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)

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.
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PMID:Identification and characterization of CD39/vascular ATP diphosphohydrolase. 895 60

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.
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PMID:Biological consequences of thrombin receptor deficiency in mice. 897 1

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.
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PMID:Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation. 908 58

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.
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PMID:Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin. 910 82

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.
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PMID:Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. 916 95

The endothelial cell protein C receptor (EPCR) binds protein C and facilitates activation by the thrombin-thrombomodulin complex. EPCR also binds activated protein C (APC) and inhibits APC anticoagulant activity. In this study, we detected a soluble form of EPCR in normal human plasma. Plasma EPCR appears to be approximately 43, 000 D, and circulates at approximately 100 ng/ml (98.4+/-27.8 ng/ml, n = 22). Plasma EPCR was purified from human citrated plasma using ion exchange, immunoaffinity, and protein C affinity chromatography. Flow cytometry experiments demonstrated that plasma EPCR bound APC with an affinity similar to that previously determined for recombinant soluble EPCR (Kdapp = 30 nM). Furthermore, plasma EPCR inhibited both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage Factor Xa clotting assay. The physiological function of plasma EPCR is uncertain, but if the local concentrations are sufficiently high, particularly in disease states, the present data suggest that the soluble plasma EPCR could attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of APC.
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PMID:Identification of functional endothelial protein C receptor in human plasma. 921 19

Platelets, when released as anuclear cells by their precursor megakaryocytes, already carry soluble proteolytic fragments of the amyloid precursor protein (APP) within their alpha-granules and intact APP in the alpha-granule membranes. In response to activation signals elicited by physiologic stimuli such as thrombin, platelets release their granules' soluble contents and translocate granule membrane-bound proteins to the plasma membrane. Because platelets carry >90% of the circulation's APP, activated platelets have been implicated as origins of the beta-amyloid peptide fragment of APP (A beta), whose deposition in the cerebrovasculature is characteristic of Alzheimer's disease. We have therefore studied the APP contents and proteolytic processing in resting DAMI human megakaryocytic cells, along with the consequences of the activation of these cells by thrombin, comparing the results in each case to those with human platelets. Resting and PMA-differentiated DAMI cell contents were examined by Western blotting, immunoprecipitation, or metabolic labeling with sulfur 35-labeled methionine during culture, while plasma membrane-bound APP was evaluated by flow cytometry. Activation was followed by changes in cytoplasmic calcium concentration ((Ca++)in) and in membrane potential. Like platelets, DAMI cells exhibited a thrombin dose-dependent delta(Ca++)in, and membrane potential change; in contrast to the surface of a platelet, the surface of an agranular resting DAMI cell expresses granule-membrane proteins (APP and CD63) that appear on platelets only after activation. DAMI cell culture with 35S-labeled methionine confirmed that megakaryocytes synthesize large amounts of APP, of slightly higher molecular weight, and degrade their APP extensively before platelets are formed.
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PMID:Stimulus responses and amyloid precursor protein processing in DAMI megakaryocytes. 924 60

The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S4. FRET was observed when Fl-FPR-APC was titrated in the presence of Ca2+ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles (kappa2 = 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 A. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC.protein S complex would be 84 A above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.
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PMID:Protein S alters the active site location of activated protein C above the membrane surface. A fluorescence resonance energy transfer study of topography. 931 8

Tissue factor is a membrane-bound glycoprotein that functions in the extrinsic pathway of blood coagulation by acting as a cofactor for factor VII, and the resulting complex leads to thrombin production in vivo. The purpose of the present study is to determine whether macrophages express tissue factor in human coronary atherosclerotic plaques. We examined directional coronary atherectomy specimens from 24 patients with unstable angina and 23 with stable exertional angina. In these specimens, macrophages were detected in 22 (92%) of 24 patients with unstable angina versus 12 (52%) of 23 with stable exertional angina (P = .003). The percentage of macrophage infiltration area was significantly larger in patients with unstable angina than in those with stable exertional angina (17 +/- 3% versus 6 +/- 2%, P = .008). The immunohistochemical double staining revealed the expression of tissue factor on macrophages in 18 (75%) of 24 patients with unstable angina versus 3 (13%) of 23 with stable exertional angina (P < .0001). Thrombus was identified in 20 (83%) of 24 patients with unstable angina versus 12 (52%) of 23 with stable exertional angina (P = .02). Fibrin deposition was mainly observed around macrophages expressing tissue factor in the patients with unstable angina. We have shown that tissue factor expression on macrophages was more frequent in coronary atherosclerotic plaques in patients with unstable angina. Tissue factor expressed on macrophages may play an important role in the thrombogenicity in coronary atherosclerotic plaques of these patients.
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PMID:Tissue factor expression on macrophages in coronary plaques in patients with unstable angina. 935 94

Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes in lipid packing, and changes in protein structure.
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PMID:Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex. 937 Apr 58


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