Investigation of the Mechanistic Events Governing Platelet Release.

Abstract 4590

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions from which platelets release. While the mechanics of proplatelet elongation have been studied, the terminal steps of platelet production remain poorly understood. To understand the mechanics of platelet release, we developed a novel gradient sedimentation protocol to isolate released proplatelets. This has enabled us to define and quantify different stages in platelet maturation and identify a new intermediate stage in platelet production—the preplatelet. Preplatelets are anucleate discoid particles considerably larger (2-10μm) than platelets that have the capacity to reversibly convert into barbell-shaped proplatelets. Video-microscopy of GFP-tubulin expressing cells reveals that preplatelets elongate and twist into barbell forms by a process mediated by microtubule-based forces. α-Granules and dense granules present in the preplatelet translocate between platelet-sized swellings during barbell proplatelet formation, and ultimately concentrate at the terminal loops. Fission releases two mature platelets from the barbell ends, and isolated proplatelets will mature into bona fide platelets in culture over a 5-day period. Correspondingly, mature platelets are generated within 2 hours post-transfusion of fluorescently-tagged proplatelets into recipient mice. Released proplatelets were plated on extracellular matrix proteins (fibrinogen, laminin, fibronectin, collagen, matrigel) or a BSA control, and cultured for 24 hours to recapitulate the bone marrow vascular niche and determine whether these cell-substrate interactions mediate platelet release. Fibrinogen and laminin binding increased platelet release roughly 4-fold, suggesting cell-substrate interactions in the bone marrow vascular niche and surrounding vasculature are critical for the final stages or platelet production. These findings confirm that proplatelets mature into multiple individual platelets both in vivo/vitro, and begin to define the mechanistic events leading to platelet production.