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)

Preincubation of human platelets with two pairs of immunologically identical monoclonal antibodies (mab) directed against epitopes on the membrane glycoprotein IIb-IIIa complex - two of them (Gi3 and Gi5) precipitating glycoproteins IIb-IIIa, and two (Gi4 and Gi6) recognizing glycoprotein IIIa - induced slightly different inhibitory effects on platelet function. Gi3 and Gi5 blocked platelet aggregation and 14C-serotonin release from platelets induced by ADP, collagen, thrombin, arachidonic acid, ionophore A 23187, platelet activating factor and ristocetin (in the presence of divalent ions). Ristocetin-induced platelet agglutination (in the presence of EDTA) was not inhibited by these mab. Gi4 had no effect on platelet aggregation and release. Gi6 had the same effect on platelet function as Gi3 and Gi5, but only when incubated with Zw(a) (PlAl)-positive platelets with the exception that collagen-induced aggregation was not affected. We conclude that the similar, though not identical, effects on platelet function induced by mab against different epitopes on the glycoprotein complex IIb-IIIa indicate an "unspecific" interference with platelet membrane function possibly due to steric hindrance by mab of the fibrinogen receptor or by inhibition of conformational changes in the GP IIb-IIIa complex necessary for exposure of the fibrinogen binding site.
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PMID:Monoclonal antibodies against human platelet membrane glycoproteins IIb-IIIa. II. Different effects on platelet function. 240 15

In this study we have used two new monoclonal antibodies, designated LJP5 and LJP9, as well as a previously described one, AP2, all specific for the platelet membrane glycoprotein (GP)IIb/IIIa complex. None of them reacted with dissociated GPIIb or GPIIIa. The monovalent Fab fragment of both LJP5 and LJP9 bound to unstimulated platelets in a saturable manner, but binding was markedly decreased after platelets had been incubated at 37 degrees C in the absence of added extracellular calcium. The binding of LJP9 was not affected by AP2, but was blocked by excess LJP5. On the contrary, the binding of LJP5 was blocked in the presence of both AP2 and LJP9. Thus, these antibodies bound to distinct epitopes of GPIIb/IIIa. At saturation, the binding to unstimulated platelets was between 2.41 and 10.9 X 10(4) molecules/platelet for LJP5 and between 3.47 and 9.1 X 10(4) molecules/platelet for LJP9 (range of 11 and 10 experiments, respectively). Binding increased up to 50% after thrombin stimulation. The estimated association constant, Ka, was 2.7 X 10(7) M-1 for LJP5 and 3.85 X 10(7) M-1 for LJP9. Both LJP5 and LJP9 partially inhibited the association of 45Ca2+ with the surface of unstimulated platelets. Moreover, both antibodies blocked the binding of von Willebrand factor (vWF) to stimulated platelets, whereas only LJP9, but not LJP5, blocked fibrinogen binding. LJP9 was also a potent inhibitor of platelet aggregation, whereas LJP5 was without effect in this regard. The results of the present study demonstrate that independent modulation of vWF and fibrinogen binding to stimulated platelets can be attained with monoclonal antibodies directed against distinct epitopes of GPIIb/IIIa.
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PMID:Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody. 241 25

Recent evidence suggests the presence in resting platelets of centrally located compartments of glycoprotein (GP) IIb-IIIa. We have employed an experimental procedure which dissociates and antigenically denatures the surface compartment of GP IIb-IIIa and allows internal compartments of GP IIb-IIIa to be studied immunochemically and functionally in intact platelets. When gel-filtered platelets are incubated with 0.25 mM EGTA at 37 degrees C for 30 min, and then supplemented for 30 min with 5 mM calcium, they lose their ability to bind GP IIb-IIIa complex-specific monoclonal antibody Fab fragments. However, when such platelets are subsequently stimulated with thrombin, GP IIb-IIIa-specific Fabs are again able to bind in large amounts to the platelet surface, in concert with the appearance of substantial amounts of receptors for fibrinogen and fibronectin. In immunoprecipitation experiments, we have found that this thrombin-displayed pool of GP IIb-IIIa originates from a pool that is not labeled by lactoperoxidase-catalyzed radioiodination of intact resting platelets. In immunofluorescence experiments, we have found that EGTA-incubated platelets contain a large sequestered internal pool of GP IIb-IIIa which upon thrombin stimulation is translocated to the platelet surface. Additional experiments suggest that this centrally located compartment may be surface connected in resting platelets and that it is accessible to some extracellular proteins, but not others.
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PMID:Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. 242 67

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.
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PMID:Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study. 243 32

We prepared a heterohybrid cell line that secretes a human IgM monoclonal autoantibody that recognizes an antigen found on thrombin-activated or stored platelets. The surface expression of the epitope recognized by this autoantibody, 5E5, increases with time as platelets age in vitro, suggesting that it may represent a senescence or activation-specific antigen. 5E5 binds to the purified platelet membrane glycoprotein (GP) IIb-IIIa complex in an enzyme-linked immunosorbent assay (ELISA). In an immunoblot technique, 5E5 binds to a protein with an apparent mol wt of 95,000, which is identical to that of GPIIIa under nonreduced conditions. In crossed immunoelectrophoresis (CIE), the predominant antigen recognized by 5E5 is contained in the GPIIb-IIIa precipitin arc. An additional precipitin arc recognized by 5E5 is often observed only on gels derived from lysates of platelets stored under blood bank conditions for greater than 3 days. These findings illustrate the usefulness of human monoclonal antibodies for the identification of membrane neoantigens expressed as a result of platelet activation or revealed as platelets age in vitro.
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PMID:A human monoclonal autoantibody recognizes a neoantigen on glycoprotein IIIa expressed on stored and activated platelets. 243 50

Vitronectin is a glycoprotein that mediates cell adhesion and spreading in a number of cell culture systems. Liposomes containing platelet glycoproteins IIb-IIIa complex have been shown to bind vitronectin-coated surfaces through an Arg-Gly-Asp cell attachment mechanism. We examined the expression of the binding sites for vitronectin on the surface of intact, resting platelets and following stimulation. 125I-Labeled vitronectin bound specifically in a saturable manner to platelets treated with physiological concentrations of thrombin. The binding reached saturation at 100 nM concentration, and, at saturation, approximately 5000 specific binding sites were detected per platelet. The binding was divalent cation-dependent and only partially reversible after complete saturation. A synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited vitronectin binding to platelets. A monoclonal antibody against platelet glycoprotein IIb-IIIa complex also inhibited the binding of vitronectin to stimulated platelets. These data suggest that platelets possess an inducible divalent cation-dependent receptor for vitronectin and that the glycoprotein IIb-IIIa complex is involved in the expression of the vitronectin receptor.
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PMID:Exposure of binding sites for vitronectin on platelets following stimulation. 244 35

A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.
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PMID:Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody. 246 42

PADGEM protein, a platelet alpha granule membrane glycoprotein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion and platelet activation. PADGEM protein is expressed on the surface of activated platelets but not on the surface of resting platelets. Human erythroleukemia (HEL) cells contain platelet alpha granule-like organelles, alpha granule proteins, and express platelet membrane glycoproteins GPIIb/IIIa and GPIb. We demonstrate that HEL cells express a protein that has a molecular weight identical to that of PADGEM and binds to anti-PADGEM antibodies. The exposure of HEL cells in culture to dimethylsulfoxide (DMSO) increased the number of cells expressing PADGEM. Fluorescence activated flow cytometric analysis demonstrated an increase in mean surface expression of PADGEM in DMSO-exposed cells compared to noninduced cells. Total cell content of PADGEM was increased 5.3-fold after DMSO exposure, as determined by radioimmunoassay. Direct binding experiments with the monoclonal anti-PADGEM antibody KC4 demonstrated specific, saturable, and time-dependent interaction of KC4 with HEL cells. A Kd of 7 nM was estimated. There were 14,000 surface binding sites per cell in noninduced cells and 24,000 surface binding sites per cell in DMSO-induced HEL cells. Surface expression of PADGEM protein on HEL cells was not increased with platelet agonists, including thrombin, epinephrine, ADP, nor cytokines, including IL-1, IL-2, tissue necrosis factor. The presence of PADGEM protein in HEL cells should facilitate the elucidation of the function of PADGEM protein.
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PMID:PADGEM protein in human erythroleukemia cells. 246 41

Vitronectin is a plasma glycoprotein that has regulatory activity in the complement and the coagulation systems, in cell-cell and cell-substrate interactions, and in monocyte/macrophage function. Because of its potential to participate in several of the processes of inflammation and repair, the association of vitronectin with platelets was investigated. Immunochemical studies demonstrated that the majority of the platelet associated vitronectin was intracellular, while a relatively modest amount was localized to the ectoplasmic portion of the plasma membrane. Analysis by Western blot showed that the electrophoretic mobility of platelet associated vitronectin was indistinguishable from that of vitronectin isolated from plasma. In response to thrombin, approximately 1 microgram of vitronectin was released into the supernate of 10(9) platelets, while somewhat less than one-half of the total platelet vitronectin remained cell associated. The binding of vitronectin to platelets was investigated by comparing the capacity of unlabelled vitronectin and fibronectin to inhibit binding of radiolabelled fibronectin to thrombin stimulated platelets. On a weight basis, inhibition by the two proteins was equivalent, suggesting that vitronectin competes with fibronectin for binding to platelet glycoprotein IIb/IIIa. These results demonstrate that vitronectin is a platelet specific protein which, because of its multifunctional properties, may participate in physiological and pathophysiological events associated with thrombosis and haemostasis.
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PMID:Vitronectin (S protein) is associated with platelets. 246 74

Using a newly developed murine monoclonal antibody (MoAb), TM83, against glycoprotein IIIa (GPIIIa) of human platelets, we have analyzed the relationship between platelet fibrinogen binding and conformational changes in GPIIIa under EDTA treatment. Crossed radioimmunoelectrophoresis demonstrated that TM83 reacted with only the GPIIb/IIIa complex but also with GPIIIa alone. TM83 dose-dependently inhibited both thrombin-induced aggregation and fibrinogen binding to activated platelets. 125I-TM83 bound to an average of 20,890 +/- 1,600 (mean +/- SE, n = 12) sites on a resting platelet, with Kd = 2.06 nmol/L in the presence of Ca2+. When platelets were incubated in 2 nmol/L EDTA-containing medium, pH 7.4, at 22 degrees C for 30 minutes, binding of TM83 decreased to 70% of the control level. The decreased binding was fully recovered to the control level when the platelets were resuspended in Ca2+-containing medium. These platelets retained their aggregability. In contrast, when platelets were incubated in 2 mmol/L EDTA-containing medium, pH 7.4, at 37 degrees C for 30 minutes, TM83 binding to the platelets markedly decreased to 7% of the control, which could only be recovered to 40% of the control by replacing the medium with calcium-containing medium; these platelets lacked thrombin-induced aggregability. These findings suggest that the epitope for TM83 may be located near the fibrinogen binding site on GPIIIa and that its conformation is dependent on Ca2+ ions.
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PMID:Calcium ions and the conformation of glycoprotein IIIa that is essential fibrinogen binding to platelets: analysis by a new monoclonal anti-GP IIIa antibody, TM83. 246 95


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