Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.
...
PMID:Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity. 609 90

The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the 125I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the 125I-Factor IXa was bound to ATIII by 1 min. The distribution of 125I-Factor IXa in mouse plasma was similar. The clearance of 125I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the 125I-Factor IXa was bound to ATIII. Organ distribution studies with 125I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.
...
PMID:Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors. 620 16

The clearance of (125)I-thrombin and diisopropylphosphoryl-(125)I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was approximately 90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-(125)I-thrombin resulted in the rapid reappearance of the DIP-(125)I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-(125)I-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to high-affinity binding sites, since dissociation constants (K(D)) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively. In contrast to DIP-thrombin, approximately 75% of the radioactivity associated with active thrombin remained in the circulation at 1 min. By 10 min 55% of (125)I-thrombin had been removed from the circulation, and essentially all of the radioactivity can be accounted for in the liver. Sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of plasma samples taken after injection of (125)I-thrombin demonstrated that all of the active thrombin was converted to covalent thrombin-antithrombin III complex by the time of initial sampling (30 s). The in vitro conversion of (125)I-thrombin to thrombin-antithrombin III complex was considerably slower (50+/-5% conversion at 30 s). The simultaneous injection of excess unlabeled DIP-thrombin inhibited the rate of formation of (125)I-thrombin-antithrombin III complex formation in vivo (but not in vitro), which suggests that the binding of active thrombin to the high affinity binding sites is required for the rapid inactivation of thrombin in vivo. We propose that (a) thrombin in the circulation binds to active site-independent high-affinity binding sites on the endothelial cell surface; (b) the inactivation of thrombin by antithrombin III is faster in vivo than in vitro because the high-affinity binding sites, present in a high concentration in the microcirculation, catalyze the reaction; (c) thrombin-antithrombin III complexes are selectively removed by the liver.
...
PMID:Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. Possible role in the inactivation of thrombin by antithrombin III. 625 9

The reaction of anticoagulation system upon perfusion of humorally isolated (with retained innervation) carotid sinus of a rabbit by alpha-,beta/gamma-, DIP-alpha-thrombin and prethrombin I was studied. DIP-alpha-thrombin without clotting activity was shown to initiate like alpha-thrombin the reflex reaction of anticoagulation system characterized by a sharp increase in non-enzymatic fibrinolysis (by 225%) and total fibrinolytic activity of blood (by 51%). Prethrombin I (thrombin precursor) is also capable of exciting the function of anticoagulation system characterized by an increase in non-enzymatic fibrinolysis (by 82%) and total fibrinolytic activity (by 36%). Furthermore, perfusion of prethrombin I or alpha-thrombin at almost the same molar concentrations resulted in the similar degree of anticoagulation system effector reaction. Reflex response of anticoagulation system was not observed upon perfusion of carotid sinus by beta/gamma-thrombin that has high esterase but little if any clotting activity that appears to be due to molecular changes in the macromolecular binding site region. These data support the suggestion that the effect of anticoagulation system excitation is due to interaction of the macromolecular binding site in the structure of alpha-thrombin with anticoagulation system chemoreceptors.
...
PMID:Role of macromolecular binding site of thrombin molecule in excitation of anticoagulation system. 628 64

The mechanism of platelet activation by thrombin was investigated using suspensions of human platelets. The assumption that the thrombin-platelet reaction shows characteristics of an agonist-receptor interaction was corroborated by an analysis of the relation between thrombin concentration and yield of 14C-serotonin secretion. However, silent thrombin binding sites that decrease the free thrombin concentration interfere with this interaction. When the influence of these binding sites is eliminated by a decrease in platelet concentration or by saturation with DIP-thrombin the concentration-response relationship can be interpreted by the adsorption isotherm of Langmuir. The binding sites interfering with the thrombin receptor interaction are identical with the high affinity binding sites of platelets. For the yield of secretion the velocity of thrombin addition is important. The platelet reaction decreases by slowing the rate of thrombin addition. In comparison with the reaction occurring at 37 degrees C, in that occurring at 0 degree C only the rate but not the yield of receptor stimulation is diminished. The results are in accordance with the assumption that the yield of platelet activation is determined by the equilibrium binding of thrombin. It is suggested that at 37 degrees C the processes of thrombin binding to the receptor, of receptor modification and of cellular response are immediately coupled with one another and that the rate-limiting step is the binding of thrombin to the receptor.
...
PMID:[Thrombin-stimulated release of serotonin from blood platelets]. 674 5

Previous studies have shown that thrombin binds to the extracellular endothelial matrix and remains biologically active. In the present study, the role of matrix-bound alpha-thrombin in thrombus formation was investigated by utilizing a model system of thrombogenesis. Plastic cover-slips coated with either matrix-bound alpha-thrombin or matrix-bound active site inactivated thrombin (DIP-alpha-thrombin) were positioned in parallel-plate perfusion chambers and subsequently exposed to non-anticoagulated human blood at a venous wall shear rate of 100/s. The blood was drawn directly from an antecubital vein by a roller pump placed distally to the perfusion chamber. The thrombotic deposits on the matrix, fibrin deposition and platelet thrombus volume, were morphologically evaluated. Matrix-bound alpha-thrombin enhanced the fibrin deposition and thrombus volume on matrices of non-stimulated endothelium with 91% (P < 0.001) and 94% (P < 0.05), respectively. In contrast, binding of DIP-alpha-thrombin to matrices of stimulated endothelium reduced the fibrin deposition by 33% (P < 0.05), but had no effect on the platelet thrombus volume. Translocation of thrombin molecules from upstream matrix areas to binding sites farther downstream on the matrix was indicated in experiments with matrices of stimulated endothelium, which showed enhanced fibrin deposition on downstream areas. Our findings are compatible with a prominent role for matrix-bound alpha-thrombin in thrombogenesis, and in particular on endothelial matrices without tissue factor. The role of matrix-bound alpha-thrombin on tissue factor containing matrices appears less prominent, although it is significant.
...
PMID:alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood. 784 12

Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent manner. The peak increase in [Ca2+]i in response to 100 microM TRP-14 or 0.1 microM alpha-thrombin was similar (i.e., 931 +/- 74 nM and 1032 +/- 80 nM, respectively), which was followed by a slow decrease with t1/2 values of 0.73 and 0.61 min, respectively. Extracellular Ca2+ chelation with 5 mM EGTA abolished the sustained increases in [Ca2+]i induced by either TRP-14 or alpha-thrombin. alpha-thrombin (0.1 microM) increased transendothelial [125I]albumin permeability, whereas TRP-14 (1-100 microM) had no effect. Coincubation of 100 microM TRP-14 with 1 microM DIP-alpha-thrombin also did not increase permeability over control values. Stimulation of BPAEC with 0.1 microM alpha-thrombin induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane indicative of PKC activation, whereas TRP-14 had no effect at any concentration. TRP-14 at 100 microM desensitized BPAEC to thrombin-induced increases in [Ca2+]i and transendothelial permeability. The Ca2+ desensitization was reversed after approximately 60 min, and this recovery paralleled the recovery of the permeability response. These findings indicate that the TRP-14-induced Ca2+ mobilization in the absence of PKC activation is insufficient to increase endothelial permeability. In contrast, the increase in endothelial permeability after alpha-thrombin occurred in conjunction with Ca2+ mobilization as well as PKC activation. TRP-14 pretreatment prevented the alpha-thrombin-induced increase in endothelial permeability secondary to desensitization of the Ca2+ signal. The results suggest that combined cytosolic Ca2+ mobilization mediated by TRP-14 and PKC activation mediated by a TRP-14-independent pathway are dual signals responsible for the thrombin-induced increase in vascular endothelial permeability.
...
PMID:Thrombin receptor peptide inhibits thrombin-induced increase in endothelial permeability by receptor desensitization. 838 91

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS-induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.
...
PMID:Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. 855 86

In addition to its pivotal role in the coagulation cascade, thrombin is mitogenic for fibroblasts and endothelial cells, and activates a number of inflammatory cells including monocytes and T-lymphocytes. To determine if other immune functions are modulated by thrombin and if this modulation is direct or indirect, we investigated whether highly purified human alpha-thrombin affects natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Thrombin enhanced NK cell-mediated cytotoxicity by more than 60% and enhanced IL-2 production and NK 3.3 cell responsiveness to IL-2. Unexpectedly, thrombin and the receptor activating "tethered ligand" domain of the thrombin receptor (TRP-7:SFLLRNP) inhibited LAK cell-mediated cytotoxicity by 50%. DIP-thrombin (a proteolytically inactive form of alpha-thrombin) had no inhibitory activity, suggesting that proteolytic activation of thrombin receptor is requisite for inhibition. These results indicate that cell-mediated cytotoxicity may be enhanced by thrombin through a mechanism involving stimulation of cytokine production and NK cell responsiveness, but that activation of thrombin receptor may also inhibit cytotoxic effects of LAK cells. The role of this dual regulation in processes of cell surveillance, would healing, and inflammation remains to be determined.
...
PMID:Thrombin modulation of natural killer activity in human peripheral lymphocytes. 880 4

In bovine pulmonary artery endothelial cells, ionic currents and the concentration of free intracellular Ca2+ ([Ca2+]i) were measured with a combined patch clamp and Ca(2+)-fluorimetric method (Fura-2). Volume-activated Cl-currents (ICl,vol) were activated by a 13 or 28% decrease in tonicity. Thrombin, 1 U/ml, strongly potentiated ICl,vol preactivated by low hypotonicity (13% HTS) but had no effect on ICl,vol preactivated by stronger hypotonic challenges (28% HTS). The thrombin-induced potentiation was not affected by buffering [Ca2+]i at 50-100 nmol/l and omitting extracellular Ca2+. A peptide agonist of the thrombin receptor, SFLLRN, also potentiated ICl,vol, while an enzymatically inactive thrombin analogue, DIP-thrombin, was without effect. These result suggest that proteolytic activation of the thrombin receptor sensitises the activation of ICl,vol in endothelial cells in a Ca(2+)-independent mechanism.
...
PMID:Thrombin potentiates volume-activated chloride currents in pulmonary artery endothelial cells. 904 79


<< Previous 1 2 3 4 Next >>

---