Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release - PubMed (original) (raw)

Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release

Darin A Trier et al. Infect Immun. 2008 Dec.

Abstract

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of > or = 10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5'-phosphonucleotide derivative (a specific P2X(1) antagonist), and cangrelor (a specific P2Y(12) antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A(2), cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X(1)/P2Y(12) receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.

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Figures

FIG. 1.

FIG. 1.

Influence of platelet ratio on antistaphylococcal efficacy. Logarithmic values of platelet-to-staphylococcus ratios ranged from 4 (10,000:1) to −3 (1:1,000). The limit of assay detection was considered to be 5% ± 2.5% killing (41). Compared with the viability of controls not exposed to platelets, significant reductions in the viability of exposed bacteria occurred at platelet-to-S. aureus ratios of ≥10:1 (*, P ≤ 0.05). Platelet-to-bacterium ratios of 10,000:1 and 1,000:1 were not significantly different in antistaphylococcal efficacy. As indicated, the levels of killing of the initial S. aureus inoculum were significantly different for platelet-to-S. aureus ratios of log10 4 and log10 2 (P < 0.05) or log10 4 and log10 1 (P < 0.01).

FIG. 2.

FIG. 2.

Inhibition of platelet antistaphylococcal efficacy. As detailed in Materials and Methods, platelets (PLT) were preexposed to antagonists, washed, and mixed with S. aureus bacteria at a ratio of 1,000:1 (108 platelets:105 bacteria; found to yield >95% staphylocidal efficacy). Geometric means of data from a minimum of three independent experiments are shown. *, P of <0.05 versus platelets alone; ‡, P of <0.05 versus antagonist-exposed platelets.

FIG. 3.

FIG. 3.

Influence of plasma on platelet anti-S. aureus response antagonism. As detailed in Materials and Methods, platelets (PLT) were mixed with S. aureus bacteria at a ratio of 1,000:1. Geometric mean values reflect data from a minimum of three independent experiments. Consistent with other data presented herein (e.g., Fig. 2), PND and CNG inhibited the platelet antistaphylococcal response in the presence of plasma. *, P of <0.05 versus platelets alone.

FIG. 4.

FIG. 4.

Effects of platelet surface adhesin antagonists on antistaphylococcal responses. Platelets pretreated with anti-surface receptors were mixed with S. aureus bacteria at a ratio of 1,000:1 (platelet-to-bacterium ratio, log10 3). Geometric means from a minimum of three independent experiments using the PMP-susceptible strain ISP479C are shown. Consistent with other data presented in this study, exposure to platelets alone achieved a significant reduction in CFU (a decrease of log 1.43 ± 0.28 CFU equates to 96.7% ± 9.5% killing; *, P of <0.05 compared with the results for the control inoculum). In contrast, none of the adhesin receptor antagonists investigated significantly interfered with the antistaphylococcal responses compared with the response of platelets alone. NS, not statistically significantly different.

FIG. 5.

FIG. 5.

Release of PMPs in response to S. aureus. Comparative RP-HPLC chromatograms are shown for platelet (PLT) supernatants following exposure to S. aureus alone (A), PAP (P2Y1 ADP receptor antagonist) and S. aureus (B), or PND (P2X receptor-specific antagonist) and S. aureus (C). The predominant constituents liberated from platelets (PLTs) in response to S. aureus ISP479C (log platelet-to-organism ratio, 3) correspond to _N_-serine and _N_-aspartate versions of PMP-1 (A, peaks 1 and 2). Peaks 3 and 4 are consistent with platelet basic peptide and its derivative, connective tissue-activating peptide 3 (CTAP-3). This profile is characteristic of previously documented PMPs or PKs (37, 38, 42, 46). Arrows in panel C indicate the expected major PMP/PK elution peaks in response to S. aureus, compared with those in the absence of platelet inhibitors (A).

FIG. 6.

FIG. 6.

Model of platelet antistaphylococcal response mechanisms. As supported by current data, the model illustrates how platelets may be activated to respond in parallel pathways that promote staphylocidal efficacy. (A) At the cellular level, the interaction with S. aureus evokes distinct responses in resting platelets: the liberation (and putative processing) of PMPs and PKs, which exert direct microbicidal effects on the organism, and the secretion of adenosine nucleotides (ADP/ATP), triggering a recursive cascade for the activation of adjacent platelets. Note that inhibitors of the ADP/ATP platelet activation pathway preclude the platelet staphylocidal response. (B) Detailed aspects of the model at the molecular level are illustrated. The degradation of extracellular ADP by APY or the inhibition of P2X or P2Y12 adenosine nucleotide receptors by SUR (a general P2 inhibitor), PND (a high-affinity P2X1 inhibitor), or CNG (a high-affinity P2Y12 inhibitor) specifically prevents platelet (PLT) staphylocidal efficacy. In contrast, the antagonism of P2Y1, phospholipase C (PLC), TXA2, or COX pathways or the CD41, CD42b, or CD62P platelet adhesin receptor did not impede the staphylocidal responses of platelets. Thus, the antistaphylococcal efficacy of platelets involves a self-amplifying and recursive sense/response mechanism: (1) direct or indirect interactions of platelets and S. aureus (SA); (2) platelet activation, with autocrine or intercrine P2X1 or P2Y12 receptor-mediated signal transduction prompting granule mobilization; (3) the degranulation and liberation of ADP/ATP from δ-granules; (4) the deployment of direct antimicrobial effector molecules (PMPs and PKs) from α-granules; and (5) the adenosine nucleotide-mediated activation of adjacent platelets, with the ensuing amplification of antimicrobial responses. The observed pattern of relationships between platelet-to-S. aureus exposure ratios and staphylocidal efficacy is suggestive of a threshold platelet ratio necessary to sustain an intercrine platelet cascade required to achieve PMP/PK concentrations sufficient for staphylocidal efficacy.

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