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)

The levels of glycoprotein (GP) Ib and GPV and phospholipase C activity were measured in platelets from three Bernard-Soulier syndrome patients. The patients' platelets had 46%, 46%, and 24% of control levels of GPIb alpha and 43%, trace, and 13% of control levels of GPV as determined by immunoblot analysis. Stimulation by thrombin, trypsin, the thromboxane analogue U46619, and the combination of U46619 and trypsin caused the formation of [32P]phosphatidic acid, an index of phospholipase C activity, in [32P]orthophosphate-prelabeled platelets. With all agonists, however, the formation of [32P]phosphatidic acid was markedly reduced in Bernard-Soulier syndrome platelets compared with control platelets. These data indicated a postreceptor defect in phospholipase C activation in Bernard-Soulier syndrome platelets and confirmed earlier observations of potential proteolytic and nonproteolytic mechanisms of platelet activation.
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PMID:Phospholipase C activity in platelets from Bernard-Soulier syndrome patients. 821 96

Polytransfused patients often develop platelet-reactive antibodies (PRAb). These give positive reactions in the platelet immunofluorescence test (PIFT) and may be either lymphocytotoxic (LCTAb) or platelet-specific antibodies (PSAb). The latter may be detected in the PIFT using chloroquine-treated platelets (Chl-PIFT) or by immunoblotting. Serial samples from 106 multiply transfused patients with bone marrow failure were screened by PIFT using a microplate method and flow-cytometric analysis. PSAb activity was confirmed by Chl-PIFT. In 45 (42%) of the patients studied PSAb were detected; 37 (35%) formed LCTAb and 19 (51%) had co-existent PSAb. Sera from 25 of 27 patients with a positive Chl-PIFT, retested by immunoblotting, recognised determinants of Mr 82-160 kD on whole platelets. A large group became sensitised to a component of Mr 105-115 kD reduced (99 kD non-reduced) with similar electrophoretic mobility to GPIIIa using a monoclonal anti-GPIIIa and two human polyclonal anti-HPA-1a sera; some also produced anti-GPIIb. The largest group recognised a determinant of Mr 80-83 kD, probably glycoprotein V (GPV). Three sera were immunoblotted against thrombin-treated platelets and the results confirmed GPV specificity.
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PMID:Antibodies to platelet glycoprotein V in polytransfused patients with haematological disease. 848 49

We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes. Ultra-thin sections of the cells were labeled with antibodies directed against glycocalicin, GPIb, GPIX, and GPV. We have shown that a significant and reproducible labeling for the three GPs was associated with the alpha-granule membrane, accounting for approximately 10% of the total labeling. Furthermore, GPIb labeling appears Willebrand factor (vWF). After thrombin activation, vWF remained close to the limiting membrane of the open canalicular system (OCS), suggesting an early association of both receptor and ligand. Plasma membrane and alpha-granule labeling was virtually absent from the Bernard-Soulier platelets (characterized by a GPIb deficiency), thus proving the specificity of the reaction. In Gray platelets (storage granule deficiency syndrome), the small residual alpha-granules were also occasionally labeled for GPIb, GPIX, and GPIX. Cultured megakaryocytes that displayed the classical GPIb distribution, eg, demarcation and plasma membranes, exhibited also a discrete labeling associated to the alpha-granules. In conclusion, this study shows that, evenly for these three GPs, the alpha-granule membrane mirrors the plasma membrane composition. This might occur through an endocytotic process affecting each plasma membrane protein to a different extent and could have a physiologic relevance in further presentation of a receptor bound to its alpha-granule ligand to the platelet surface.
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PMID:Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and V. 860 28

In this study, we show that the platelet surface expression of glycoprotein (GP) V is regulated by two independent mechanisms. While confirming that both thrombin and neutrophil elastase proteolyse GPV, we show that neutrophil cathepsin G, thrombin receptor activating peptide (TRAP), and a combination of ADP and epinephrine can each result in a decrease in the platelet surface expression of GPV by a nonproteolytic mechanism: a cytoskeletal-mediated redistribution of platelet surface GPV to the surface-connected canalicular system (SCCS). Four independent lines of evidence documented the nonproteolytic nature of this decrease in the platelet surface expression of GPV. First, flow cytometric studies showed that cathepsin G, TRAP, and ADP/epinephrine decreased the platelet surface expression of GPV without changing the total platelet content of GPV. Second, immunoelectron microscopy directly demonstrated translocation of GPV from the platelet surface to the SCCS. Third, the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV were fully reversible. Fourth, cytochalasin B, an inhibitor of actin polymerization, completely inhibited the cathepsin G-, TRAP-, and ADP/epinephrine-induced decreases in platelet surface GPV. The cytoskeletal-mediated redistribution of GPV occurred in a whole blood milieu and at physiologic temperatures (37 degrees C) and extracellular calcium concentrations (2 mmol/L). This study also defines the diverse effects on GPV, GPIb, and GPIX of multiple important platelet agonists. Cathepsin G proteolysed platelet surface GPIb alpha, but redistributed platelet surface GPIX and GPV to the SCCS. Thrombin proteolysed platelet surface GPV, but redistributed platelet surface GPIb and GPIX to the SCCS. Both TRAP and ADP/epinephrine redistributed platelet surface GPIb, GPIX, and GPV to the SCCS. Elastase proteolysed platelet surface GPIb alpha and GPV, but, unlike the other agonists tested, neither proteolysed nor redistributed platelet surface GPIX. The experiments with TRAP showed that activation of the seven-transmembrane domain thrombin receptor can result in translocation of GPIb, GPIX, and GPV to the SCCS independently of the GPIb-mediated pathway of thrombin-induced platelet activation. This study also provides two additional lines of support for the recent report that GPV is noncovalently complexed with GPIb and GPIX in the platelet surface membrane. First, although only the GPIb alpha subunit of this putative complex is known to be directly linked to the platelet cytoskeleton via actin-binding protein, cytochalasin B inhibited the ADP/epinephrine-, cathepsin G-, and TRAP-induced decrease in platelet surface GPV. Second, triple labeling flow cytometric experiments showed that, on each individual platelet, the ADP/epinephrine-induced decrease and subsequent return of the platelet surface expression of GPV occurred simultaneously with the decrease and subsequent return of the platelet surface expression of GPIb. In summary, the platelet surface expression of GPV is regulated by two independent mechanisms: proteolysis and a reversible, cytoskeletal-mediated redistribution to the SCCS.
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PMID:The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system. 860 29

Varicella zoster infection in children can be complicated by acute idiopathic thrombocytopenic purpura (ITP). To determine the etiologic mechanism of this thrombocytopenia, we studied three children with clinically diagnosed varicella infection. Immunoblot analysis of these patients' anti-platelet antibodies identified a unique band at 85 kD. Characterization of this protein revealed that it was platelet surface glycoprotein V (GPV) because it was not affected by a disulfide bond reduction but was cleaved by thrombin. Bernard-Soulier syndrome (BSS) platelets deficient in GPIb-IX and GPV did not react with the sera from our varicella-infected study patients. There was no apparent cross-reactivity between anti-varicella antibody and patients' anti-GPV Ig. We report here the first cases of GPV as the target antigen in autoimmune thrombocytopenia.
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PMID:Varicella-associated thrombocytopenia: autoantibodies against platelet surface glycoprotein V. 888 92

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.
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PMID:Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage. 912 30

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ib alpha, GP Ib beta, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ib alpha, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ib alpha was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.
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PMID:Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site. 919 58

Three different separation methods, all using centrifugation, are routinely used to prepare therapeutic platelet concentrates from human donor blood. Platelet concentrates derived from platelet-rich plasma (PRP-PC), buffy coat (BC-PC) and apheresis (AP-PC) were investigated at the end of production, and over an 8 d storage period. Change in platelet surface markers were measured by flow cytometry, using fluorescein-conjugated antibodies to fibrinogen, P-selectin (CD62P), GPIIb-IIIa (CD41), GPIb alpha (CD42b) and GPV (CD42d), and fluorescein-conjugated Annexin V was used to measure expression of anionic phospholipid. All concentrates showed some changes during preparation but PRP-PC underwent the greatest changes with significantly higher levels of P-selectin (P<0.001) and bound Annexin V (P=0.001) than AP-PC or BC-PC, and lower levels of GPIb alpha (P=0.002) and GPV (P<0.001). These changes were attributable to component separation rather than venesection. These markers all continued to change on storage with a strong positive correlation between the changes seen during production and those after 5 d storage. PRP-PC continued to show the greatest changes whereas BC-PC showed the least. Fibrinogen was bound to 40-50% of platelets in all preparations and this did not alter significantly on storage whereas total expression of GPIIb-IIIa remained unchanged throughout. There was no evidence that the platelet surface changes were thrombin-mediated and leucocyte depletion of BP-PC by filtration had no effect on the changes. It is proposed that the deterioration of platelet concentrates during storage may be related to activation occurring during preparation. 'Whole blood' flow cytometry using a panel of fluorescein-labelled reagents provides an informative method for evaluating platelet concentrates.
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PMID:Activation during preparation of therapeutic platelets affects deterioration during storage: a comparative flow cytometric study of different production methods. 923 69

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.
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PMID:Synthesis, assembly, and intracellular transport of the platelet glycoprotein Ib-IX-V complex. 981 57

We have studied down-regulation and redistribution of glycoprotein V (GPV) and its fragment GPVf2, a subunit of a receptor for von Willebrand factor (VWF), on the surface membrane of thrombin and thrombin receptor activating peptide (TRAP) stimulated platelets by using a newly developed GPVf2-specific monoclonal antibody (1D9). Immunoelectron-microscopical studies revealed that about 50% each of total GPV and GPIX were expressed on the surface membrane of the resting human platelets, and about 83% of GPlbalpha was expressed on the surface. In thrombin-stimulated platelets, the surface GPIbalpha, GPIX and GPV, after hydrolysis by thrombin, was converted to GPVf2, translocated from the surface to the intraplatelet pool and then again redistributed to the surface. In TRAP-stimulated platelets, GPIbalpha, GPIX and GPV, without conversion to GPVf2, were translocated from the surface to the intraplatelet pool and then returned to the surface. Ristocetin-induced agglutinations of both the thrombin- and TRAP-stimulated platelets were lowered during the decreased surface expressions of GPIbalpha, GPIX and GPV/GPVf2 and then normalized when these GPs were again redistributed onto the surface, indicating that the redistributed GPIb/IX/Vf2 complex on the surface can act as a VWF receptor as efficiently as an intact GPIb/IX/V.
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PMID:Down-regulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human platelets. 1002 12


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