Stimulation Of DNA Synthesis In Mouse Fibroblasts By Purified Porcine Platelet Secreted Proteins

Abstract

Human platelets secrete proteins which stimulate multiplication of fibroblast and smooth muscle cells and appear to be involved in the development of atherosclerosis. We have studied the effect of two purified porcine platelet secreted proteins on DNA synthesis in mouse fibroblasts (3T3 cells). Material released from washed platelets stimulated by thrombin was fractionated with 0.04 M ZnSO4, followed by chromatography on Sephacryl S-200 and heparin-agarose. A number of proteins were eluted from heparin-agarose with a gradient of increasing NaCl concentration including Heparin Binding Protein (HBP) and Platelet Factor 4 (PF4), eluted at 0.7 M and 1.1 M NaCl respectively. Both proteins were homogenous in immunoelectrophoresis and in SDS polyacrylamide gel electrophoresis. Their apparent molecular weights were 6,000 and 14,000 respectively. They showed no proteolytic activity using fibrinogen and D-Phe-Pip-Arg-pNa (S2238) as substrates. At concentrations 500-5000 ng/ml, HBP stimulated 3H-thymidine incorporation into subconfluent 3T3 cells made quiescent by serum deprivation. At the latter concentration the effect was similar to that induced by 3-5% fetal bovine serum. Addition of HBP (5000 ng/ml) also stimulated proliferation of 3T3 cells plated in 1% of fetal bovine serum. In both assays porcine platelet factor 4 was at least 10 times less active than HBP. Mitogenic activities of human platelet basic protein and of human platelet derived growth factor were potentiated by addition of fetal bovine or human serum. By contrast, no synergistic effect was found between these sera or porcine serum and HBP (500 ng/ml) when tested by 3H-thymidine incorporation assay. A possible role of secreted porcine platelet proteins in the development of atherosclerosis in swine is under investigation.