Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes

Abstract

Summary. Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34 + cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin aIIbb3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIba by the metalloproteinase mocarhagin. Membraneassociated VWF was detected exclusively on proplateletforming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.

Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions

“…CD34 ϩ cells were separated and cultured as described previously (27). Skin human fibroblasts were cultured in DMEM supplemented with 10% FBS.…”

“…Adhesion-To analyze MK adhesion onto type I collagen, 12-mm glass coverslips were coated with 25 g/ml type I collagen as described previously (27). Where indicated, type I collagen was incubated with 20 g/ml DDR1-Fc for 4 h at 4°C (29,30) or with an unrelated IgG as control.…”

“…Analyses by conventional fluorescence and confocal microscopy were performed with an Olympus BX51 microscope using a 20ϫ/0.5 UPlanF1 objective (Olympus; Watford, UK) and a TCS SPII confocal laser scanning microscopy system equipped with a DM IRBE inverted microscope using a 40ϫ oil NA 1.25 objective (Leica; Bensheim, Germany), as described previously (27,28). To analyze matrix structures in different conditions, glass coverslips were coated with type I collagen, as described previously (27), in the presence or absence of DDR1-Fc, for 16 h at 37°C in PBS and then observed by tapping-mode atomic force microscopy on a Digital Instruments multimode NanoScope III/a SPM (Digital Instruments, Santa Barbara, CA) with Olympus OTR 8 oxide-sharpened silicon nitride probes.…”

“…Where indicated, cells were preincubated with FcR blocking solution for 15 min, according to the manufacturer's instructions, before being seeded. Adhering cells were washed with PBS, and after being fixed and stained with anti-CD61 antibody and Hoechst 33258, they were counted by fluorescence microscopy (27).…”

“…Immunofluorescence, Confocal, and Atomic Force Microscopy Analysis-To evaluate DDR1 distribution on the MK surface, cells were fixed and stained with anti-DDR1 and anti-CD61 antibodies both diluted 1:100 as described previously (27). Analyses by conventional fluorescence and confocal microscopy were performed with an Olympus BX51 microscope using a 20ϫ/0.5 UPlanF1 objective (Olympus; Watford, UK) and a TCS SPII confocal laser scanning microscopy system equipped with a DM IRBE inverted microscope using a 40ϫ oil NA 1.25 objective (Leica; Bensheim, Germany), as described previously (27,28).…”