Platelet basic protein (PBP) and platelet factor 4 (PF4) are localized in platelet alpha granules and are secreted during platelet stimulation. They have heparin binding and heparin neutralizing activities. They belong to the family of proteins encoded by small inducible genes. These proteins play an important role in cell growth, maturation, inflammation and wound healing. The NH2 terminal domain of PBP is susceptible to limited proteolysis resulting in formation of low affinity platelet factor 4 (LA- PF4), beta-thromboglobulin (betaTG) and neutrophil activating peptide 2 (NAP2). We intend to study the interaction with cells of PF4, PBP, LA-PF4, betaTG, NAP2 and of a number of synthetic and natural peptides derived from these proteins in order to identify domains endowed with biological functions. We will study mechanism of neutrophil activation by these proteins; we will compare activities of NAP2 with that of interleukin 8 and we will investigate whether both peptides bind to the same receptor on neutrophils. We will study the significance of the primary amino acid sequence and secondary structure of NAP2 for expression of its biological activity. We will investigate the effects of PF4, PBP and their derivatives on megakaryocyte maturation in vitro and on the development of megakaryocytes and on platelet production in mice in vivo. In order to verify our hypothesis that heparin binding sites of PF4 play a major role in hepatic clearance of this protein we will investigate clearance from the circulation, uptake by the liver and by cultured hepatocytes of PF4 and its analogues. We will also compare effects of protamine-heparin complexes, of PF4-heparin complexes and of PF4 derived peptide-heparin complexes on complement activation in human serum and on complement activation, neutrophil sequestration, formation of thromboxane A2 and the occurrence of lung edema in the rat. Our preliminary data indicates that in contrast to protamine sulfate, reversal of heparin anticoagulation by PF4 does not cause any detectable adverse effects. The proposed experiments are relevant in view of a possible replacement of protamine sulfate with recombinant PF4 or PF4-derived peptides for heparin neutralization in surgical patients.
