrombin injection and insult. A low dose of thrombin did not induce a major deleterious effect in the acute phase of the infarct development after mild transient ischemia. © 2006 Wiley-Liss, Inc. [source] The molecular basis of factor V and VIII procofactor activationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2009R. M. CAMIRE Summary., Activation of precursor proteins by specific and limited proteolysis is a hallmark of the hemostatic process. The homologous coagulation factors (F)V and FVIII circulate in an inactive, quiescent state in blood. In this so-called procofactor state, these proteins have little, if any procoagulant activity and do not participate to any significant degree in their respective macromolecular enzymatic complexes. Thrombin is considered a key physiological activator, cleaving select peptide bonds in FV and FVIII which ultimately leads to appropriate structural changes that impart cofactor function. As the active cofactors (FVa and FVIIIa) have an enormous impact on thrombin and FXa generation, maintaining FV and FVIII as inactive procofactors undoubtedly plays an important regulatory role that has likely evolved to maintain normal hemostasis. Over the past three decades there has been widespread interest in studying the proteolytic events that lead to the activation of these proteins. While a great deal has been learned, mechanistic explanations as to how bond cleavage facilitates conversion to the active cofactor species remain incompletely understood. However, recent advances have been made detailing how thrombin recognizes FV and FVIII and also how the FV B-domain plays a dominant role in maintaining the procofactor state. Here we review our current understanding of the molecular process of procofactor activation with a particular emphasis on FV. [source] Vascular and dendritic cell coagulation signaling in sepsis progressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009W. RUF Summary., The intrinsic signaling networks of the coagulation pathways have recently emerged as crucial determinants for survival in sepsis and systemic inflammatory response syndromes. Protease activated receptor (PAR) 1 is central to both lethality promoting and vascular protective signaling. In the vascular anticoagulant pathway, EPCR/aPC-PAR1 signaling prevents vascular leakage and genetic or acute deficiencies in this pathway promote lethality. In addition, coagulation signaling acts directly on cells of the innate immune system. Dendritic cell (DC) thrombin-PAR1 signaling is coupled to the migration promoting sphingosine 1 phosphate receptor 3 (S1P3). Thrombin generated in the lymphatic compartment perturbs DCs to promote systemic inflammation and disseminated intravascular coagulation in severe sepsis. Signaling-selective aPC variants and selective modulators of the S1P receptor system attenuate sepsis lethality, suggesting novel therapeutic approaches that can be employed to rebalance alterations in the coagulation signaling pathways in severe inflammatory disorders. [source] Critical roles for thrombin in acute and chronic inflammationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2009D. CHEN Summary., Thrombin can amplify inflammation induced by other stimuli, either through ischemia (consequent upon thrombosis), indirectly through generation of downstream mediators such as activated protein C, or directly via signals through protease activated receptors (PAR). This paper will summarize recent data from our laboratory indicating that thrombin is required to initiate CCR2-dependent leukocyte recruitment and that it is the principal determinant of the outcome after vascular injury, via PAR-1 activation of a distinct subset of smooth muscle cell progenitors. In both, tissue factor (TF) initiates thrombin generation and the thrombin acts locally, exemplifying that the initiation phase can generate autocrine or paracrine signalling molecules. Thrombin is an important constituent of innate immunity, able to amplify and modify responses to invading pathogens or tissue damage. With novel anti-thrombin therapeutics and agents to target PAR, a new understanding of the importance of thrombin may allow the development of innovative anti-inflammatory strategies. [source] Protease-activated receptor-induced Akt activation , regulation and possible functionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2007J. C. RESÉNDIZ Summary.,Background:,Thrombin induces the activation of the platelet serine/threonine kinase Akt. Akt activation is dependent on its phosphorylation at Thr308 and Ser473. The mechanism by which thrombin induces Akt phosphorylation is controversial, as is the role of Akt in platelet function. Objectives:,To investigate how protease-activated receptors (PARs) stimulate Akt and the role that Akt plays in human platelet function. Methods:,Platelets were stimulated through PAR1 or PAR4. Specific inhibitors were used to evaluate, by Western blotting, signaling pathways regulating Akt phosphorylation, and the role of activated Akt was evaluated by aggregometry and flow cytometry. Results:,Phospholipase C (PLC) controls Akt phosphorylation elicited by PARs. Stimulation of PAR1 or PAR4 resulted in rapid Akt phosphorylation, independently of secreted ADP and phosphatidylinositol-3-kinase (PI3K) activation. Akt phosphorylation ,60 s after PAR1 stimulation became entirely dependent on the purinergic receptor P2Y12 and the activation of PI3K. In contrast, PAR4 partially sustained Akt phosphorylation independently of P2Y12 and PI3K for up to 300 s. Pharmacologic inhibition of Akt reduced P-selectin expression and fibrinogen binding in platelets stimulated through PAR1, and delayed platelet aggregation in response to submaximal PAR1 or PAR4 stimulation, although aggregation at 300 s was unaffected. Conclusions:,Platelet PAR stimulation causes rapid Akt phosphorylation downstream of PLC, whereas with continuous stimulation, ADP and PI3K are required for maintaining Akt phosphorylation. Activated Akt regulates platelet function by modulating secretion and ,IIb,3 activation. [source] Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphataseJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2006B. PATEL Summary.,Background:,Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. Objectives:,We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. Methods:,Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. Results:,We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. Conclusions:,Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1. [source] Thrombin induces GPIb-IX-mediated fibrin binding to ,IIb,3 in a reconstituted Chinese hamster ovary cell modelJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2006D. PABÓN Summary.,Background: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. Objectives: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. Methods: We used Chinese hamster ovary (CHO) cells stably expressing human ,IIb,3 and/or GPIb-IX complexes (CHO- ,IIb,3 -IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. Results: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO- ,IIb,3 -IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against ,IIb,3 and GPIb,. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3, adaptor and calcium,calmodulin-dependent events are involved. Conclusions: The present data support a significant role of GPIb-IX and ,IIb,3 receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin. [source] Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damageJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2006P. RAIVIO Summary.,Background: Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia,reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown. Objectives: We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage. Methods: One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma prothrombin fragment F1+2 and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day. Results: Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F1+2 during early reperfusion associated with postoperative pulmonary vascular resistance index. F1+2 at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.001) and TnT (r = 0.44, P < 0.001) at 18 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F1+2 during reperfusion than others (P < 0.002). Logistic regression models identified F1+2 during reperfusion to independently associate with postoperative myocardial damage (odds ratios 2.5,4.4, 95% confidence intervals 1.04,15.7). Conclusions: Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage. [source] Critical role of ADP interaction with P2Y12 receptor in the maintenance of ,IIb,3 activation: association with Rap1B activationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2006T. KAMAE Summary.,Objective:,Platelet integrin ,IIb,3 plays a crucial role in platelet aggregation, and the affinity of ,IIb,3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which ,IIb,3 maintains its high-affinity state. Methods and results:,Washed platelets adjusted to 50 × 103 ,L,1 were stimulated with 0.2 U mL,1 thrombin or 5 ,m U46619 under static conditions. After the completion of ,IIb,3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated ,IIb,3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 ,m AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained ,IIb,3 activation by ,92% and ,38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 ,L,1 also disrupted the sustained ,IIb,3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5,-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL,1 thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions:,Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of ,IIb,3 activation. [source] Thrombin and PAR-1 stimulate differentiation of bone marrow-derived endothelial progenitor cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2006S. T. TARZAMI Summary., Endothelial progenitor cells (EPCs) from the bone marrow play an important role in vascular response to injury and ischemia. The mediators involved in the mobilization, recruitment, proliferation and differentiation of EPCs are not fully understood. In this study, the role of coagulation factor thrombin and protease-activated receptor-1 (PAR-1) on bone marrow-derived cell proliferation and differentiation was investigated. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and plated on fibronectin-coated flasks. Cell characteristics, proliferation and the expression of endothelial cell markers were determined using immunohistochemistry, thymidine uptake and fluorescence activated-cell sorting (FACS), respectively. The results show that thrombin stimulated enrichment of bone marrow cells with endothelial morphology, exhibiting acetylated-low-density lipoprotein (LDL) uptake and isolectin staining. Thrombin or PAR-1-activating peptide produced a 2- to 3-fold increase in the total number of cells as well as an increase in vascular endothelial (VE)-cadherin-positive cells. Thrombin treatment of VE-cadherin-negative cells prepared after cell sorting resulted in the generation of 3- to 4-fold higher VE-cadherin-positive cells than the untreated cultures. Increase in VE-cadherin-positive cells was inhibited by hirudin and efegatran. These results provide first evidence for a novel activity of thrombin and PAR-1 on bone marrow progenitor cell proliferation and EPC differentiation, and suggest their potential role in vascular regeneration and recanalization of thrombus. [source] The role of thrombin in gliomasJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2005Y. HUA Summary.,Background:,In a previous study we found that intracerebral infusion of argatroban, a specific thrombin inhibitor, reduces brain edema and neurologic deficits in a C6 glioma model. Objectives:,To examine the role of thrombin in gliomas and whether systemic argatroban administration can reduce glioma mass and neurologic deficits and extend survival time in C6 and F98 gliomas. Methods:,The presence of thrombin in human glioblastoma samples and rat C6 glioma cells (in vitro and in vivo) was assessed using immunohistochemistry. The effect of thrombin on C6 cell proliferation in vitro was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The role of thrombin in vivo was assessed in rat C6 and F98 glioma cell models using argatroban, a thrombin inhibitor. The effects of argatroban on tumor mass, neurologic deficits and survival time were investigated. Results:,Thrombin immunoreactivity was found in cultured rat C6 glioma cells and human glioblastomas. Thrombin induced C6 cell proliferation in vitro. In C6 glioma, argatroban reduced glioma mass (P < 0.05) and neurologic deficits (P < 0.05) at day 9. In F98 glioma, argatroban prolonged survival time (P < 0.05). Conclusion:,These results suggest that thrombin plays an important role in glioma growth. Thrombin may be a new therapeutic target for gliomas. [source] Activation of the small GTPase Rap2B in agonist-stimulated human plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2004F. Greco Summary., The activation of the small GTPase Rap2B in resting and agonist-stimulated human platelets was investigated. Both thrombin, that stimulates heterotrimeric G-protein-coupled receptors, and the GPVI ligand convulxin, that activates a tyrosine-kinase based signaling pathway, were able to induced the rapid and sustained binding of GTP to Rap2B. Similarly, a number of other agonists tested, previously known to activate the highly related protein Rap1B, were also able to stimulate Rap2B. In contrast, platelet antagonists that increase the intracellular concentration of cAMP did not signal to Rap2B. Thrombin- and convulxin-induced activation of Rap2B was not dependent on thromboxane A2, did not require the interaction of the protein with the cytoskeleton, and was not regulated by integrin ,IIb,3 -dependent outside-in signaling. When secreted ADP was neutralized, activation of Rap2B induced by thrombin, but not by convulxin, was significantly reduced. ADP itself was found to induce the rapid and sustained binding of GTP to Rap2B, and this effect was predominantly mediated by stimulation of the Gi-coupled P2Y12 receptor. Activation of Rap2B promoted by both thrombin and convulxin was regulated by intracellular Ca2+, while protein kinase C was found to be involved in convulxin- but not in thrombin-induced activation of Rap2B. Moreover, Rap2B activation induced by thrombin, but not by convulxin, was totally dependent on phosphatidylinositol 3-kinase activity. These results demonstrate that the small GTPase Rap2B is involved in platelet activation, and outline some important differences between the regulation of highly related GTPases Rap2B and Rap1B in human platelets. [source] Factor VIIa-mediated tenase function on activated platelets under flowJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2004M. S. Goel Summary.,Background: Tissue factor (TF) and/or active factor (F)VIIa may be stored inside resting platelets. Objectives: The objective of this study was to examine if platelets, following activation of GPVI, could support tenase and prothrombinase activity without any exogenously added tissue factor. Methods: Thrombin (IIa) formation on gel-filtered platelets with added factors or the clotting of platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (CTI) (to inhibit factor XIIa) was studied in well plate assays with a fluorogenic thrombin substrate or in flow assays by fibrin visualization. Results: Pretreatment of convulxin (CVX)-stimulated, fibrinogen-adherent, gel-filtered platelets with anti-TF, anti-FVII/VIIa, or 1 nm PPACK [inhibitor of FVIIa, factor XIa and factor (F)IIa] delayed fibrin deposition on platelets perfused with PFP/CTI at 62.5 s,1. Anti-TF or anti-FVII/VIIa also attenuated thrombin generation in plate assays using recalcified PRP/CTI treated with CVX. Anti-TF or anti-FVII/VIIa (but not inhibited factor IXa) delayed the burst in thrombin production by gel-filtered platelets suspended in prothrombin and CVX by 14 min and 40 min, respectively. Anti-FVII/VIIa completely eliminated thrombin generation on fibrinogen-adherent, gel-filtered platelets pretreated with 10 µm PPACK and 10 µm EGR-CK [inhibitor of factor (F)Xa], rinsed, and then supplemented with CVX, prothrombin, and FX. Addition of anionic phospholipid to PFP/CTI or to a mixture of prothrombin, FX, and recVIIa was not sufficient to generate detectable tenase activity. Lastly, isolated, unactivated neutrophils suspended in FX, FII and recVIIa supported a very low level of thrombin generation sensitive to antagonism of P-selectin, CD18, and TF. Conclusions: Activated platelets supported tenase and prothrombinase activity by elevating the function or level of FVIIa and exposing active FVIIa or FVIIa-cofactor(s), distinct from anionic lipid, that may be, in part, TF. [source] Thrombin induces neoangiogenesis in the chick chorioallantoic membraneJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2003M. Caunt Summary., Most tumors have constitutively active tissue factor on their surface, capable of generating thrombin in the surrounding environment, and thrombosis is associated with cancer. Thrombin is known to induce a malignant phenotype by enhancing tissue adhesion and cell growth in vitro and in vivo in mice. Because tumors require angiogenesis for growth, we examined whether thrombin induces neoangiogenesis in a physiologically intact in vivo model. Thrombin (0.1 U mL,1) induced neoangiogenesis in the chick chorioallantoic membrane over a 24,72-h period by approximately 2,3-fold. This was inhibited by the potent thrombin inhibitor, hirudin and shown to have its mode of action by ligation of the thrombin protease-activated receptor, PAR-1. The thrombin receptor activation peptide, SFLLRNPNDKYEPF (200 µm) also enhanced neoangiogenesis c. 2,3-fold. Thrombin-induced neoangiogenesis was accompanied by the induction of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) mRNA at 24,48 h (approximately 2-fold) as determined by semi-quantitative reverse transcriptase-polymerase chain reaction. Thrombin-induced neoangiogenesis was inhibited to baseline level by the specific angiogenesis receptor inhibitors KDR-Fc (vs. VEGF) and Tie-2-Fc (vs. Ang-1 and Ang-2), as well as the non-specific angiogenesis inhibitor thrombospondin-1. Thrombin-induced neoangiogenesis was also inhibited to baseline level by agents known to inhibit thrombin receptor signaling in other cells: G-coupled protein receptor inhibitor, pertussis toxin (40 pg per egg), protein kinase C inhibitor, bisindolylmaleimide (1 µm per egg), MAP kinase inhibitor, PD980598 (10 µm per egg) and PI3 kinase inhibitor, LY294002 (0.25 µm per egg). Thus angiogenesis is stimulated by thrombosis, which could help explain the enhancement of experimental tumorigenesis by thrombin. [source] Progress in the design of low molecular weight thrombin inhibitorsMEDICINAL RESEARCH REVIEWS, Issue 1 2005Stuti Srivastava Abstract Intravascular thrombosis and its complication, embolism, is a leading cause of morbidity and mortality throughout the world. Past few decades have seen a great deal of progress in the development of antithrombotic agents, though the current treatment options are limited to heparin, LMW heparins, and warfarin. Detailed understanding of the biochemical and biophysical mechanisms of activation and regulation of blood coagulation have helped in developing specific inhibitors of enzymes, especially thrombin, within the coagulation cascade. Thrombin plays a central role in the coagulation cascade and so has become the primary target for the development of antithrombotic drugs. The review covers the main pharmacological aspects of haemostasis and thrombosis and provides an update on low molecular weight thrombin inhibitors along with the limitations of the prevalent antithrombotic agents. Recent developments in small molecule inhibitors of Protease Activated Receptor-1 (PAR-1) which can be helpful for the treatment of thrombotic and vascular proliferative disorders, have also been discussed. © 2004 Wiley Periodicals, Inc. [source] Roxithromycin inhibits transforming growth factor-, production by cultured human mesangial cellsNEPHROLOGY, Issue 6 2006HIDEAKI YAMABE SUMMARY: Background: Transforming growth factor-, (TGF-,) plays an important role in progression of renal injury. However, few materials which inhibit TGF-, have been known. Roxithromycin (ROX), macrolide antibiotics, is known to have anti-inflammatory, immunomodulatory and tissue reparative effects besides its bacteriostatic activity, although the exact mechanism of its anti-inflammatory and immunomodulatory effects was not defined. We examined the effect of ROX on production of TGF-, and type IV collagen by cultured human mesangial cells (HMC). Methods: Human mesangial cells were incubated with several concentrations of ROX and TGF-, and type IV collagen levels in the culture supernatants were measured by enzyme-linked immunoassay. Amount of TGF-, mRNA was also quantified by using a colourimetric mRNA quantification kit and semiquantitative reverse transcriptase polymerase chain reaction. We also examined the effect of ROX on tyrosine kinase, MAP kinase and NF-,B stimulated by thrombin. Results: Roxithromycin (0.1,10.0 µg/mL) inhibited TGF-, production by HMC in a dose- and time-dependent manner without inducing cell injury. ROX (10.0 µg/mL) also inhibited mRNA expression of TGF-, in HMC. Thrombin (5 U/mL) stimulated TGF-, production by HMC and ROX significantly inhibited the stimulating effect of thrombin on TGF-, production. ROX also inhibited the increment of type IV collagen production stimulated by thrombin. ROX (10.0 µg/mL) suppressed the thrombin-induced NF-,B activation, although ROX did not inhibit the activation of tyrosine kinase and MAP kinase by thrombin. Conclusion: Roxithromycin has an inhibitory effect on TGF-, production by HMC possibly via inhibition of NF-,B. ROX may be a potential agent for the treatment of glomerulosclerosis. [source] Functional protease-activated receptors in the dorsal motor nucleus of the vagusNEUROGASTROENTEROLOGY & MOTILITY, Issue 4 2010H. Wang Abstract Background, Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). Methods, DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca2 + ]i) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. Key Result, Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca2 + ]i expressed as ,F/F0 of 229 ± 14% and 137 ± 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum ,F/F0 change of 258 ± 12% and 242 ± 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 ,m) decreased the maximal change in ,F/F0 induced by PAR-1 activation from 140 ± 17% to 21 ± 3%, while the PAR-2-mediated maximal change in ,F/F0 decreased from 185 ± 21% to 19 ± 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in ,F/F0 due to PAR-1 and PAR-2 activation by 72 ± 13% and 71 ± 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. Conclusions & Inferences, Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3. [source] Reversible Photoswitching of a G-QuadruplexANGEWANDTE CHEMIE, Issue 36 2009Shinzi Ogasawara Dr. Halt finden: Die Bildung von G-Quadruplexen lässt sich über die reversible cis-trans -Photoisomerisierung der photochromen Nucleobase 8-Fluorenylvinyl-2,-desoxyguanosin regeln (siehe Bild: Fluoren,rot, Guanin,grün). Durch den Wechsel zwischen einem sehr stabilen Quadruplexzustand und einem unstrukturierten Zustand kann das Binden an Thrombin (rosa) gesteuert werden. [source] Blood,brain barrier breakdown and repair by Src after thrombin-induced injuryANNALS OF NEUROLOGY, Issue 4 2010Da-Zhi Liu PhD Objective Thrombin mediates the life-threatening cerebral edema that occurs after intracerebral hemorrhage. Therefore, we examined the mechanisms of thrombin-induced injury to the blood,brain barrier (BBB) and subsequent mechanisms of BBB repair. Methods Intracerebroventricular injection of thrombin (20U) was used to model intraventricular hemorrhage in adult rats. Results Thrombin reduced brain microvascular endothelial cell (BMVEC) and perivascular astrocyte immunoreactivity,indicating either cell injury or death,and functionally disrupted the BBB as measured by increased water content and extravasation of sodium fluorescein and Evans blue dyes 24 hours later. Administration of nonspecific Src family kinase inhibitor (PP2) immediately after thrombin injections blocked brain edema and BBB disruption. At 7 to 14 days after thrombin injections, newborn endothelial cells and astrocytes were observed around cerebral vessels at the time when BBB permeability and cerebral water content resolved. Delayed administration of PP2 on days 2 through 6 after thrombin injections prevented resolution of the edema and abnormal BBB permeability. Interpretation Thrombin, via its protease-activated receptors, is postulated to activate Src kinase phosphorylation of molecules that acutely injure the BBB and produce edema. Thus, acute administration of Src antagonists blocks edema. In contrast, Src blockade for 2 to 6 days after thrombin injections is postulated to prevent resolution of edema and abnormal BBB permeability in part because Src kinase proto-oncogene members stimulate proliferation of newborn BMVECs and perivascular astrocytes in the neurovascular niche that repair the damaged BBB. Thus, Src kinases not only mediate acute BBB injury but also mediate chronic BBB repair after thrombin-induced injury. ANN NEUROL 2010;67:526,533 [source] Injections of Blood, Thrombin, and Plasminogen More Severely Damage Neonatal Mouse Brain Than Mature Mouse BrainBRAIN PATHOLOGY, Issue 4 2005Mengzhou Xue MD The mechanism of brain cell injury associated with intracerebral hemorrhage may be in part related to proteolytic enzymes in blood, some of which are also functional in the developing brain. We hypothesized that there would be an age-dependent brain response following intracerebral injection of blood, thrombin, and plasminogen. Mice at 3 ages (neonatal, 10-day-old, and young adult) received autologous blood (15, 25, and 50 ,l respectively), thrombin (3, 5, and 10 units respectively), plasminogen (0.03, 0.05, and 0.1 units respectively) (the doses expected in same volume blood), or saline injection into lateral striatum. Forty-eight hours later they were perfusion fixed. Hematoxylin and eosin, lectin histochemistry, Fluoro-Jade, and TUNEL staining were used to quantify changes related to the hemorrhagic lesion. Damage volume, dying neurons, neutrophils, and microglial reaction were significantly greater following injections of blood, plasminogen, and thrombin compared to saline in all three ages of mice. Plasminogen and thrombin associated brain damage was greatest in neonatal mice and, in that group unlike the other 2, greater than the damage caused by whole blood. These results suggest that the neonatal brain is relatively more sensitive to proteolytic plasma enzymes than the mature brain. [source] | |||||||||||||||||||||||||||||||||||||