Thrombocidins, microbicidal proteins from human blood platelets, are C-terminal deletion products of CXC chemokines

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their α-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis,Escherichia coli, Staphylococcus aureus, andLactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

During the last decade, antibacterial proteins have been recognized as effector molecules in the innate immune system of widely divergent animal species (1-5). The cationic nature of the vast majority of these proteins is thought to be crucial to target and disrupt microbial membranes (6). Based on their primary structure, antibacterial proteins are classified in four groups. The largest group found thus far is formed by the β-stranded proteins, containing 4–6 conserved cysteines interlinked by disulfide bridges. Defensins are probably the best studied members of this group. Other classes consist of amphipathic α-helical proteins, proline-rich coiled proteins, and looped or cyclic proteins (6, 7).

The antibacterial proteins found in man are distributed over a variety of tissues and cell types. They have been found in leukocytes, most abundantly in polymorphonucleated neutrophils, where they are thought to be involved in the killing of engulfed bacteria (8). More recently, cationic antibacterial peptides have also been found in various epithelial tissues (9). Enteric defensins are produced and secreted by human (10, 11) and mouse (12, 13) Paneth cells. β-Defensins, first isolated from bovine neutrophils (14) and epithelial tissue of tongue and trachea (15-17), have recently been identified in human airway (18, 19) and urogenital epithelial tissue (20), as well as in plasma (21) and skin epithelial cells (22). Expression of some of the epithelial proteins was found to be elevated after injury or contact with lipopolysaccharide or bacteria (22-26), which indicates their functionality in nonspecific host defense.

In addition to the cell types mentioned above, human and rabbit blood platelets are known to store antibacterial proteins (27-33). These antibacterial proteins are released from platelet α-granules in vitro after activation with thrombin (27) and were designated thrombocidins (34). In vivo, direct contact of platelets with bacteria causes aggregation and activation of platelets (35). The subsequently released antibacterial proteins most likely are involved in the elimination of adherent bacteria (36). Dankert et al.(27, 36) showed that antibacterial proteins released from thrombin-activated platelets were involved in the clearance of viridans streptococci from cardiac vegetations in the rabbit experimental infective endocarditis (IE)1model. Viridans streptococci with low susceptibility to these proteins persisted in vegetations, whereas highly susceptible bacteria were rapidly eliminated (36). Similarly, strains ofStaphylococcus aureus and Candida albicansinsusceptible to rabbit platelet microbicidal proteins (PMPs) caused more severe experimental IE than did PMP-susceptible strains (37, 38). Furthermore, thrombocytopenic rabbits (39) or rabbits with antibodies neutralizing their platelet bactericidal proteins (40) were more susceptible to streptococcal IE than control rabbits. The present study was undertaken to gain insight into the structure, activity, and mechanism of action of antimicrobial proteins present in human platelets.