Biochemical Properties of Human Platelet Factor 4

Human platelet factor 4 (PF4) was isolated from outdated platelet concentrates to evaluate its biochemical properties. Platelets were washed in saline, lysed by freeze-thawing, and centrifuged to remove particulates from the lysate. PF4 was adsorbed on heparin immobilized on Sepharose. The yield of PF4, eluted in molar saline, was 9.3 ug/ml platelets. Polyacrylamide electrophoresis indicated a single-chain protein with an apparent molecular weight of 9,600. Amino acid composition indicated a 92 residue chain having 2 disulfide bridges and free of MET, PHE, and TRP. Electrophoresis of equal molar ratios of heparin and PF4 in the presence of 0.1% SDS resulted in the appearance of a slow migrating band having an apparent molecular weight of 20,000. The isolated PF4 was poorly soluble in the absence of heparin or at low salt concentrations. Elution profiles of gel and membrane filtration studies were indicative of molecular aggregation. PF4 appeared to be totally resistant to tryptic and chymotryptic hydrolysis but not to pepsin. However, PF4 bound to heparin was resistant to pepsin. Reduced-alkylated PF4 retained its ability to bind heparin but lost its resistance to tryptic or chymotryptic hydrolysis. The presence of heparin did not alter the peptide pattern following tryptic or pepsin hydrolysis of reduced-alkylated PF4 while the peptide pattern after chymotrypsin was modified. Peptides released after chymotryptic hydrolysis of reduced-alkylated PF4 had no apparent affinity for heparin unless heparin was present originally. These peptides after chymotryptic hydrolysis of heparin-bound-reduced-alkylated PF4 may serve as models for the design of small, synthetic heparin antagonists. (Supported by USPHS NIH grant #HL-20317-01 and the Charles DeVlieg Foundation.)