Proteolysis of fibrinogen deposits enables hydrogen peroxide-stimulated polymorphonuclear leukocytes to spread in an acidified environment

Abstract

Polymorphonuclear leukocytes might be expected to employ functional regulatory systems adapted to an acidified environment, such as found in the inflammatory sites where polymorphonuclear leukocytes act in host defense. We previously reported the unusual characteristics of phorbol 12-myristate 13-acetate (PMA)-induced polymorphonuclear leukocyte spreading over immobilized fibrinogen at acidic pH, including extracellular Ca 2+ requirement and independence of protein kinase C (PKC) activity. In the present study, we found that PMA-induced spreading was strongly inhibited at pH 6.0 by the serine protease inhibitor phenylmethanesulfonylfluoride at pH 6.0 but was only mildly inhibited at pH 7.2 and not inhibited at pH 8.0; furthermore, PMA-stimulated polymorphonuclear leukocytes markedly digested immobilized fibrinogen only at pH 6.0. In experiments without stimulation by PMA, we found that at pH 6.0 polymorphonuclear leukocytes were able to spread over fibrinogen surfaces pre-digested by neutrophil serine proteases; this process required extracellular Ca 2+ and stimulation by hydrogen peroxide (H 2O 2). Pharmacological studies demonstrated the involvement of Src family protein tyrosine kinases, but not PKC, in H 2O 2-induced spreading over pre-digested fibrinogen surfaces; this was also the case for PMA-induced spreading at pH 6.0 but not at pH 7.2 or 8.0. These results suggest that PMA-induced polymorphonuclear leukocyte spreading depends on serine protease-mediated fibrinogenolysis in an acidic milieu, but that other mechanisms operate at neutral/alkaline pH.