Antimicrobial peptides from human platelets

Abstract

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin beta-4 (Tbeta-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tbeta-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.

FIG. 1.

Purification of human platelet antimicrobial peptides. (A) Bio-Gel P-60 size exclusion chromatography. An acetic acid extract from human platelets was loaded onto a 4.8- by 60-cm Bio-Gel P-60 column and eluted with 30% acetic acid at a flow rate of 30 ml/h. Several fractions, encompassed by fractions 17 to 48, demonstrated antibacterial activity upon initial screening assays. These fractions were further purified by RP-HPLC. (B) RP-HPLC purification. RP-HPLC conditions were a 1.0- by 25-cm Vydac C₄ column that was equilibrated in 0.1% TFA-water at a flow rate of 2.5 ml/min. Linear gradients of acetonitrile containing 0.1% TFA were applied over 60 min (0 to 40%) and 40 min (40 to 100%). The following samples were injected onto the column: supernatant from nonstimulated human platelets (I), supernatant from thrombin-stimulated human platelets (II), and fractions 17 to 48 from the Bio-Gel P-60 column (III). Seven active peptides were obtained from peaks 1 to 7 in panel II and identified as shown in Fig. 2. Corresponding peaks 3 to 7 in panels II and III were identified to be identical. Peak A (panels I and II) and peak D (panel III) were identified as human serum albumin and dioctylphthalate, respectively.

FIG. 2.

Analytical RP-HPLC of HPAPs. Samples (0.5 nmol) of each of the peaks, 1 to 7, isolated for Fig. 1B were injected onto a 0.4- by 25-cm Vydac C₁₈ column and eluted at a flow rate of 1.0 ml/min in the same solvent system as that described for Fig. 1B. A linear gradient of 0 to 40% acetonitrile containing 0.1% TFA was applied over 60 min. Peak identification (also see Table 1): 1, FP-A; 2, FP-B; 3, Tβ-4; 4, PBP; 5, CTAP-3; 6, RANTES; 7, PF-4. The methods for identification of these peptides are shown in Table 1.

FIG. 3.

Antimicrobial activities of HPAPs as influenced by pH. Purified peptides (0.5 nmol) were pipetted into wells produced in agarose-glucose plates preseeded with corresponding organisms. The plates were buffered with MES (pH 5.5 or 6.5) or PIPES (pH 7.5). After incubation, antimicrobial activity was assessed by measurement of the diameters of the clear zones of inhibition (in millimeters). ▵, FP-A; ▪, FP-B; □, Tβ-4; ▾, PBP; ▿, CTAP-3; •, RANTES; ○, PF-4.

FIG. 4.

Dose-dependent antimicrobial activities of HPAPs. Purified peptides FP-A (A), FP-B (B), Tβ-4 (C), PBP (D), CTAP-3 (E), RANTES (F), and PF-4 (G) in the concentration ranges indicated were loaded into wells of MES-buffered (pH 5.5) glucose-agarose plates preseeded with specific organisms. ○, E. coli ML35; •, S. aureus 502A; ▿, C. albicans 16820; ▾, C. neoformans 271A. Other conditions were identical to those described for Fig. 3.

FIG. 5.

Microbicidal activity of PF-4, CTAP-3, PBP, and Tβ-4. Two million CFU of the indicated organism per milliliter was incubated with 0 to 20 nmol of peptide per ml in 50 μl (final volume) of MES buffer (2 mM, pH 5.5) for PF-4 (A and B), CTAP-3 (C and D), and PBP (E and F) or PIPES buffer (10 mM, pH 7.50) for Tβ-4 (G and H). After 1.0 h of incubation at 37°C, samples of each incubation were serially diluted in 10 mM sodium phosphate buffer (pH 7.4) and plated in duplicate on nutrient agar plates. Surviving CFU were enumerated following incubation (24 to 48 h) at 37°C. ○, E. coli ML35; •, S. aureus 502A; ▿, C. albicans 16820; ▾, C. neoformans 271A.

FIG. 6.

Microbicidal activity of PF-4 and CTAP-3 in combination. Assay conditions were identical to those described for Fig. 5A, except that sodium acetate buffer (2 mM, pH 5.5) was used. Dose-dependent killing of E. coli by PF-4 (○) and CTAP-3 (•) and the synergistic effect of CTAP-3 and PF-4 (▾) are shown. Synergy was demonstrated in incubations containing increasing concentrations of CTAP-3 supplemented with 0.1 nmol of PF-4 per ml. Each data point represents the mean of two independent experiments.