Inhibition of platelet aggregation by a protein factor present in rat peripheral polymorphonuclear leukocyte supernatant

Abstract

In the present investigation, effect of rat peripheral polymorphonuclear leukocyte (PMNL) supernatant was investigated on platelet aggregation. Rat PMNLs suspended in Hanks balanced salt solution (HBSS, pH 7.4) were incubated at 37° C for different time intervals and cell-free supernatant was obtained by centrifugation. Supernatant was found to inhibit adenosine diphosphate (ADP), arachidonic acid (AA), and calcium ionophore-induced platelet aggregation. The inhibitory effect of PMNL supernatant on platelet aggregation was not blocked by methylene blue (10 μM) or adenosine deaminase (5 U ml^-1) pretreatment, suggesting that the inhibitory effect of the supernatant on aggregation was not mediated by nitric oxide (NO) or ecto-ADPase. The effect of PMNL supernatant on platelet aggregation was abolished by preheating the supernatant at 95° C for 5 minutes. Pretreatment of the supernatant with protease inhibitors abolished the inhibitory effect of supernatant on platelet aggregation suggesting that the factor may be a protein or peptide with protease activity. Partial purification of biologically active factor by fine particle liquid chromatography (FPLC) by using Superose 6B column yielded a peak with a molecular weight of approximately 30 kDa having antiaggregatory activity. The results obtained suggest that rat peripheral PMNLs release yet another factor(s) that inhibits platelet aggregation. The factor is a heat labile protein with a molecular weight of approximately 30 kDa.