Platelet regulating properties of insulin revisited

Abstract

Disturbances in platelet responsiveness in diabetes mellitus (DM) lead to platelet-dependent complications in the vasculature. Our studies showed that insulin inhibits platelet activation by inhibiting ADP- and thrombin-induced Ca2+ levels. Ca2+ is under control of cAMP that is a potent endogenous platelet inhibitor. ADP and thrombin lower the level of cAMP by activating the G-protein Giα2. After insulin binding, the insulin receptor becomes activated and phosphorylates the insulin receptor substrate-1 (IRS-1). Insulin alone does not change the level of Ca2+ or cAMP. Upon activation, IRS-1 associates with and inactivates Giα2 by tyrosine phosphorylation. Giα2 is coupled to the P2Y12 receptor of ADP that support platelet responses by a number of agonists such as thrombin and collagen. The insulin-induced inactivation of Giα2 results in the stabilization of cAMP levels in the presence of ADP and thrombin and thereby to inhibition of platelet responsiveness.

By analogy, insulin would interfere with platelet responses that are under control of cAMP or its effector protein kinase A (PKA). Inhibition of PKA or adenylyl cyclase that produces cAMP, abolishes insulin-induced inhibition. The most profound effect of cAMP is the regulation of platelet adhesion to collagen, which is exposed to the circulation after vascular injury. The adhesion of platelets to collagen is the first phase of hemostasis, followed by platelet aggregation resulting in the formation of a platelet plug. Our studies demonstrated that insulin inhibits the formation of the platelet plug, but also the exposure of phosphatidylserine (PS) that propagates the coagulation cascade. Thus insulin regulates thrombus formation.

We confirmed the reported platelet hyperresponsiveness in DM2. DM2 platelets (1) were more rapidly activated by collagen resulting in an increased thrombus formation, (2) thrombus formation was unresponsive to insulin, (3) showed a decreased sensitivity to antagonism of the Giα2-coupled P2Y12 receptor of ADP, an increased down-regulation of cAMP by ADP, and signs of increased P2Y12 signaling, (4) were characterized by a disturbed Ca2+ homeostasis, and ADP and collagen-induced Ca2+ levels that are unresponsive to insulin. Insulin unresponsiveness has been described in cell models of insulin resistance and has been attributed to a reduced IRS-1 activity. Indeed, IRS-1 activity in platelets was impaired in DM2 platelets. We suggest that the loss of regulation of Giα2 activity by impaired insulin signaling via IRS-1 in contributes to the development of platelet-dependent complications in DM.

IRS-1 associated PI3-K activity and PKB phosphorylation potently upregulates glucose transport in platelets through GLUT3 (Km of about 1.4 mM), which is present in plasma- and intracellular α-granule membranes. RT-PCR analysis reveals the absence of GLUT4. Inhibition of glucose transport inhibits platelet responses. Insulin induces PKB-dependent glucose uptake by lowering the Km for glucose without increasing the GLUT3 copy number at the plasma membrane. At physiological glucose conditions (5 mM), stimulation of glucose uptake by insulin disappears. Glucose uptake at this condition depends on a Ca2+- and PKB-dependent increase in GLUT3 copy number at the PM. Our studies suggest that PKB-dependent glucose transport through GLUT3 in platelets is regulated by changes in surface expression and affinity modulation.