Platelet aggregation induced by serotype polysaccharides from Streptococcus mutans

Abstract

Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I(2) (PGI(2)) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI(2). RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.

FIG. 1.

S. mutans-induced rabbit platelet aggregation and inhibition of aggregation detected by aggregometry. Representative platelet aggre-gation responses to S. mutans strain GS-5, Xc, or Xc24R. The traces show the effects of different doses of bacteria on platelet aggregation. A short lag time (around 1.5 min) was detected in each response. Dose-dependent aggregation response in PRP from a rabbit to S. mutans strain GS-5 (A) at a dose of 2.3 × 10⁷ CFU (left panel), 5.8 × 10⁷ CFU (center panel), or 1.1 × 10⁸ CFU (right panel); to strain Xc (B) at a dose of 3.5 ×10⁷ CFU (left panel), 8.9 × 10⁷ CFU (center panel), or 1.2 × 10⁸ CFU (right panel); and to strain Xc24R (C) at a dose of 3.8 × 10⁷ CFU (left panel), 7.5 × 10⁷ CFU (center panel), or 1.5 × 10⁸ CFU (right panel).

FIG. 2.

(A) Plasma-dependent aggregation response in PPP from a rabbit to S. mutans strain GS-5 at a dose 7 × 10⁷ CFU. No aggregation was observed unless plasma was added (4 min later) to a concentration of 2% (left panel), 5% (middle panel), or 10% (right panel). (B) Essential role of anti-serotype c IgG on aggregation response in PPP from a rabbit to S. mutans strain Xc at a dose 10⁸ CFU. No aggregation was observed in PS after the addition of Xc (left panel) at a dose of 10⁸ CFU (labeled X) and dPPP (labeled d). But aggregation was induced by the addition of anti-serotype c IgG (62.5 μg/ml; labeled G). No aggregation was detected after the addition of dPPP and anti-serotype c IgG (center panel). No aggregation was observed when Xc24R (labeled R) was added in the presence of dPPP and anti-serotype c IgG (right panel).

FIG. 3.

Preadsorption of plasma induced aggregation in PPP. Representative traces of aggregation induced by S. mutans at different doses. (A) Strain Xc was preincubated with plasma, washed, and then added to PPP at a dose of 5.8 × 10⁷ CFU (left panel), 1.2 × 10⁸ CFU (left center panel), 2.4 × 10⁸ CFU (right center panel), or 4.7 × 10⁸ CFU (right panel). (B) Strain Xc24R, defective in the synthesis of RGPs, was preincubated with plasma, washed, and then added to PPP at a dose of 6.2 × 10⁷ CFU (left panel), 2.2 × 10⁸ CFU (center panel), or 4.1 × 10⁸ CFU (right panel).

FIG. 4.

Binding of S. mutans RGPs to rabbit platelets determined by flow cytometry. Cell wall polysaccharide extracts containing serotype c-specific RGPs were incubated with rabbit PPP and subsequently labeled with anti-serotype c-specific rabbit IgG. Mean fluorescence intensities are noted. (A) Unstimulated platelets only. (B) Background fluorescence (rabbit IgG and anti-rabbit FITC). (C through E) Cell wall polysaccharides were added to final concentrations of 0.5, 1.0, and 2.0 mg/ml, respectively.

FIG. 5.

Rabbit platelet shape changes induced by cell wall polysaccharides with and without RGPs. Rabbit platelets in PS preparations deprived of plasma components were incubated with cell wall polysaccharides extracted from S. mutans strain Xc (panels A and B), strain Xc24R (panels C and D) or ADP (panels E and F). Activated platelets, characterized by a shape change from discoid to irregular with multiple protrusions, were readily detectable in panel C.

FIG. 6.

Human platelet shape change induced by cell wall polysaccharides with and without RGPs. Human platelets in PS preparations deprived of plasma components were incubated with cell wall polysaccharides extracted from S. mutans strain Xc (panels A and B) or strain Xc24R (panels C and D). Unstimulated platelets exhibited a typical discoid shape with a uniform size (panels E and F). Panels B, D, and F show portions of the corresponding top panels at higher magnification.

FIG. 7.

Induction and inhibition of human platelet aggregation detected by aggregometry. (A) Representative platelet aggregation response from one donor to S. mutans strain Xc at a dose of 1.2 × 10⁸ CFU. A longer lag time (around 13 min) was detected. (B) The aggregation was inhibited completely by the addition of PGI₂. (C through E) Aggregation induced by RGPs at a concentration of 0.5 mg/ml from three donors. (F) Aggregation triggered by RGPs in panel E could be blocked completely by pretreatment with PGI₂.