Elevated Cytosolic Cyclic AMP Inhibits Platelet Adhesion to Von Willebrand Factor by Disengaging the Glycoprotein Ib-IX-V Complex From Lipid Rafts
Authors: Munday Adam D , Penkala Rebecca A , Davizon Pavel D , Pierce Clyde J , Hobbs William E , Kulman John D , Thomas Wendy E , Lopez Jose A. 
Published: October 2019

Abstract
Abstract 3204
A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function.

Disclosures:
No relevant conflicts of interest to declare.