■肌醇1,3,4,5-四磷酸(IP4)在大多数细胞中由肌醇1,4,5-三磷酸(IP3)通过IP3-激酶(ITPK)形成。它的功能是未知的,但已被建议参与Ca2+进入,IP3法规,和磷酸肌醇3-激酶拮抗作用。
■为了更好地阐明IP4的功能,我们测试了ITPK(GNF362)对血小板的特异性抑制剂,IP4直接在透化血小板中的作用及其对磷脂酰肌醇3,4,5-三磷酸(PIP3)与血小板中含有pleckstrin同源(PH)结构域蛋白结合的作用。
■在全血中利用人血小板形成血栓,在富含血小板的血浆和洗涤的悬浮液中聚集,对于Ca2+研究,或重新悬浮在高K和低Na缓冲液中进行透化实验。AKT-Ser473的磷酸化和Rap1-GTP形成通过蛋白质印迹和使用PIP3珠的PIP3结合来测量。
■GNF362增强低浓度ADP刺激的血小板聚集,胶原蛋白,凝血酶,U46619和胶原涂层毛细血管中的血栓形成。GNF362诱导了Ca2+浓度的瞬时升高,IP3的基础水平升高,并增加了激动剂升高的Ca2的峰高。在透化血小板中,IP4抑制GTPγS诱导的AKT-Ser473磷酸化形成和血小板聚集。IP4降低了GTPγS刺激的Rap1-GTP水平,并有效降低了PIP3珠子对RASA3和BTK的提取。
■ITPK和IP4是血小板功能的负调节因子。包含PH结构域的蛋白质的IP4调节可以代表在血栓形成期间可以控制血小板活化的途径。
UNASSIGNED: Inositol 1,3,4,5-tetrakisphosphate (IP4) is formed from inositol 1,4,5-trisphosphate (IP3) by IP3 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca2+ entry, IP3 regulation, and phosphoinositide 3-kinase antagonism.
UNASSIGNED: To better elucidate a function for IP4, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP4 directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding to pleckstrin-homology (PH) domain-containing proteins in platelets.
UNASSIGNED: Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca2+ studies, or resuspended in high K+ and low Na+ buffers for permeabilization experiments. Phosphorylation of AKT-Ser473 and Rap1-GTP formation were measured by Western blotting and PIP3 binding using PIP3 beads.
UNASSIGNED: GNF362-enhanced platelet aggregation stimulated by low concentrations of ADP, collagen, thrombin, U46619, and thrombus formation in collagen-coated capillaries. GNF362 induced a transient elevation of Ca2+ concentration, elevated basal levels of IP3, and enhanced the peak height of Ca2+ elevated by agonists. In permeabilized platelets, IP4 inhibited GTPγS induced formation of AKT-Ser473 phosphorylation and platelet aggregation. IP4 reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP3 beads.
UNASSIGNED: ITPK and IP4 are negative regulators of platelet function. IP4 regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.