New synthesis of a platelet-specific protein: platelet factor 4 synthesis in a megakaryocyte-enriched rabbit bone marrow culture system

The site of synthesis of platelet-specific proteins remains to be established. With the use of short-term megakaryocyte-enriched cultures, direct evidence was obtained to show that megakaryocytes synthesize the platelet-specific protein, platelet factor 4. A megakaryocyte-enriched fraction of rabbit bone marrow for culture was obtained by centrifugal elutriation and cultured with [3H]leucine. Newly synthesized 3H-platelet factor 4 was sought by copurification with added carrier rabbit platelet factor 4, using heparin agarose affinity chromatography and immunoprecipitation with specific goat anti-rabbit platelet factor 4 antisera. SDS PAGE of the washed immunoprecipitates demonstrated a [3H]leucine-containing peak which migrated identically with purified homogeneous rabbit platelet factor 4. A second, slightly larger molecular-weight protein was identified in the gels also, suggesting that rabbit platelet factor 4 may be synthesized as a larger molecular-weight precursor in rabbit megakaryocytes. These results provide direct evidence that the platelet-specific protein, platelet factor 4, is synthesized in rabbit megakaryocytes before it is packaged into alpha-granules for release in circulating platelets.