Membrane permeabilization by thrombin-induced platelet microbicidal protein 1 is modulated by transmembrane voltage polarity and magnitude

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, catio nic peptide generated from rabbit platelets when they are exposed to thromb in in vitro. IL has potent microbicidal activity against a broad spectrum o f bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bil ayers (as artificial bacterial membrane models) suggested that membrane per meabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Payer, Infect. Immun. 64:3758-3764, 1996; M, R. Yeaman, A. S. Payer, S. P, Koo, W. Foss, and P, M, Sullam, J. Clin. Investig. 101:178-187, 1998) , Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by t PMP-1 by using artificial planar lipid bilayer membranes. We assessed the i nfluence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentration s of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at -90 mV than at +90 mV. It is no teworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfu lly initiated at -90 mV but not at +90 mV, Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage a nd tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentr ation (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was mo re dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytopla smic membrane may involve distinct induction and propagation stages of memb rane permeabilization which, in turn, are modulated by transmembrane potent ial, as well as peptide concentration.