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Purified System (purified + system)
Selected AbstractsCleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010Y. WU Summary.,Background and objective:,Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. Methods and results:,We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and ,v,3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of ,v,3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. Conclusions:,HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction. [source] The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular diseaseJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007P. Y. KIM Summary.,Background:,Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known.Objectives:,These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen.Methods:,The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, kcat and Km values for plasmin action on fibrin were determined.Results:,The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 ,M and did not increase further as the fibrinogen concentration was raised to 20 ,M. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 ,M. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent Km and kcat values for plasmin were 1.1 ± 0.6 ,M and 28 ± 2 min,1, respectively. Km values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 ± 0.2 ,M in the purified system and 2.1 ± 0.9 ,M in plasma.Conclusion:,As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen. [source] Complete inhibition of fibrinolysis by sustained carboxypeptidase B activity: the role and requirement of plasmin inhibitorsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007J. B. WALKER Summary.,Background:,The antifibrinolytic effect of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and carboxypeptidase B (CPB) displays threshold behavior. When CPB was used to simulate conditions mimicking continuous TAFIa activity, it affected the lysis of plasma clots differently to clots formed from a minimal fibrinolytic system comprising fibrinogen, plasminogen and ,2 -antiplasmin. Whereas CPB saturably prolonged clot lysis in the purified system, the effect of CPB did not appear saturable in plasma clots. Methods:,To rationalize this difference, we investigated the effects of ,2 -antiplasmin, ,2 -macroglobulin, antithrombin and aprotinin on CPB-mediated antifibrinolysis. Results:,CPB alone prolonged fibrinolysis in a saturable manner and the efficacy of CPB increased with decreasing tissue-type plasminogen activator (t-PA) concentration. The inhibitors by themselves did not halt fibrinolysis and the potency of each inhibitor in the absence of CPB mirrored their solution-phase plasmin inhibitory potentials: ,2 -antiplasmin , aprotinin >> ,2 -macroglobulin >> antithrombin. With both CPB and inhibitor present, a synergistic effect was observed. The antifibrinolytic sensitivity to CPB was related to the plasmin inhibitory potential of the inhibitor. Conclusions:,Fibrinolysis could be completely inhibited by ,2 -antiplasmin, ,2 -macroglobulin and antithrombin, but not aprotinin, in the presence of CPB, and occurred only when the irreversible inhibitor or pool of inhibitors were in excess of plasminogen. Western blot analysis indicated that the CPB-mediated shutdown of fibrinolysis was a result of plasminogen consumption prior to clot lysis. The CPB concentration required for fibrinolytic shutdown was dependent on t-PA concentration and the inhibitory potential of the irreversible inhibitor pool. [source] Phospholipid content, expression and support of thrombin generation of neonatal plateletsACTA PAEDIATRICA, Issue 2 2009Heike Bernhard Abstract Aim: Newborns have, despite low clotting factors and poor in vitro platelet function, a well functioning haemostasis. We investigated whether phospholipids (PL) in neonatal platelets differ from those in adult platelets in their exposure on the platelet surface, and their effect on thrombin generation. Methods: The effect of newborn and adult platelets on thrombin generation (TG) was measured by means of calibrated automated thrombography (CAT), and in a purified system. In addition, clotting times were measured. Phosphatidylserine (PS)-exposure was measured by flow cytometry. The amount of PL was determined by means of mass-spectrometry (Materials and Methods section in Supporting Information online). Results: In comparison with adults the clotting times in platelet-rich plasma of newborns were less shortened by adding calciumionophore. No differences in the support of TG between neonatal and adult platelets were found by means of CAT. In the purified system, TG was increased by ionophor-stimulated platelets but no difference was evident between newborn and adult platelets. Flow cytometric analysis showed no difference between adult and newborn platelets. Results of mass-spectrometry showed a very similar pattern of phospholipid-content of adult and newborn platelets. Conclusion: Our results do not provide any evidence that a different phospholipid-expression of neonatal platelets may alter TG in neonates. [source] Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activityCONTRAST MEDIA & MOLECULAR IMAGING, Issue 4 2010Lorenzo Tei Abstract Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd,DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material. Copyright © 2010 John Wiley & Sons, Ltd. [source] Direct anticoagulant activity of protein S-C4b binding protein complex in Heerlen heterozygotes and normals,JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2004M. J. Heeb Summary.,Background:,Plasma protein S normally circulates free (40%) or complexed with C4b-binding protein (PS-C4BP); only free protein S is a cofactor for activated protein C during factor (F) Va inactivation. Protein S-Heerlen lacks a carbohydrate group, leading to low plasma free protein S levels, but normal levels of PS-C4BP. Objectives:,Because protein S-Heerlen is not associated with thrombosis, we investigated whether PS-C4BP is directly anticoagulant in plasma and whether PS-Heerlen-C4BP has enhanced direct anticoagulant activity. Methods:,An assay for protein S direct activity was applied to Heerlen-heterozygous plasmas. Free and complexed protein S were repeatedly isolated from normal and Heerlen-heterozygous plasmas and tested for direct anticoagulant activity in prothrombinase assays and in plasma. Results:,Heerlen-heterozygous plasmas were deficient in free and total protein S antigen but had normal to high protein S direct anticoagulant activity. Purified Heerlen-heterozygous PS-C4BP was 7-fold more potent than normal PS-C4BP in inhibiting full prothrombinase activity, and 22-fold more potent in inhibiting prothrombin activation in the absence of FVa; it also specifically prolonged plasma clotting times 14-fold more than normal PS-C4BP. Heerlen-heterozygous PS-C4BP did not compete for limiting phospholipids any better than normal PS-C4BP. However, ligand blots and surface plasmon resonance studies showed that Heerlen-heterozygous PS-C4BP bound more avidly to FXa than did normal PS-C4BP (apparent Kd = 4.3 nm vs. 82 nm). Conclusions:,Plasma-derived PS-C4BP has direct anticoagulant activity in plasma and in purified systems. Enhanced direct activity of PS-Heerlen-C4BP may compensate for low free protein S levels and low cofactor activity in individuals with protein S-Heerlen. [source] | ||||||||||