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Thrombin Activation (thrombin + activation)

Distribution by Scientific Domains

Medical Sciences	80%

Selected Abstracts

Thrombin-activatable carboxypeptidase B cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis

ARTHRITIS & RHEUMATISM, Issue 10 2009
Shadi A. Sharif
Objective Osteopontin (OPN) is a proinflammatory cytokine that plays an important role in the pathogenesis of rheumatoid arthritis (RA). OPN can be cleaved by thrombin, resulting in OPN-R and exposing the cryptic C-terminal ,4,1 and ,9,1 integrin,binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also called thrombin-activatable fibrinolysis inhibitor, removes the C-terminal arginine from OPN-R, generating OPN-L and abrogating its enhanced cell binding. We undertook this study to investigate the roles of OPN-R and OPN-L in synoviocyte adhesion, which contributes to the formation of invasive pannus, and in neutrophil survival, which affects inflammatory infiltrates in RA. Methods Using specifically developed enzyme-linked immunosorbent assays, we tested the synovial fluid of patients with RA, osteoarthritis (OA), and psoriatic arthritis (PsA) to determine OPN-R, OPN-L, and full-length OPN (OPN-FL) levels. Results Elevated levels of OPN-R and OPN-L were found in synovial fluid samples from RA patients, but not in samples from OA or PsA patients. Increased levels of OPN-R and OPN-L correlated with increased levels of multiple inflammatory cytokines, including tumor necrosis factor , and interleukin-6. Immunohistochemical analyses revealed robust expression of OPN-FL, but only minimal expression of OPN-R, in RA synovium, suggesting that cleaved OPN is released into synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an antiapoptotic effect on neutrophils. OPN-R augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to ,4,1, whereas OPN-L did not. Conclusion Thrombin activation of OPN (resulting in OPN-R) and its subsequent inactivation by thrombin-activatable CPB (generating OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA by regulating neutrophil viability and reducing synoviocyte adhesion. [source]

Thrombin activation of proteinase-activated receptor 1 potentiates the myofilament Ca2+ sensitivity and induces vasoconstriction in porcine pulmonary arteries

BRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2010
Jun Maki
Background and purpose:, Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery. Experimental approach:, The cytosolic Ca2+ concentrations ([Ca2+]i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery. Key results:, Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR1) antagonist, while it was mimicked by PAR1 -activating peptide (PAR1AP), but not PAR4AP. The thrombin-induced contraction was associated with a small elevation of [Ca2+]i and an increase in MLC20 phosphorylation. Thrombin and PAR1AP induced a greater increase in tension for a given [Ca2+]i elevation than that obtained with high K+ -depolarization. They also induced a contraction at a fixed Ca2+ concentration in ,-toxin-permeabilized preparations. Conclusions and implications:, The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR1 and thereby increasing the sensitivity of the myofilament to Ca2+. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH. [source]

von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2003
J. J. Briedé
Summary., Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet,fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s,1 requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca2+]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 ± 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 ± 14%) along with a transient elevation in [Ca2+]i from 0.05 µmol L,1 up to 1.1 ± 0.2 µmol L,1. Platelet adhesion to fibrin at a shear rate of 50 s,1 is mediated by thrombin but not by fibrin-bound VWF. The [Ca2+]i of these thrombin-activated platelets was elevated (0.2 ± 0.1 µmol L,1), but only a minority of the platelets (11 ± 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 ± 23%) exposed PS and had peak values of [Ca2+]i of about 0.6 µmol L,1. Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF,platelet interaction. [source]

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization

BRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004
Marie-Hélène Rodriguez
Summary Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue-specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B-domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte-produced FVIII. [source]

Synthesis of Novel Peptide Inhibitors of Thrombin-induced Platelet Activation

CHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2006
Fernanda M. Burke
Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome (ACS). Platelet activation by thrombin, a serine protease, occurs by binding to and cleavage of the extracellular N-terminal domains of protease-activated receptors 1 and 4 (PAR1 and PAR4). The proteolysis of the PARs exposes new tethered ligands that then signal through transmembrane domains to initiate platelet activation as a downstream effect. A pentapeptide cleavage product of bradykinin with the sequence Arg-Pro-Pro-Gly-Phe serves as a thrombin inhibitor by blocking , - and , -thrombin-induced platelet aggregation. Analogs of RPPGF have been prepared that result in improved inhibition of thrombin activation of platelets. Specific amino acid residues required for activity against platelet aggregation have been identified, and a lead compound, rOicPaPhe(p -Me)-NH2 (FM19), has been developed. FM19, which completely inhibits threshold , -thrombin-induced platelet aggregation at a concentration of 16 ± 4 ,m, represents an important lead compound in the development of inhibitors of thrombin-mediated platelet aggregation for treatment of ACS. [source]