Extracellular matrix stimulation of guinea pig megakaryocyte proplatelet formation in vitro is mediated through the vitronectin receptor

We have used an in vitro culture system to study the role of extracellular matrix (ECM) in the fragmentation of guinea pig bone marrow megakaryocytes (MK) and the formation of proplatelet fragments from these cells. Proplatelet formation is stimulated by culturing the cells on a hydrated type I collagen gel in the presence of serum. MK express integrin proteins alpha₅, alpha₆, beta₁, and the alpha(v) beta₃ complex as demonstrated by immunofluorescence. A monoclonal antibody, LM 609, to the alpha(v) beta₃ vitronectin receptor blocked proplatelet formation, whereas the monoclonal antibodies to the beta₁, alpha₅, and alpha₆ integrin proteins did not inhibit proplatelet formation. The tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits proplatelet formation; however, there was no inhibition by the fibronectin receptor-specific peptide GRGDdSP. The fibrinogen gamma chain peptide HHLGGAKQAGDV, which binds to the platelet membrane glycoprotein complex IIb-IIIa but not to the vitronectin receptor (VnR), did not inhibit proplatelet formation, nor did two different laminin peptides. In the absence of serum, 5.7% of MK spontaneously formed processes or fragmented. The addition of 50 μg/ml of vitronectin to serum-free cultures increased proplatelet formation to 21.5% of the MK, equal to cultures with 10% serum. Stimulation of proplatelet formation by vitronectin in serum-free cultures was inhibited by LM 609. Antibody staining with anti-bovine vitronectin antibody showed that MK contain intracellular vitronectin. These data show that guinea pig MK express alpha₅, alpha₆, beta₁, and alpha(v) beta₃ integrins. However, only the MK vitronectin receptor and its interaction with vitronectin plays an essential role in proplatelet formation in vitro.