HBD-2-a member of β-defensin family of antimicrobial peptides-is known to permeabilize cell membranes of susceptible cells, but the mechanism of such interactions is poorly understood. In our study, we used a hemolytic model to explore the kinetic properties of HBD-2 interactions with membranes of human erythrocytes. We ran hemolytic assays with a wide range of both HBD-2 and erythrocyte concentrations, as well as varying pH values, incubation times, and osmotic strengths; each in the presence or the absence of inhibitory substances such as proteins and salts. The results show that HBD-2 cell membrane permeabilization is both dose-and time-dependent (with plateau effect observed in each case), and inversely dependent on erythrocyte concentration. HBD-2 interactions with cell membranes highly depend on pH value and the presen ce of inhibitors but are not affected by tested osmotic strength range. Our findings suggest that interactions of HBD-2 with cell membranes are mainly electrostatic in nature and are limited by released cell content. We developed a speculative model of such interaction based on our results. K e y w o r d s: human beta-defensin 2, hemolytic activity, membrane permeabilization.

To investigate an ability of HBD-2 to induce aggregation of erythrocytes. Methods. Aggregation assay, turbidimetry, light microscopy. Results. We have identified aggregation of erythrocytes in the presence of at least 12.5 μM HBD-2 that was confirmed by light microscopy. Effect was strongly depended on HBD-2 concentration but was masked by hemolysis of erythrocytes. Also, aggregation was diminished in presence even 10 mM of NaCl that may indicate electrostatic nature of interactions between HBD-2 and erythrocytes. Conclusions. HBD-2 can induce the salt-sensitive aggregation of erythrocytes in a dose-dependent manner.