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)

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune-mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ-CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.
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PMID:Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age. 247 8

The activation of protein C by thrombin is thought to occur at the endothelial cell surface in the presence of an essential membrane glycoprotein cofactor, thrombomodulin. In the present study it is demonstrated that, in the presence of hirudin, the most potent known inhibitor of thrombin, human protein C can be activated by human factor Xa (20 nM), but by a thrombomodulin-independent mechanism requiring only the presence of Ca2+ and phospholipid vesicles bearing a high proportion of negative charges (30-75% phosphatidylserine, depending on the conditions). At an optimal concentration of phosphatidylserine/phosphatidylcholine (1:1, w/w) of 75 microM, the apparent Km was 1 microM with a kcat. of 1 min-1. At 25 microM-phospholipid the Km was unchanged and the kcat. was 0.67 min-1. At either lipid concentration, increasing the density of negative charges by the adjunction of sulphated polysaccharides, like pentosan polysulphate or standard heparin at optimal concentrations of 2-5 micrograms/ml and 5-10 micrograms/ml respectively, resulted in a 4-fold increase of the kcat. without affecting the Km. Sulphated polysaccharides alone were poor promoters of protein C activation by factor Xa. In any case the presence of Ca2+ was essential, the dependence being sigmoidal with Hill coefficients ranging from 1.4 to 2.0. No significant activation of 4-carboxyglutamic acid-domainless protein C, a chymotrypic derivative lacking the phospholipid-binding domain, could be detected in the presence of phospholipids and Ca2+, with or without pentosan polysulphate. In a large molar excess, other phospholipid-binding entities like prothrombin fragments F1 or F1+2 could inhibit protein C activation by factor Xa, but pentosan polysulphate exerted a clear protective effect. Factor Xa irreversibly inhibited at its active centre, but not di-isopropyl phosphoro-thrombin, behaved as an inhibitor but in a more complex manner than simple Michaelis-Menten kinetics. Among several derivatives of pentosan polysulphate or of heparin which were tested, those having the higher degree of sulphation and/or molecular mass were the most efficient in enhancing the rate of activation of protein C by factor Xa in the presence of phospholipids. These results suggest that human factor Xa, at physiological concentrations, could activate human protein C in the presence of anionic phospholipids and that this activation could be potentiated by therapeutic concentrations of sulphated polysaccharides.
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PMID:Activation of human protein C by blood coagulation factor Xa in the presence of anionic phospholipids. Enhancement by sulphated polysaccharides. 247 15

The human blood platelet membrane glycoprotein Ib (GPIb) functions as a receptor for von Willebrand factor and thrombin. The gene (gpIb alpha) encoding the GPIb alpha-chain was cloned from a genomic cosmid library. The promoter region of this gene was characterized by sequencing two BamHI fragments including 2.8 kb of the 5' flanking region where several Alu repeated elements and purine-rich sequences were found. Possible cis-regulatory elements were identified by comparing the gpIb alpha gene with established consensus sequences known to function as binding sites for transcription factors. To obtain further information on possible megakaryocyte-specific promoter or enhancer sequences, the gpIb alpha promoter region was compared with other genes expressed in platelets that are known so far. The gpIb alpha gene was found to be located on chromosome 17 in region 17p12-ter, by in situ hybridization.
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PMID:The 5' flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Ib alpha. 262 81

In Yersinia pestis KIM, there are 11 Yops (yersinial outer membrane proteins) encoded by the low-Ca2+ response virulence plasmid pCD1. Only Yops M and N are found in easily detectable amounts in the culture medium. In this study, we located and characterized the yopM gene to obtain clues about its role in the virulence of Y. pestis. Rabbit antibody was raised against Yops M and H, copurified from the supernatant of Y. pseudotuberculosis 43(pGW600, pCD1 yopE::Mu dI1[Apr lac]). This antiserum was adsorbed with an Escherichia coli clone that strongly expressed YopH. The resulting YopM-specific antibody was used to screen a HindIII library of pCD1. HindIII-F and several subclones from it expressed YopM in E. coli minicells. A DNA fragment of 1.39 kilobases from HindIII-F was sequenced and found to contain a 367-amino-acid open reading frame capable of encoding a protein with molecular mass (41,566 daltons) and isoelectric point (4.06) similar to those of YopM. The +1 site of the yopM gene was determined by primer extension. The DNA sequence contained repeating structures: 11 pairs of exact direct repeats, two exact inverted repeats, and three palindromes, ranging from 10 to 42 bases in size. One consensus 14-amino-acid sequence was repeated six times in the predicted protein sequence. The YopM sequence shares some significant homology with the von Willebrand factor- and thrombin-binding domain of the alpha chain of human platelet membrane glycoprotein Ib. These findings suggested a testable hypothesis for the function of YopM.
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PMID:The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. 267 Aug 88

Intracellular calcium levels in the platelets of patients with myeloproliferative disorders (MPD) were measured by the Aequorin method. In the patients with MPD, the maximum intracellular calcium level [Ca2+]i was significantly lower and the time required to reach the peak calcium level was significantly longer than in normal controls after thrombin (0.5 U/ml) and collagen (2.5 micrograms/ml) stimulation. The rate of increase in intracellular calcium levels after thrombin (0.5 U/ml) stimulation in the presence of various concentrations of extracellular calcium was lower than in the normal controls, which suggests that the low level of intracellular [Ca2+]i after stimulation is caused mainly by a low Ca2+ influx. These results suggest the relationship between abnormalities of membrane glycoprotein and the impaired calcium influx of MPD platelets.
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PMID:Low intracellular calcium concentration in the platelets after stimulation in patients with myeloproliferative disorders. 274 45

Endogenous calcium-activated neutral protease (CAP, calpain), the major proteolytic activity in human platelets, cleaves a number of proteins. Preferential substrates are actin-binding protein, P 235, and a Mr 83,000 component. A fourth substrate completely degraded by CAP in platelet lysate within seconds is a Mr 70,000 protein. This polypeptide has been shown to influence the electrophoretic behaviour of membrane glycoprotein Ib during isoelectric focusing probably by forming a complex. This in vitro effect suggests that cleavage of the Mr 70,000 protein by CAP might modulate glycoprotein Ib receptor function. Proteolysis of known substrates by CAP during thrombin-induced platelet aggregation could not be detected.
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PMID:Endogenous calcium-activated neutral protease in human platelets. Identification of a Mr 70,000 substrate. 284 60

Human protein C is the precursor of a serine proteinase in plasma which contains nine 4-carboxyglutamic acid residues and functions as a potent anticoagulant. It is activated by thrombin in the presence of an essential endothelial-cell-membrane glycoprotein cofactor, thrombomodulin. In a purified human system, vitamin K-dependent proteins such as factor X, prothrombin and prothrombin fragment 1 were able to inhibit protein C activation by the thrombin-thrombomodulin complex, using either detergent-solubilized thrombomodulin or thrombomodulin reconstituted into vesicles consisting of phosphatidylcholine and phosphatidylserine (1:1, w/w). Factors VII and IX and protein S were much less efficient. Prothrombin fragment 1 behaved as a non-competitive inhibitor with apparent Ki values of 4 microM in the absence, and of 2-2.5 microM in the presence, of phospholipids. Heat decarboxylation of fragment 1 abolished its ability to interfere in protein C activation, and high phospholipid concentrations could attenuate its inhibitory effect and were responsible for a gradual loss of the non-competitive character. Fragment 1 also inhibited the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues, without any influence from phospholipids. At high thrombin concentrations, with respect to thrombomodulin, the inhibitory effect of fragment 1 was diminished. Fragment 1, at 3.8 microM, inhibited by 50% the activation of protein C (0.1 or 0.3 microM) by thrombin. These results suggest that the 4-carboxyglutamic acid domain of vitamin K-dependent proteins can act as a modulator of the protein C anticoagulant pathway through two distinct types of interaction. The functional 4-carboxyglutamic acid domain would be necessary to allow the enhancement of protein C activation in the presence of anionic phospholipids and it could recognize a phospholipid-independent binding site on the thrombin-thrombomodulin complex.
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PMID:Interference of blood-coagulation vitamin K-dependent proteins in the activation of human protein C. Involvement of the 4-carboxyglutamic acid domain in two distinct interactions with the thrombin-thrombomodulin complex and with phospholipids. 285 94

Human platelets contain a single membrane glycoprotein which is susceptible to thrombin proteolysis, glycoprotein V. We have purified 1 mg of glycoprotein V from 10(13) platelets using a combination of gel filtration, hydroxylapatite and ion-exchange chromatographies. Glycoprotein V has a blocked amino-terminus. Following proteolysis by human alpha-thrombin, a major fragment, termed glycoprotein Vf1, had the sequence Gly-Pro-Phe-X-Arg-Pro-Ala-Ala-Asp-Glu-Ser-Val-Glu-Ala-Pro-Val-Asn-Gln-Al a-Glu- Ala-Pro-. The purified glycoprotein was not a substrate for human gamma-thrombin. Glycoprotein V contained 17.5% carbohydrate, with the majority of the carbohydrate consisting of neutral hexoses. Deglycosylated glycoprotein V had a molecular weight of 57.5 kDa compared to the glycosylated protein's 82 kDa and the deglycosylated protein was recognized by polyclonal antibodies raised against glycoprotein V. Immunoelectrophoresis of human and rat platelets and megakaryocytes gave a single immunoreactive band, with the rat glycoprotein having a slightly larger molecular mass. Glycoprotein V is most likely an integral membrane protein.
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PMID:Platelet membrane glycoprotein V: characterization of the thrombin-sensitive glycoprotein from human platelets. 292

Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane.
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PMID:Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib. 293 70

Treatment of platelets with human leukocyte elastase causes a rapid loss in response to von Willebrand factor and a biphasic loss in response to thrombin, first rapid then more slowly. The rapid phase corresponds to cleavage of a 45-kDa glycopeptide from the extracellular end of membrane glycoprotein GPIb. Longer treatment removes 80-kDa and 90-kDa glycopeptides and a glycopeptide corresponding to the major part of GPV. The 45-kDa and 90-kDa species could be obtained by elastase treatment of glycocalicin, the major proteolytic cleavage product of GPIb. Polyclonal rabbit antibodies against the purified 45-kDa glycopeptide had the same effect on the action of von Willebrand factor and thrombin on platelets as cleavage of GPIb by elastase. These results indicate that both the von Willebrand factor and thrombin binding sites on GPIb are located in the same region on the outside of the molecule. Thrombin activation of platelets involves at least two receptors, one on the 45-kDa glycopeptide, which when occupied causes an increase in the speed of response of the platelets to the cleavage of the second. GPV, a candidate for the second receptor, is a hydrophobic glycoprotein that is cleaved from the platelet membrane by several proteases. Proteases that do not activate platelets but degrade the second receptor remove larger fragments from GPV than do proteases such as thrombin or trypsin which activate platelets.
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PMID:Structure and function of platelet membrane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelets response to von Willebrand factor and thrombin. 293 56


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