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

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

Calpains are Ca2+-dependent serine proteases that can regulate protein kinase C-mediated cellular events by cleaving the membrane-bound native enzyme to yield an activated cytosolic fragment. Inhibition of calpain by leupeptin may cause enhancement or inhibition of cellular functions depending on the nature of the protein kinase C reaction involved. We have studied the effects of leupeptin on platelet responses (aggregation, secretion, thromboxane B2 formation and intracellular Ca2+ and pH changes) induced by either thrombin, collagen or phorbol 12-myristate 13-acetate (TPA), which are known to activate protein kinase C by different mechanisms. Only thrombin-induced responses were inhibited by leupeptin. This suggests that the inhibitory effect of leupeptin is not due to antagonism of calpain in this system, but to direct interference with the proteolytic effect of thrombin.
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PMID:Leupeptin does not affect the normal signal transduction mechanism in platelets. 292 Aug 37

The role of platelet plasma membrane in mediating cellular response to external stimuli was investigated by studying phosphatidylinositol turnover and structural physical molecular alterations. Phosphoinositide turnover was evaluated by phosphatidylinositol breakdown and phosphatidic acid (PA) synthesis. The membrane structure was investigated by measuring fatty acid chain organization, flexibility and movements using the spin label method. When platelets are stimulated by thrombin, a rapid phosphatidylinositol bisphosphate (PIP2) breakdown is observed, accompanied by an immediate PA synthesis. At the same time a membrane-bound calcium liberation within the cell is revealed by the decrease in chlortetracycline fluorescence, and the dense granule constituent serotonin is released from the cell. These events result in disorganization of the platelet membrane as estimated by the decrease in order parameter. All these thrombin-induced effects occur in the presence of EDTA, thus being independent of external calcium and aggregation. By contrast ionophore A 23187 does not induce any PIP2 breakdown nor structural disorganization and the dense granule release measured at 10 seconds is only very weak. These results were confirmed by studies on pathological platelets in which PIP2 breakdown was normal when release was normal, such as in Glanzman thrombasthenia, or did not occur when no granule release was measurable as in Gray-platelet syndrome. It is suggested that membrane-bound calcium has a structural rather than a functional role.
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PMID:Molecular membrane organization in normal and pathological platelets: changes in inositide metabolism and membrane fluidity. 299 7

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.
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PMID:Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets. 301 59

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)-polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X-100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N-ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.
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PMID:Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation. 302 23

Phospholipase A2 was solubilized from rat platelet membrane by 1 M KCl and purified to near homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC. The characteristics of the purified membrane-bound enzyme were compared with those of phospholipase A2 released from thrombin-stimulated rat platelets (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 625-631). The molecular weights, elution profiles on reversed-phase HPLC, and NH2-terminal sequences were identical for the two enzymes. Other characteristics of the two enzymes, such as specific activity, substrate specificity, pH optimum, Ca2+ requirement, heat lability, and sensitivity to p-bromophenacyl bromide were also indistinguishable. These findings suggest that both enzymes share a common structure.
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PMID:Purification and characterization of membrane-bound phospholipase A2 from rat platelets. 337 90

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].
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PMID:Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. 347 Jul 89

Platelets were obtained from patients with various hyperlipidemias [type II, type V, lecithin-cholesterol acyltransferase (LCAT) deficiency] and hypolipidemias (abetalipoproteinemia, Tangier disease) to ascertain relationships among plasma lipids, platelet lipids, thrombin binding and thrombin-induced platelet aggregation, and to compare these data with those previously obtained on stimulus-response coupling in platelets following in vitro modification of membrane microviscosity. Washed platelets were studied for their ability to bind 125I-thrombin in the range of 10(-10) to 10(-6) mol/L (10 mU/mL to 100 U/mL) and to aggregate with thrombin at concentrations less than 10(-9) mol/L (100 mU/mL). The values for binding and aggregation in eight patients from six kindred with familial hypercholesterolemia, taken as a group, fell in the low normal range. If divided into two groups, patients with overt cardiovascular disease bound normal amounts of thrombin but were more responsive to it, whereas patients without overt cardiovascular disease bound lower amounts of thrombin but gave an aggregation response in the normal range. These results suggest that platelet hyperresponsiveness in familial hypercholesterolemia arises from an alteration in the coupling mechanism between thrombin binding and response such that platelets from patients with familial hypercholesterolemia are able to respond with lower receptor occupancy than is the case with normal platelets. Thrombin binding and aggregation were within normal ranges for platelets from abetalipoproteinemia patients (N = 4) and type V hyperlipoproteinemia (N = 2), although in the latter case the response appeared to be less at very low thrombin concentrations (less than 30 mU/mL). Thrombin binding was elevated in Tangier disease (N = 3) but with lower responsiveness at lower thrombin concentrations. Thrombin binding was also elevated in LCAT deficiency (N = 2), and one patient showed increased and another showed decreased aggregation responses. In general, increased plasma cholesterol levels resulted in increased stimulus-response coupling (type II), whereas increased triglyceride levels resulted in decreased coupling (type V, Tangier), and there was no apparent alteration in the coupling mechanism with overall reduction in plasma lipid levels as in abetalipoproteinemia.
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PMID:Thrombin binding and response in platelets from patients with dyslipoproteinemias: increased stimulus-response coupling in type II hyperlipoproteinemia. 352 9

A membrane-bound, Ca2-dependent complex of the cofactor factor Va and the enzyme factor Xa comprises the prothrombinase coagulation complex, which catalyzes the proteolytic conversion of prothrombin to thrombin. In normal hemostasis, the platelet is presumed to supply the surface membrane and thus constitutes the site at which an enzymatically functional complex assembles and thrombin generation occurs. Factor Va, the two subunit protein produced by thrombin activation of factor V, is an essential, nonenzymatic cofactor of the prothrombinase complex. Factor Va performs its cofactor role in part by binding to the platelet membrane and functioning as the membrane receptor for factor Xa in a 1:1 stoichiometric complex of high affinity (Kd = 10(-10) M). Factor Va also appears to participate in the binding of prothrombin to the enzymatic complex. Because deletion of factor Va from the prothrombinase complex decreases the rate of thrombin generation by four orders of magnitude, the essential role it plays is easily understood. Therefore, in the evaluation of factor Va function in the prothrombinase complex, the ability of factor Va to support various binding interactions with the platelet, factor Xa, and prothrombin must be considered. Factor Va can be made available from two potential blood compartments: the plasma and platelets. Approximately 80 per cent of the total blood factor V circulates in plasma whereas the remaining 20 per cent is contained within platelet granules. The relative contribution of plasma versus platelet factor V to factor Va binding interactions in the prothrombinase complex are not clearly defined. However, data from our laboratory and several others suggest that factor V stored and released from platelets is of utmost importance in maintaining normal hemostasis. A discussion of these data relative to congenital and acquired deficiencies of both plasma and platelet factor V is the subject of this report.
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PMID:Abnormal formation of the prothrombinase complex: factor V deficiency and related disorders. 380 20

A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.
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PMID:Human prothrombinase complex assembly and function on isolated peripheral blood cell populations. 397 83

We describe four patients with impaired platelet aggregation and 14C-serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.
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PMID:Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation. 608 37


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