INTERACTION OF MEMBRANE GLYCOPROTEIN GPIIb AND Ilia WITH CYTOSKELETAL PROTEINS DURING PLATELET ACTIVATION

Experiments were designed and performed to analyse the cytoskeleton assembly and the interaction of glycoprotein (GP)IIb, IIIa and cytoskeletal proteins during platelet activation. A23187 stimulated 125I labeled platelets were solubilised with Triton X-100 solution and centrifuged. The insoluble fraction were analysed by two dimensional electrophoresis and the soluble fraction were fractionated with 5-25% sucrose gradient centrifugation and analysed by SDS PAGE. In Triton X-100 insoluble fraction, high molecular weight protein fraction(MW > 106) was present after stimulation which were consisted of actin binding protein(ABP), myosin heavy chain(MHC), actin and GPIIb and IIIa. And some of the ABP and MHC formed dimer. ABP and actin in this fraction were decreased with 1 mM CaCl2 treatment but the reduction of ABP was inhibited by leupeptm. In Triton X-100 soluble fraction after stimulation, some of the ABP, MHC, P235 protein, actin and small amount of GPIIb, IIIa were sedimented in the same high density fraction but most proteins were sedimented as a monomer form or GPIIb-IIIa complex form. The GPIIb, IIIa incorporation in high molecular weight protein fraction or high density fraction was absent in Ca++ chelating condition or the presence of competitive fibrinogen binding inhibitor which blocked the platelet aggregation. It is concluded that cytoskeletal proteins and GPIIb, IIIa are assembled each other and formed high molecular weight protein fraction or dimer formation during activation. In stimulated platelets these assembled cytoskeletal proteins containing GPIIb, IIIa were also found in Triton X-100 soluble fraction as a precursor of high molecular weight fraction in Triton X-100 insoluble fraction. The binding of fibrinogen to GPIIb-IIIa complex induce the linkage of GPIIb, IIIa to assembled cytoskeletal proteins.

Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells.

The regulation of the synthesis of muscle-specific proteins has been examined in BC3H1 cells, a smooth muscle-like cell line isolated by Schubert et al. (J. Cell Biol., 1974, 61: 398-413.). The synthesis of both creatine kinase and the acetylcholine receptor appear to be under dual control, a positive control due to cell-cell contact which increases the rate of synthesis of this protein, and a negative signal, elicited by serum components, that decreases the rate of synthesis of these proteins. Induction of muscle-specific proteins in BC3H1 cells is a reversible process and can be arrested after partial induction has taken place by the addition of serum or high-molecular-weight protein fraction from serum to these cells. The high-molecular-weight protein fraction from serum is not by itself mitogenic for Bc3H1 cells and cannot be replaced by a variety of known hormones (mitogenic factors).