Authi Kalwant S, Khan Sabeeya, Gibbins Jonathan M, Brain Susan D
School of Cardiovascular and Metabolic Medicine and Sciences, BHF Centre for Research Excellence, London, UK.
Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK.
Res Pract Thromb Haemost. 2024 Jan 26;8(1):102326. doi: 10.1016/j.rpth.2024.102326. eCollection 2024 Jan.
Inositol 1,3,4,5-tetrakisphosphate (IP) is formed from inositol 1,4,5-trisphosphate (IP) by IP 3-kinase (ITPK) in most cells. Its function is unknown but has been suggested to be involved in Ca entry, IP regulation, and phosphoinositide 3-kinase antagonism.
To better elucidate a function for IP, we tested a specific inhibitor of ITPK (GNF362) on platelets, the effects of IP directly in permeabilized platelets and its effect on phosphatidylinositol 3,4,5-trisphosphate (PIP) binding to pleckstrin-homology (PH) domain-containing proteins in platelets.
Human platelets were utilized in whole blood for thrombus formation, in platelet-rich plasma and washed suspensions for aggregation, and for Ca studies, or resuspended in high K and low Na buffers for permeabilization experiments. Phosphorylation of AKT-Ser and Rap1-GTP formation were measured by Western blotting and PIP binding using PIP beads.
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 Ca concentration, elevated basal levels of IP, and enhanced the peak height of Ca elevated by agonists. In permeabilized platelets, IP inhibited GTPγS induced formation of AKT-Ser phosphorylation and platelet aggregation. IP reduced GTPγS-stimulated Rap1-GTP levels and potently reduced extraction of RASA3 and BTK by PIP beads.
ITPK and IP are negative regulators of platelet function. IP regulation of PH domain-containing proteins may represent a pathway by which platelet activation may be controlled during thrombosis.
在大多数细胞中，1,3,4,5-四磷酸肌醇（IP）由1,4,5-三磷酸肌醇（IP）经IP3激酶（ITPK）形成。其功能尚不清楚，但有人认为它参与钙内流、IP调节和磷脂酰肌醇3激酶拮抗作用。
为了更好地阐明IP的功能，我们在血小板上测试了ITPK的特异性抑制剂（GNF362），直接在透化血小板中检测IP的作用及其对血小板中磷脂酰肌醇3,4,5-三磷酸（PIP）与含pleckstrin同源（PH）结构域蛋白结合的影响。
使用人血小板进行全血血栓形成实验、富含血小板血浆和洗涤悬浮液的聚集实验以及钙研究，或将其重悬于高钾和低钠缓冲液中进行透化实验。通过蛋白质印迹法测量AKT-Ser的磷酸化和Rap1-GTP的形成，并使用PIP珠进行PIP结合实验。
GNF362增强了低浓度ADP、胶原、凝血酶、U46619刺激的血小板聚集以及胶原包被毛细管中的血栓形成。GNF362诱导钙浓度短暂升高，提高IP的基础水平，并增强激动剂引起的钙升高峰值。在透化血小板中，IP抑制GTPγS诱导的AKT-Ser磷酸化形成和血小板聚集。IP降低GTPγS刺激的Rap1-GTP水平，并有效减少PIP珠对RASA3和BTK的提取。
ITPK和IP是血小板功能的负调节因子。IP对含PH结构域蛋白的调节可能代表了一种在血栓形成过程中控制血小板活化的途径。
