Arya Maneesh, López José A, Romo Gabriel M, Dong Jing-Fei, McIntire Larry V, Moake Joel L, Anvari Bahman
Department of Bioengineering, Rice University, Houston, Texas 77005, USA.
Lasers Surg Med. 2002;30(4):306-12. doi: 10.1002/lsm.10044.
Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction.
STUDY DESIGN/MATERIALS AND METHODS: Canine GP Ibalpha does not normally bind to human vWf, and thus canine-human GP Ibalpha chimeras were constructed by sequentially replacing human GP Ibalpha structural regions with their canine counterparts. Chinese hamster ovary (CHO) cells, which are frequently used to express platelet GP complexes, were transfected with the chimeric proteins. Optical tweezers (lambda = 830 nm) were used to investigate bond strengths between vWf and different GP Ibalpha canine-human chimeras. Since vWf does not bind GP Ibalpha without high shear stress, the compounds botrocetin and ristocetin were used to induce binding between human vWf and the chimeras.
All human-canine GP Ibalpha chimeras bound to vWf in the presence of botrocetin. Replacement of the N-terminal flanking sequence and the first leucine-rich repeat resulted in lower GP Ibalpha/vWf bond strengths than the wild-type human GP Ibalpha/vWf bond strength (P < 0.05). Chimeras lacking the second leucine-rich repeat did not adhere to vWf with ristocetin acting as modulator.
The N-terminal flanking sequence and the first leucine-rich repeat of GP Ibalpha were found to be important but not necessary for GP Ibalpha to adhere to vWf. The second leucine-rich repeat was found to be critical for GP Ibalpha to bind vWf and could potentially be used in the development of a novel recombinant anti-thrombotic drugs.
血栓形成始于血小板受体糖蛋白（GP）Ib-IX-V复合物与内皮下或血浆中其黏附配体血管性血友病因子（vWf）的黏附。GP Ib-IX-V的vWf结合结构域位于该复合物的GP Iα亚基中，且包含一个富含亮氨酸的重复区域。为确定哪些富含亮氨酸的重复序列对vWf/GP Iα相互作用至关重要，使用光镊研究了不同富含亮氨酸重复序列的黏附情况。
研究设计/材料与方法：犬类GP Iα通常不与人vWf结合，因此通过用人源对应区域依次替换犬类GP Iα结构区域构建了犬-人GP Iα嵌合体。常用的用于表达血小板GP复合物的中国仓鼠卵巢（CHO）细胞用嵌合蛋白进行转染。使用光镊（波长λ = 830 nm）研究vWf与不同犬-人GP Iα嵌合体之间的结合强度。由于在没有高剪切应力的情况下vWf不与GP Iα结合，因此使用蛇毒因子和瑞斯托霉素诱导人vWf与嵌合体之间的结合。
在蛇毒因子存在的情况下，所有犬-人GP Iα嵌合体均与vWf结合。N端侧翼序列和第一个富含亮氨酸的重复序列被替换后，GP Iα/vWf的结合强度低于野生型人GP Iα/vWf的结合强度（P < 0.05）。缺乏第二个富含亮氨酸重复序列的嵌合体在瑞斯托霉素作为调节剂时不与vWf黏附。
发现GP Iα的N端侧翼序列和第一个富含亮氨酸的重复序列对于GP Iα与vWf的黏附很重要，但并非必需。发现第二个富含亮氨酸的重复序列对于GP Iα结合vWf至关重要，并且可能用于开发新型重组抗血栓药物。
