Publications by authors named "Kouns W"

Autoantibodies against platelet glycoprotein (GP) GPIIb/IIIa have been demonstrated in patients with autoimmune thrombocytopenic purpura. Recently, it has been shown that plasma autoantibodies from some patients bind to the cytoplasmic domain of GPIIIa. Our aim was to evaluate further the binding specificity of these plasma autoantibodies.

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Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen.

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Platelet glycoprotein (GP) IIb-IIIa complex (alpha IIb beta 3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to < or = 10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes.

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During platelet activation, the glycoprotein (GP) complex IIb-IIIa (alpha 11b beta 3-integrin) changes its conformation, resulting in binding of adhesive proteins of RGD containing an amino acid sequence as well as in expression of new ligand-induced binding sites (LIBS) on the GPIIb-IIIa molecule. Like its F(ab)-fragments, the monoclonal antibody CRC54, whose epitope is located in the N-terminal part of the GPIIIa molecule, binds to no more than 10% of GPIIb-IIIa on the resting platelet surface. However, the binding of CRC54 increases considerably during activation of platelets by thrombin, platelet adhesion on plastic, GPIIb-IIIa interaction with RGDS-peptide as well as during dissociation of the complex in the presence of EDTA.

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The binding reaction between purified human platelet glycoprotein IIb-IIIa and fibrinogen was investigated by real-time measurements using the surface-plasmon-resonance sensor technology. In these experiments, either glycoprotein IIb-IIIa or fibrinogen was immobilized on a sensor surface. The time-dependent change in surface coverage that occurred immediately upon contact with a solution of the complementary protein was then detected.

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The specificity of tetrafibricin was examined by comparing its activities on GPIIb/IIIa and on the vitronectin receptor (alpha v beta 3) with those of Arg-Gly-Asp-Ser (RGDS) on the same receptors. Tetrafibricin, which inhibited fibrinogen-GPIIb/IIIa binding 10 times more potently than RGDS, was three orders of magnitude less potent compared to RGDS on the inhibition of fibrinogen binding to alpha v beta 3. Furthermore, tetrafibricin potently inhibited platelet adhesion to both fibrinogen and von Willebrand factor.

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One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex.

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Arg-Gly-Asp (RGD) is an amino acid sequence in fibrinogen recognized by platelet glycoprotein (GP) IIb/IIIa. Recently, it was found that RGD peptide binding to GPIIb/IIIa leads to conformational changes in the complex that are associated with the acquisition of high-affinity fibrinogen-binding function. In this study, we found that tetrafibricin, a novel non-peptidic GPIIb/IIIa antagonist, induced similar conformational changes in GPIIb/IIIa as did RGD peptides.

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Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469).

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Peptides derived from a sequence within the loop structure of human platelet glycoprotein (GP) IIIa (integrin beta 3) were previously shown to inhibit fibrinogen binding to purified GPIIb-IIIa. In this study a series of peptides based on the GPIIIa sequence 211-221 (SVSRNRDAPEG) was synthesized. The most active peptide was determined to be RNRDA, and its inhibitory potency was 4-fold greater (IC50 = 4.

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Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 43-5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 43-5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 43-5054 was highly selective GPIIb-IIIa inhibitor.

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This study characterized conformational states of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and regions of the molecule required for fibrinogen binding. Platelet lysates were passed sequentially over concanavalin A and aminoethylglycine (Aeg)RGDS affinity columns. Approximately 10% of the total GPIIb-IIIa bound to the Aeg-RGDS column.

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Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin.

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Following platelet activation, surface receptors for fibrinogen are exposed. On the activated platelet, glycoprotein IIb-IIIa (GPIIb-IIIa) serves as the receptor for fibrinogen. However, the molecular mechanisms which regulate GPIIb-IIIa fibrinogen receptor exposure are unknown.

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The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content.

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We have previously used the IV-3 monoclonal antibody specific for Fc gamma RII to demonstrate that platelet activation by CD9 monoclonal antibodies such as ALB-6 is mediated by the Fc gamma RII. Here, we show that platelet activation following addition of a monoclonal antibody directed against GPIIb/IIIa, P256 is completely blocked by IV-3, as monitored by serotonin release, calcium and pH modifications. However, aggregation was only partially inhibited.

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This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion.

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Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin.

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