Zheng Yongwei, Adams Tamara, Zhi Huiying, Yu Mei, Wen Renren, Newman Peter J, Wang Demin, Newman Debra K
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin, United States of America; Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Science, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin, United States of America.
PLoS One. 2015 Mar 20;10(3):e0119739. doi: 10.1371/journal.pone.0119739. eCollection 2015.
Receptor-mediated platelet activation requires phospholipase C (PLC) activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct β, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT), PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbβ3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbβ3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbβ3 ligation. Consistently, αIIbβ3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbβ3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbβ3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2.
受体介导的血小板激活需要磷脂酶C（PLC）活性来升高细胞内钙并诱导肌动蛋白细胞骨架重组。PLC可分为结构不同的β、γ、δ、ε、ζ和η亚型。有两种PLCγ亚型（PLCγ1、PLCγ2），它们对于酪氨酸激酶依赖性受体的激活至关重要。血小板同时表达PLCγ1和PLCγ2。尽管已证明PLCγ2在血小板激活中起主导作用，但PLCγ1的贡献程度尚未得到评估。为了确定PLCγ1和PLCγ2对血小板激活的相对贡献，我们构建了条件性PLCγ1缺陷、野生型（WT）、PLCγ2缺陷和PLCγ1/PLCγ2双缺陷小鼠，并测量了血小板对不同激动剂的反应能力。我们发现，PLCγ2缺陷消除了αIIbβ3依赖性血小板铺展、GPVI依赖性血小板聚集以及在剪切条件下胶原包被表面上的血栓形成，这两者均依赖于GPVI和αIIbβ3。添加外源性ADP克服了PLCγ2缺陷血小板在固定化纤维蛋白原上的铺展缺陷，表明PLCγ2是响应αIIbβ3连接而进行颗粒分泌所必需的。同样，在没有PLCγ2的情况下，αIIbβ3介导的颗粒内容物释放受损。相比之下，PLCγ1缺陷的血小板在纤维蛋白原上正常铺展并释放颗粒内容物，表现出正常水平的GPVI依赖性聚集，并在胶原包被表面上正常形成血栓。有趣的是，强制表达PLCγ1可完全恢复PLCγ2缺陷血小板的GPVI依赖性聚集和αIIbβ3依赖性铺展。我们得出结论，通过GPVI和αIIbβ3的血小板激活利用PLCγ2，因为PLCγ1水平不足以支持反应性，但如果以PLCγ2通常达到的水平表达，PLCγ1可以恢复反应性。
