Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon.
Knight Cardiovascular Institute, Oregon Health & Science University, Portland, Oregon.
Am J Physiol Cell Physiol. 2021 May 1;320(5):C902-C915. doi: 10.1152/ajpcell.00296.2020. Epub 2021 Mar 10.
Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αβ on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.
脾酪氨酸激酶 (Syk) 和布鲁顿酪氨酸激酶 (BTK) 在血小板生理学中发挥着关键作用，促进血小板糖蛋白 VI (GPVI) 和 GPIIb/IIIa 受体下游细胞内免疫受体酪氨酸基激活基序 (ITAM) 介导的信号转导。针对 Syk 和 BTK 的小分子酪氨酸激酶抑制剂 (TKI) 已被开发为抗肿瘤和抗炎治疗药物，并作为抗血小板药物引起了人们的兴趣。在这里，我们研究了 12 种不同的 Syk 和 BTK 抑制剂对 GPVI 介导的血小板信号转导和功能的影响。这些抑制剂包括四种 Syk 抑制剂，Bay 61-3606、R406（ fostamatinib）、entospletinib 和 TAK-659；四种不可逆 BTK 抑制剂，ibrutinib、acalabrutinib、ONO-4059（ tirabrutinib）和 AVL-292（spebrutinib）；以及四种可逆 BTK 抑制剂，CG-806、BMS-935177、BMS-986195 和 fenebrutinib。在体外，针对 Syk 或 BTK 的 TKI 可降低血小板对胶原的黏附、致密颗粒分泌和α颗粒分泌，以响应 GPVI 激动剂交联胶原相关肽 (CRP-XL)。同样，这些 TKI 降低了 CRP-XL 刺激下血小板表面活化整合素 αβ 的百分比，如 PAC-1 结合所测定。虽然所有测试的 TKI 都抑制了 GPVI 介导的激活后磷脂酶 C γ2 (PLCγ2) 的磷酸化，但其他靠近磷酸肌醇 3-激酶 (PI3K) 和蛋白激酶 C 的下游信号事件则受到不同的影响。此外，可逆 BTK 抑制剂对纤维蛋白原上 GPIIb/IIIa 介导的血小板铺展的影响较小，并在纤维蛋白原上血小板铺展时改变了 PI3K 围绕微管的组织。选择性 TKI 还抑制了胶原在生理流动条件下形成血小板聚集体。总之，我们的结果表明，针对 Syk 或 BTK 的 TKI 抑制了血小板的核心功能反应，但可能会对血小板中 Syk 和 BTK 下游关键系统中的蛋白活性和组织产生不同的影响。
