| Budget Amount *help |
¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1986: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1985: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
Eighteen monoclonal antibodies against human platelets were raised. Extensive serological analyses confirmed that all 18 antibodies reacted only with human cells of thrombocytic lineage. Four different molecules were identified by immunoprecipitation and SDS-PAGE analysis with these antibodies: GPIIb-IIIa as detected with the four antibodies HPL 1-4, GPIb with the twelve antibodies HPL7-18, and two mutually distinct, newly defined molecules, gp78 and p151, were detected with the two remaining antibodies, HPL 5 and HPL 6, respectively.
The effects of 18 monoclonal antibodies ( <alpha> -GPIb, <alpha> -GPIIb-IIIa, <alpha> -gp78, and <alpha> -p151) on platelet function were examined. Four anti-GPIIb-IIIa antibodies and twelve anti-GPIb antibodieswere classifies into two groups depending on their binding specificities in assays of antibody binding blocking. Of the four anti-GPIIb-IIIa antibodies, three completely inhibited platelet aggregation induced with ADP or collagen, while the other ca
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used only partial inhibition. The first three antibodies showed identical specificity in antibody binding blocking assays. Of the twelve anti-GPIb antibodies, 4 completely inhibited platelet aggregation induced with ristocetin, 2 inhibited it partially, and the other 6 antibodies did not inhibit it at all. No direct correlation was observed between the ability of the antibodies to inhibit platelet aggregation and their binding specificities. The monoclonal antibodies anti-gp78 and anti-p151 did not affect any of the platelet functions examined.
GPIIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using HPL 2. GPIIb and IIIa were further separated in the presence of SDS by HPLC. Two cycles of this procedure yielded almost comlete separation of homogeneous preparations of GP IIb and IIIa. Each protein was then digested with lysyl endopeptidase, which cleaves at the corboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column. Comparison of the elution profiles showed no obvious homology between the two proteins. Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer. Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified. Less
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