Rand M L, Packham M A, Taylor D M, Yeo E L, Gemmell C H, Patil S, Lam S C
Department of Biochemistry, The Hospital for Sick Children, Toronto, Canada.
Thromb Haemost. 1999 Dec;82(6):1680-6.
The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.
人纤维蛋白原γ链羧基末端的HHLGGAKQAGDV（H12）序列以及α链中的RGD序列是可溶性纤维蛋白原与活化的人血小板上糖蛋白IIb-IIIa（GPIIb-IIIa）结合的潜在识别位点。因此，添加H12或含RGD的肽可抑制人血小板的聚集以及纤维蛋白原与人血小板的结合。相比之下，我们之前报道RGDS对兔血小板的这些功能抑制作用相对较小。在本研究中，我们发现H12以剂量依赖性方式抑制ADP和凝血酶诱导的兔血小板聚集。变异的HHLGGAKQAGEV肽不能抑制ADP诱导的聚集，从而证明了其特异性。此外，流式细胞术分析表明，H12以剂量依赖性方式抑制FITC标记的纤维蛋白原与ADP活化的兔血小板的结合。为了检测H12与兔GPIIb-IIIa的直接相互作用，我们通过将兔血小板蛋白的辛基葡糖苷提取物应用于含有纤维蛋白原γ链序列的亲和基质上进行亲和层析。约135 kDa和约95 kDa的蛋白质被可溶性H12特异性洗脱，并且95 kDa的蛋白带被抗-LIBS1免疫印迹，抗-LIBS1是一种抗人GPIIIa的单克隆抗体。在对照样品中，GRGDSP不能从RGD亲和基质上洗脱兔血小板裂解物中可检测到的蛋白质。总的来说，我们的结果表明，H12通过与兔GPIIb-IIIa直接相互作用来抑制兔血小板的聚集以及纤维蛋白原与兔血小板的结合。这些发现表明，兔血小板可作为测试源自H12的肽模拟物功效的合适血栓形成模型。
