Abstract 12496: Plasma Growth Factors and Hormones Drive Constitutive Translation of a Broad-Spectrum Proteome in Circulating Platelets via Stimulation of Translation Initiation Signal Pathways, Necessary for Rapid Hemostasis

Abstract

Introduction: The ability of anucleate platelets to translate nascent protein from their inherited mRNAs was first demonstrated in 1967. Later studies supported these findings, but translation quickly ceased in washed ex vivo platelets, leading to a general presumption that platelet translation is not robust, and thus of limited biological significance. Agonist stimulation promotes platelet protein translation, and recent profiling studies confirmed >6000 inherited mRNAs in polysomes, associated with inducible translation of cognate protein products in thrombin-stimulated human platelets. However, relevance of post-activation translation - a slow, rate-limiting step in cell response - to rapid hemostasis is not clear. Platelets express the requisite components and show broad ribosome occupancy of mRNAs under resting conditions. Hypothesis: Plasma-borne growth factors/hormones (GFH) drive constitutive translation in circulating platelets to modulate reactivity. Methods: We employed metabolic labeling of newly synthesized proteins by bio-orthogonal non-canonical amino acid tagging in plasma-starved human platelets fed back with autologous plasma or isolated GFH (VEGF, IGF-1, insulin, EGF, HMGB1), coupled with translation initiation signaling and functional tests. Results: We found that platelets undergo constitutive translation of a broad-spectrum translatome dependent on plasma or GFH exposure. Platelets freshly isolated from platelet-rich plasma showed homeostatic activation of translation initiation signaling, evidenced by phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and downstream effectors eIF4E/4E-BP1. Plasma starvation led to a steady loss of phosphorylation of this pathway, which was fully restored within 5 min stimulation by either plasma or most GFH. Inhibition of translation with puromycin or cycloheximide suppressed thrombin-induced integrin GPIIb/IIIa activation, P-selectin exposure, and platelet aggregation in vitro , and delayed platelet clot retraction. Transfusion of puromycin or MNK inhibitor eFT-508 resulted in delayed hemostasis in a tail bleed assay in mice. Conclusions: Constitutive plasma GFH-driven translation supports hemostatic platelet function.