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Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet- poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from “postheparin plasma” by adsorption on heparin-agarose and elution with 2.0 M NaCl and “authentic PF4” isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

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Previous investigations suggested that elastolytic activity found in platelets could be due to contamination by neutrophil elastase. In the present study, the lysate of blood platelets free of detectable neutrophils was examined for elastase-like activity using tertiary-butyloxycarbonyl (tBOC)-ala-ala-pro-ala-aminomethyl coumarin (I), tBOC-ala-ala-pro-val-aminomethyl coumarin (II), and succinyl-tri-ala-p-nitroanilide (SAPNA), and for elastolytic activity using 3H-labeled dog and human lung elastins. The platelet lysate degraded I at a higher rate than II, while the reverse was true of neutrophil elastase. The rate of degradation of I, II, and SAPNA by the lysate increased with reaction time up to 20 min. The rate of I, II, and SAPNA degradation by the lysate was decreased by the presence of 0.5 M NaCl, whereas NaCl greatly potentiated their degradation by neutrophil elastase. Plasma a2-macroglobulin inhibited elastolysis by the platelet lysate, whereas plasma a1-antitrypsin did not. The lysate activity was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, elastatinal, Trasylol, and furoyl-saccharin. The optimum pH for platelet lysate activity was 8.5-9.0, as in other studies using elastin as substrate. The pH 4.5 eluate obtained after incubation of the lysate with dog lung elastin at neutral pH exhibited the same catalytic properties as the activity in the lysate. The different substrate and inhibitor specificities and the failure of IgG specific for neutrophil elastase to remove elastaselike and elastolytic activities from the lysate indicate that a unique elastase occurs in platelets.

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