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

使用单克隆抗GPIb抗体进行流式细胞术研究，在HEL-cell表面未检测到GPIb。TM60。然而，当在含有25 ng/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，发现细胞在表面上表达GPIb。从如上所述用TPA刺激的细胞中提取mRNA，并在无TPA培养基中培养24小时后收获。制备噬菌体λ gt 10 cDNA文库，并使用基于洛佩斯的报道合成的探针进行筛选。从HEL-cell λ gt 10文库中筛选出了几种米冷蛋白重组噬菌体。为了更准确地确定GPIb-链上的凝血酶结合位点，我们合成了11个不同的、部分重叠的GPIb-链类似肽，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。一种肽FT 25（#216-240）显示出最强的抑制作用（IC_<50>= 12 μ M），并且在该区域中接近FT 25的另外两种肽（#216-274）也具有抑制作用（IC_<50>小于100 μ M）。相反，区域（#216-274）之外的肽，例如DN 24（#18-41）、EP 26（#181-206）和ETP 26（#281-306）不抑制凝血酶诱导的聚集（IC_<50>大于150 μ M）。在靠近FT 25的区域（#216-274）中具有短于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区域#216-274上，核心部分在FT 25上。

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.