Human platelet factor 4, a protein that binds heparin, has been purified to apparent homogeneity and the complete amino acid sequence of the protein has been determined. The 70-residue polypeptide chain contains no methionine, tryptophan, or phenylalanine, and contains only a single tyrosyl residue. The sequence analysis demonstrates a highly negatively charged amino-terminal region. The carboxyl-terminal region of the polypeptide is unusual in that it contains a repetitive clustering of positively charged and hydrophobic pairs of amino acids; preliminary evidence suggests that this domain may play a role in the binding of heparin.Heparin is a potent anticoagulant that is used extensively in clinical medicine for the treatment of thrombotic disease (1). Platelets release-a small protein that neutralizes the anticoagulant effect of heparin (2). A plasma factor with this property was first predicted inr 1948 by Conley (3) when he noticed that patients with thrombocytopenia had increased sensitivity to heparin. Partial purification of the specific, platelet-derived protein, referred to as platelet factor 4 (4), was reported by Deutsch et al. (5), and, more recently, various investigators have published methods for isolation of the protein in an apparently homogeneous form (6-13). Platelet factor 4 is a relatively small, heat-stable protein that is insoluble in solutions of low ionic strength, but that is soluble in high ionic strength solutions and in acidic buffers (12, 13). Platelet factor 4 is released with and bound to a carrier proteoglycan molecule from which it is displaced by heparin (8, 10).Because of a possible relationship of platelet factor 4 to arterial and venous thrombosis (14)(15)(16)(17)(18)(19)(20) tained by precipitation between ammonium sulfate concentrations of 50 and 100%, by affinity chromatography on columns of heparin-E-aminocaproic acid-Sepharose prepared as described by Schmer (21), and by gel filtration through Sephadex G-100 in 1 M NaCl. These methods and the modified thrombin time used in the assay of platelet factor 4 were adapted from those reported by Levine and Wohl (12). Purified platelet factor 4 binds 27 units of heparin per mg of protein.The purified protein appears as a single band after polyacrylamide gel electrophoresis (15% acrylamide) in the presence of sodium dodecyl sulfate and is estimated to be at least 95% pure both by this criterion and by automated Edman degradation of the intact S-carboxamidomethylated protein. The purity of the preparation was also established by immunological techniques. The IgG fraction of antisera obtained from rabbits immunized with platelet factor 4 was isolated by ammonium sulfate precipitation and DEAE-cellulose chromatography (22). The purified IgG gave a single precipitin line against purified platelet factor 4 in immunodiffusion experiments, suggesting that the purified protein behaves as a single major antigenic species.

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Preventing the oxidation of hemoglobin in solution is one of the major requirements for the successful production and long-term storage of hemoglobin-based blood substitutes. To this end we have studied the effects of antioxidants on the rate of methemoglobin formation and disappearance in solutions of human and bovine hemoglobin at 4 degrees C and 37 degrees C. Ascorbate and desferal (5 mM) were observed to act as prooxidants, increasing the rate of methemoglobin formation at 37 degrees C. Trehalose, mannitol, glucose, and EDTA (5 mM) had no significant effect. Glutathione and NADH (10 mM) were the most effective antioxidants tested, causing a significant decrease in the rate of methemoglobin formation at 37 degrees C for periods of up to 50 hours. The combination of these antioxidants in bovine hemoglobin at 4 degrees C resulted in the reduction of methemoglobin levels to nearly undetectable levels in approximately 150 hours. In addition, NADH and glutathione were found to reduce methemoglobin levels to 10% over a period of 100 hours in a sample of human hemoglobin that had been stored at 4 degrees C for one year and had 60% methemoglobin. These results suggest that the prevention and reversal of methemoglobin formation during the long-term storage of hemoglobin solutions and hemoglobin-based blood substitutes may now be possible.

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