cDNA cloning of thrombin-receptor on platelet membrane glycoprotein Ib.

使用单克隆抗GPIb抗体的流式细胞术研究未在hell细胞表面检测到GPIb。TM60。然而，当hell细胞在含25ng/ml TPA的培养基中培养24小时，然后在不含TPA的培养基中培养44小时时，发现细胞表面表达GPIb。从上述经TPA刺激的细胞中提取MRNA，在不含TPA的培养基中培养24小时后收获。利用Lopez报告合成的探针制备噬菌体lambda gt10 cDNA文库并进行筛选。从hell -cell lambda gt10文库中筛选了数百万蛋白重组噬菌体。GPIb -链克隆失败。为了比先前估计的更准确地确定GPIb -链上的凝血酶结合位点，合成了11种不同的、部分重叠的肽，这些肽由11-28个类似GPIb -链的氨基酸残基组成，并观察了它们对0.02U/ml凝血酶诱导的人洗涤血小板聚集的影响。其中一个肽FT25（#216-240）的抑制作用最强（IC_<50>=12 μ M），另外两个靠近FT25的肽（#216-274）也有抑制作用（IC_<50>，小于100 μ M）。相比之下，区域外的肽（#216-274），例如DN24(#18-41), EP26（#181-206）和ETP26（#281-306）没有抑制凝血素诱导的聚集（IC_<50>大于150mu M）。在靠近FT25的区域（#216-274）中，短于18个氨基酸残基的肽对聚集没有影响。这些结果表明凝血酶结合位点存在于#216-274区域，核心部分位于FT25。

GPIb was not detected on the surface of HEL-cells with flow-cytometrical studies using a monoclonal anti-GPIb antibody. TM60. However, when HEL-cells were cultured for 24hrs in a medium containing 25ng/ml TPA and then for 44 hrs in a TPA-free medium, the cells were found to express GPIb on the surfaces. MRNA was extracted from the cells which had been stimulated by TPA as above and harvested after 24 hr-culture in a TPA-free medium. A phage lambda gt10 cDNA library was prepared and screened using probes synthesized based on Lopez's report. Several milloin recombinant phage from HEL-cell lambda gt10 library were screened. Cloning of GPIb -chain was unsuccessful.In order to determine a thrombin-binding site on GPIb -chain more accurately than previously estimated, 11 different, partially overlapping peptides consisting of 11-28 amino acid residues analogous to GPIb -chain were synthesized and their effects on 0.02U/ml thrombin-induced aggregation of human washed platelets were observed. A peptide, FT25(#216-240) showed the strongest inhibition (IC_<50>=12mu M) and two other peptides close to FT25 in the region (#216-274) also had inhibitory effects (IC_<50> of less than 100 mu M). In contrast, peptides out of the region (#216-274), for examples, DN24(#18-41), EP26(#181-206) and ETP26(#281-306) did not inhibit thrombin-induced aggregations (IC_<50> of more than 150mu M). Peptides with shorter than 18 amino acid residues in the region (#216-274) close to FT25 had no effect on the aggregation. These results suggest that thrombinbinding site exists on the region #216-274 with a core portion on FT25.