Forestier Marc, Reséndiz Julio C, Pontiggia Luca, Lassila Riitta, Beer Juerg H
Department of Medicine, University Hospital of Bern, Switzerland.
Blood Coagul Fibrinolysis. 2008 Dec;19(8):801-6. doi: 10.1097/MBC.0b013e3283177b43.
Our aim was to investigate the role of filamin cleavage and protein tyrosine phosphorylation in shear-stress-induced platelet microparticle formation and of its suppression by the monoclonal antibody (mAb) Ib-23 directed against GPIbalpha. PPACK-anticoagulated or EDTA-anticoagulated platelet-rich plasma or washed platelets were exposed to high shear stress (5000 s-1 for 5 min) in the presence of antagonists of GP Ibalpha (mAb Ib-23), of GP IIb/IIIa (abciximab) and their combination. We assessed the generation of microparticle by flow cytometry, the filamin cleavage and the protein tyrosine phosphorylation by western blotting. Microparticle formation decreased by more than 50% with mAb Ib-23 alone (P < 0.01, n = 23) but filamin cleavage was not inhibited. In contrast, abciximab did not change microparticle numbers nor filamin cleavage under the same conditions. However, when combined, the inhibitors against GP Ibalpha and GP IIb/IIIa decreased microparticle by 70% and the filamin cleavage by 20% (P < 0.05). Focal adhesion kinase and p72Syk phosphorylation was enhanced by mAb Ib-23, whereas treatment with abciximab reduced it. mAb Ib-23 inhibits platelet microparticle induced by high shear stress. The mechanism seems not to depend on filamin cleavage: abciximab allowed for full microparticle formation at similar levels of cleaved filamin, whereas the combined inhibitors reduced it. The effect of mAb Ib-23 on protein tyrosine phosphorylation supports previous data, which correlates microparticle formation with the extent of protein tyrosine dephosphorylation and mirrors the dephosphorylation by protein tyrosine phosphatase (PTP-1B) in platelets of calpain-/- knockout mice.
我们的目的是研究细丝蛋白裂解和蛋白酪氨酸磷酸化在剪切应力诱导的血小板微粒形成中的作用，以及针对糖蛋白（GP）Ibalpha的单克隆抗体（mAb）Ib-23对其的抑制作用。将PPACK抗凝或EDTA抗凝的富含血小板血浆或洗涤后的血小板，在存在GP Ibalpha拮抗剂（mAb Ib-23）、GP IIb/IIIa拮抗剂（阿昔单抗）及其组合的情况下，暴露于高剪切应力（5000 s-1，持续5分钟）。我们通过流式细胞术评估微粒的生成，通过蛋白质印迹法评估细丝蛋白裂解和蛋白酪氨酸磷酸化。单独使用mAb Ib-23时，微粒形成减少超过50%（P < 0.01，n = 23），但细丝蛋白裂解未受抑制。相比之下，在相同条件下，阿昔单抗未改变微粒数量，也未抑制细丝蛋白裂解。然而，当联合使用时，针对GP Ibalpha和GP IIb/IIIa的抑制剂使微粒减少70%，细丝蛋白裂解减少20%（P < 0.05）。粘着斑激酶和p72Syk磷酸化被mAb Ib-23增强，而用阿昔单抗处理则使其降低。mAb Ib-23抑制高剪切应力诱导的血小板微粒形成。其机制似乎不依赖于细丝蛋白裂解：阿昔单抗在细丝蛋白裂解水平相似时允许完全的微粒形成，而联合抑制剂则使其减少。mAb Ib-23对蛋白酪氨酸磷酸化的影响支持先前的数据，该数据将微粒形成与蛋白酪氨酸去磷酸化程度相关联，并反映了钙蛋白酶基因敲除小鼠血小板中蛋白酪氨酸磷酸酶（PTP-1B）的去磷酸化作用。
