Yanabu M, Ozaki Y, Nomura S, Miyake T, Miyazaki Y, Kagawa H, Yamanaka Y, Asazuma N, Satoh K, Kume S, Komiyama Y, Fukuhara S
First Department of Internal Medicine, Kansai Medical University, Osaka, Japan.
Blood. 1997 Mar 1;89(5):1590-8.
NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ib alpha, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-Fc gamma II receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the Fc gamma II receptor. Cross-linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.
NNKY5-5是一种针对糖蛋白（GP）Ibα的血管性血友病因子结合结构域的IgG单克隆抗体，在枸橼酸盐抗凝的富含血小板血浆中可诱导血小板发生微弱但不可逆的聚集（或缔合）。这种现象被定义为小聚集体形成（SAF）。水蛭素抗凝血浆中的血小板或洗涤后的血小板对单独的NNKY5-5几乎无反应，但该抗体可增强低浓度二磷酸腺苷或血小板活化因子诱导的聚集。NNKY5-5不会诱导颗粒释放或细胞内Ca2+动员。然而，NNKY5-5可导致一种64-kD蛋白的酪氨酸磷酸化以及酪氨酸激酶p72syk的激活。抗FcγII受体抗体对SAF无影响，提示NNKY5-5通过与糖蛋白Ib相互作用激活血小板。NNKY5-5的Fab'片段不会诱导SAF，但可增强其他激动剂诱导的聚集。NNKY5-5的Fab'片段可诱导p72syk的激活，提示这种激活独立于FcγII受体。用二抗使受体结合的NNKY5-5的Fab'片段交联可诱导SAF。GRGDS肽、细胞外Ca2+螯合剂以及抗GPIIb/IIIa抗体可抑制NNKY5-5诱导的SAF，但对64-kD蛋白酪氨酸磷酸化或p72syk激活无影响。包括阿司匹林和蛋白激酶C在内的各种抑制剂对SAF、蛋白酪氨酸磷酸化或p72syk激活均无影响。相反，强效酪氨酸激酶抑制剂 tyrphostin 47可抑制NNKY5-5诱导的SAF以及酪氨酸磷酸化和p72syk激活。我们的研究结果提示，NNKY5-5与GPIb的结合通过一种与p72syk激活和一种64-kD蛋白酪氨酸磷酸化相关联的机制，促进纤维蛋白原与GPIIb/IIIa之间的相互作用，从而增强血小板聚集。
