Platelet-Platelet Fusion Results from Fusing of Plasma Membranes and Is Augmented by Stimulation through PAR1 or CCR4.

Abstract

Cell-cell fusion occurs between many cell types including hematopoietic stem cells and macrophages. Fusion between platelets has been proposed based on both early and more recent studies using light and electron microscopy, but these approaches have lead to conflicting conclusions. We addressed the question of whether or not mature platelets are capable of fusing with one another using videomicroscopy, fluorescence recovery after photobleaching (FRAP), and dye transfer studies. Studies using time-lapse DIC videomicroscopy revealed a process whereby lamellipodia of two adjacent platelets grow towards each other and develop contact points. Fusion of adjacent membranes subsequently occurs between contact points until the two platelets appear as one large platelet with two granulomeres. For studies using FRAP, DiO-labeled platelets were plated densely on coverslips and a laser was used to photobleach areas of approximately 6 x 4 μm2 within collections of large platelets with multiple granulomeres. Following photobleaching, recovery of fluorescence into the bleached areas and loss of fluorescence from areas outside the bleached portions was monitored. Fluorescence recovered within the photobleached areas to 90.4 ± 2.1% of baseline, along with a concomitant loss of fluorescence from adjacent areas. In contrast, FRAP was not observed following similar photobleaching of single platelets or of adjacent platelets without morphologic evidence of fusion. Dye transfer studies were used to determine whether cytosolic mixing occurred between platelets following fusion of membranes. Platelets were labeled with CMFDA, a thiol-reactive, cell-permeant dye that associates with cytosolic proteins. The diffusion of CMFDA from labeled platelets to unlabeled platelets was quantified using digital fluorescence microscopy. Plating of unlabeled platelets with CMFDA-labeled platelets resulted in a markedly increased radius of diffusion of fluorescence and a decrease in average mean fluorescence intensity in CMFDA-labeled platelets. In contrast, mean fluorescence intensity did not change over time when CMFDA-labeled platelets were plated in buffer alone or when CMFDA-labeled platelets were fixed with paraformaldehyde prior to incubation with unlabeled platelets. Diffusion of CMFDA increased in a linear fashion by 4.3 ± 0.9-fold as the density of unlabeled platelets increased from 5 x 103 to 8 x 104 platelets/mm2. The diffusion of CMFDA increased by 7.8 ± 1.4-fold when platelets were activated using SFLLRN, a peptide that stimulates platelets via the thrombin receptor PAR1. Diffusion of CMFDA to unlabeled platelets also increased following incubation of platelets with thrombin or macrophage-derived chemokine (MDC), which stimulates through the chemokine (C-C motif) receptor 4. Time course studies using thrombin- and MDC-stimulated platelets showed that dye transfer to unlabeled platelets was detected 1 h after plating and increased in a linear manner for 6 h. No further diffusion of CMFDA occurred from 6 to 24 h incubation. These studies show that membrane fusion with mixing of cytosolic contents occurs between mature platelets. Platelet-platelet fusion is a relatively slow event and is significantly augmented by physiologic platelet agonists including thrombin and MDC. Platelet-platelet fusion may function in remodeling thrombi that form following vascular injury.