Assay of procarboxypeptidase U, a novel determinant of the fibrinolytic cascade, in human plasma.
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BACKGROUND: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin. METHODS: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-L-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals. RESULTS: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations. CONCLUSIONS: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure. (+info)
Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning.
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BACKGROUND AND PURPOSE: Edema formation after intracerebral hemorrhage has been linked to thrombin toxicity induced by the clot. However, thrombin at low concentrations actually protects neurons and astrocytes in culture from hypoglycemic and ischemic cell death. It is also known that a brief episode of brain ischemia increases neuronal tolerance to a subsequent severe ischemic episode. The objective of this study was to investigate whether pretreatment of the brain with low-dose thrombin induces tolerance to a subsequent large dose of thrombin injected into brain parenchyma. METHODS: The rat brain was preconditioned with 1 U thrombin by direct infusion into the right caudate nucleus. After thrombin pretreatment, the effects of a large dose (5 U) of thrombin on brain edema formation were studied at different intervals. We examined whether heat-shock protein (HSP) 27, HSP32, and HSP70 were induced by Western blot analysis, immunocytochemistry, and immunofluorescent double staining. RESULTS: Thrombin pretreatment significantly attenuated the brain edema that normally follows the infusion of a large dose of thrombin (79.2+/-0.4 versus 84.0+/-0.3; P<0.01). This effect was abolished by the thrombin inhibitor hirudin. Time course studies showed that the maximal effect of thrombin preconditioning (TPC) on brain edema formation was 7 days after pretreatment. This time course corresponded to marked upregulation of HSP27 in the ipsilateral brain. TPC also induced HSP32, but this effect occurred earlier than the effect on edema formation. TPC had no effect on HSP70. Immunocytochemistry and immunofluorescent double labeling showed that HSP27 and HSP32 were expressed in astrocytes after TPC. CONCLUSIONS: OFF phenomenon of thrombin-induced tolerance of the brain to edema formation may be related to HSP27 induction. (+info)
Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.
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1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine. (+info)
Role of caspase in a subset of human platelet activation responses.
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Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3-like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses, alpha granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation. (+info)
Leukocyte-endothelial cell interactions in nitric oxide synthase-deficient mice.
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Nitric oxide (NO) is known to be an important endogenous modulator of leukocyte-endothelial cell interactions within the microcirculation. We examined leukocyte rolling and adhesion under baseline conditions and following thrombin (0.25 U/ml) superfusion in the mesentery of wild-type, inducible NOS (iNOS)-deficient (-/-), neuronal NOS (nNOS) -/-, and endothelial cell NOS (ecNOS) -/- mice to further our understanding of NO and leukocyte function. Baseline leukocyte rolling (cells/min) was significantly elevated in both the nNOS -/- (30.0 +/- 4.0) and ecNOS -/- mice (67.0 +/- 12.0) compared with wild-type mice (11.0 +/- 1.4). In addition, baseline leukocyte adherence (cells/100 micrometers of vessel) was also significantly elevated in the nNOS -/- (5.2 +/- 1.0) and ecNOS -/- (13.0 +/- 1.3) compared with wild-type animals (1.3 +/- 0.5). Deficiency of iNOS had no effect on baseline leukocyte rolling or adhesion in the mesentery. Baseline surface expression of P-selectin was observed in 68.0 +/- 9.0% of intestinal venules in ecNOS -/- mice compared with 10.0 +/- 2.0% in wild-type mice. Additional studies demonstrated that administration of an anti-P-selectin monoclonal antibody (RB40. 34) or the soluble P-selectin ligand, PSGL-1, completely inhibited the increased rolling and firm adhesion response in nNOS -/- and ecNOS -/- mice. Transmigration of neutrophils into the peritoneum following thioglycollate injection was also significantly augmented in nNOS -/- and ecNOS -/- mice. These studies clearly indicate the NO derived from both nNOS and ecNOS is critical in the regulation of leukocyte-endothelial cell interactions. (+info)
Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.
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The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway. (+info)
The effect of a low molecular mass thrombin inhibitor, inogatran, and heparin on thrombin generation and fibrin turnover in patients with unstable coronary artery disease.
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AIM: This study evaluated a novel specific thrombin inhibitor, inogatran, in comparison with unfractionated heparin, with regard to markers for coagulation activity in patients with unstable coronary artery disease. METHODS AND RESULTS: In the Thrombin Inhibition In Myocardial Ischaemia (TRIM) study patients were randomized to one of three different doses of inogatran or to unfractionated heparin, given intravenously over 72 h. In a subpopulation of 320 patients, markers for coagulation activity were measured at baseline, during and after the study infusion. Prothrombin fragment 1 + 2, indicating thrombin generation, decreased in the low, medium and high dose inogatran groups and in the heparin group during the first 6 h of treatment by 12%, 15%, 21% and 26%, respectively. From 6 h to 72 h after the start of infusion the levels changed by -7%, -6%, -4% and +34%, respectively. The increase in the heparin group continued after the infusion was stopped. Thrombin-antithrombin complex, also indicating thrombin generation, decreased by 0%, 2%, 18% and 22%, respectively, during the first 6 h of treatment. During the same period soluble fibrin, an intermediate in fibrin formation, increased both in the low and medium inogatran group by 9%, while a decrease by 4% and 18%, respectively, was seen in the high dose inogatran group and in the heparin group. Fibrin dissolution, as measured by fibrin D-dimer, decreased during the first 24 h of treatment by 20%, 18%, 18% and 20%, respectively. The first 24 h after discontinuation of infusion, fibrin D-dimer increased by 6%, 23%, 25% and 44%, respectively. After 72 h, at the end of infusion, patients treated with inogatran, to a larger extent than those given heparin, had suffered from death, myocardial infarction or refractory angina pectoris. After 7 days this trend was less marked. CONCLUSION: The more pronounced decrease in thrombin generation and fibrin turnover during the first 6 h of infusion, and the later increase in thrombin generation and fibrin turnover, in the heparin group, as compared to the inogatran groups, may be related to the lower clinical event rate during infusion with heparin compared with inogatran and the recurrence of ischaemic events, early after cessation of heparin infusion. (+info)
Activation of haemostasis by exercise, mental stress and adrenaline: effects on platelet sensitivity to thrombin and thrombin generation.
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Stress-induced activation of haemostasis may be involved in the triggering of acute coronary syndromes. We compared the effects of mental stress, dynamic exercise and adrenaline infusion on platelet sensitivity to thrombin using flow-cytometric analysis of platelet fibrinogen binding in whole blood, and platelet aggregability using filtragometry ex vivo, in healthy volunteers. Furthermore, we assessed thrombin generation [prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes in plasma] and thrombin activity (fibrinopeptide A in plasma). Exercise (bicycle ergometry) enhanced thrombin-induced platelet fibrinogen binding (P<0.05) and platelet aggregability (P<0.01), and elevated F1+2, thrombin-antithrombin complexes and fibrinopeptide A (P<0.05 for all three). Adrenaline infusion enhanced thrombin-induced platelet fibrinogen binding and platelet aggregability (P<0.05), and elevated thrombin-antithrombin complexes (P<0.05), whereas F1+2 and fibrinopeptide A levels were not significantly affected. Mental stress increased platelet sensitivity to high concentrations of thrombin only, and produced small increases in levels of thrombin-antithrombin complexes. Time control experiments showed no important changes with repeated measurements during rest. Platelet responses to exercise and adrenaline were reversible, with recovery 60 min later. Thus, heavy exercise and high levels of adrenaline reversibly increased platelet aggregability and platelet sensitivity to thrombin, and enhanced thrombin formation; the effects were most pronounced during exercise. Mental stress only weakly affected these parameters. (+info)