OC 61.5 - Plasma growth factors drive constitutive translation in circulating platelets to maintain protein homeostasis and regulate platelet GPCR reactivity and the hemostatic and thrombotic balance.

Tuesday, June 27, 2023

15:45 – 16:00 ET

Room: 710A

Session Available As: On-Demand

Presenting Author(s)

Lawrence E. Goldfinger, PhD (he/him/his)

Professor
Thomas Jefferson University
Philadelphia, Pennsylvania, United States

Co-Author(s)

JW

Jeremy Wurtzel

Research Technician
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
SL

Sophia Lazar

Graduate student
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
SA

Shayan Askari

Graduate student
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
XZ

Xuefei Zhao

Graduate student
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
Jenna R. Severa, BS

Graduate student
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
James V. Michael, PhD

Research Assistant professor
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
Steven E McKenzie, MD, PhD

Professor
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
TS

Timothy J. Stalker

Assistant professor
Thomas Jefferson University
Philadelphia, Pennsylvania, United States
Peisong Ma, PhD

Assistant Professor
Thomas Jefferson University
Philadelphia, Pennsylvania, United States

Background: Mechanisms of proteostasis in anucleate circulating platelets are unknown, and may regulate platelet function.

Aims: Test the hypothesis that plasma-borne growth factors/hormones (GFH) maintain constitutive translation in circulating platelets to facilitate reactivity.

Methods: Metabolic labeling of newly synthesized proteins by bio-orthogonal non-canonical amino acid tagging (BONCAT) and translation initiation signaling in plasma-starved platelets refed with autologous plasma or isolated GFH; proteomic and functional analysis in circulating platelets in translation inhibitor-infused mice.

Results: BONCAT protein labeling and SDS-PAGE revealed constitutive synthesis of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly-isolated platelets from platelet-rich plasma showed homeostatic activation of translation initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of phosphorylation of this pathway, which was fully restored within 5-minute stimulation by plasma or GFH. Cycloheximide infusion in mice had minimal effects on protein expression in resting platelets by proteomic analysis, suggesting suppressed degradation to maintain proteostasis under these conditions. However, ribosomal and chaperone proteins, and major clients including Mtor and Akt, were downregulated, confirmed by immunoblotting. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low-dose thrombin ( < 0.1 U/mL), and low-to-saturating ADP ( < 20 μM) or thromboxane analogue ( < 5 μM), but not convulxin. ADP-induced, Akt-dependent thromboxane generation was blunted, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously-administered puromycin reduced injury-induced clot size, and delayed primary but not final tail clip hemostasis, nor hemostasis following jugular vein puncture. In contrast, these mice were protected from thrombin-induced pulmonary embolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited PE.

Conclusion(s): Constitutive plasma GFH-driven translation regulates platelet GPCR reactivity to balance hemostatic and thrombotic potential. Ribosome-associated chaperone clients Mtor/Akt manifest labile protein levels under proteostatic stress conditions.

Learning Objectives:

Upon completion, participants will be able to recognize that circulating, resting platelets maintain proteostasis by constitutive translation driven by plasma growth factors and hormones.
Upon completion, participants will be able to recognize that constitutive protein synthesis is required for platelet reactivity and regulates the balance of hemostasis and thrombotic potential.
Upon completion, participants will be able to consider the plasma environment as a key driver of platelet reactivity and function.