M type 1 and 3 group A streptococci stimulate tissue factor-mediated procoagulant activity in human monocytes and endothelial cells

Abstract

Streptococcal toxic shock syndrome (StrepTSS) is an invasive infection characterized by marked coagulopathy, multiple organ failure, and rapid tissue destruction and is strongly associated with M type 1 and 3 group A streptococci (GAS). Initiation of the coagulation cascade with formation of microvascular thrombi contributes to multiple organ failure in human cases of gram-negative bacteremia; however, little is known regarding the mechanism of coagulopathy in StrepTSS. Thus, we investigated the abilities of several strains of M type 1 and 3 GAS isolated from human cases of StrepTSS to stimulate production of tissue factor (TF), the principal initiator of coagulation in vivo. Washed, killed M type 1 and 3 GAS, but not M type 6 GAS, elicited high-level TF-mediated procoagulant activity from both isolated human monocytes and cultured human umbilical vein endothelial cells. M type 1 GAS consistently elicited higher levels of TF from monocytes than did M type 3 GAS. GAS-induced TF synthesis in monocytes did not correlate with production of tumor necrosis factor alpha or interleukin-8. Conversely, M type 3 GAS were consistently more potent than M type 1 GAS in stimulating endothelial cell TF synthesis. These results demonstrate that (i) M type 1 and 3 strains of GAS are potent inducers of TF synthesis, (ii) GAS-induced TF synthesis is not simply an epiphenomenon of cytokine generation, and (iii) induction of TF in endothelial cells and monocytes may be M type specific. In total, these findings suggest that a novel interaction between GAS and host cells contributes to the observed coagulopathy in StrepTSS.

FIG. 1.

M type 3 GAS stimulates endothelial cell PCA. The ability of GAS to elicit PCA in cultured HUVEC was investigated using a single-stage clotting assay. After cultures were grown as described in Materials and Methods, HUVEC were stimulated with 100 μl of heat-killed GAS at an A₆₅₀ of 0.5, 0.05, or 0.005 for 6 h. Strains tested were M type 1 (strains 96/004 and 94/059) or M type 3 GAS (strains 88/003, 94/017, and 93/040). LPS (5 μg/ml) served as a positive control. After washing and three freeze-thaw cycles, citrated plasma was added followed by CaCl₂. PCA (i.e., clot formation) was monitored by absorbance at 550 nm. Data shown are the absorbance values obtained 10 min after recalcification of the plasma. Data (means ± standard deviations [SD]) are representative of four experiments performed in duplicate. *, P < 0.001 by one-way analysis of variance with Tukey's test.

FIG. 2.

Endothelial cell-derived PCA induced by M type 3 GAS is that of TF. PCA in HUVEC was measured by a single-stage clotting assay as described in Materials and Methods. PCA in lysates of HUVEC stimulated for 1 or 6 h with washed, killed M type 3 GAS (A₆₅₀ = 0.5; strain 88/003) was measured in the presence of either normal plasma or factor VII-deficient plasma. Data are the means of duplicate wells (± SD) minus the mean values of unstimulated control wells.

FIG.3.

GAS elicit TF PCA and cytokine production in human peripheral blood monocytes. Human peripheral blood monocytes were stimulated for 6 h with either heat-killed M type 1 (strain 96/004) or type 3 (strain 88/003) GAS at an A₆₅₀ of 0.5, 0.05, or 0.005. LPS (5 μg/ml) served as a positive control. PCA in monocyte lysates (A) was measured in duplicate by a single-stage clotting assay as described in Materials and Methods. Data shown are the absorbance values at 10 min postrecalcification of plasma. The overlying monocyte culture medium was also tested for the presence of TNF-α (B) or IL-8 (C) by commercial ELISAs. These data (means of results from triplicate wells ± SD) are representative of three different experiments utilizing monocytes from different donors; a small but notable donor-dependent variation in TF and cytokine levels was observed (data not shown).

FIG. 4.

Monocyte-derived, GAS-stimulated PCA is that of TF. Monocytes were stimulated with heat-killed GAS of M type 1 (strain 96/004) and 3 (strain 88/003) for 6 h. Monocyte lysates were preincubated for 10 min at 37°C with either monoclonal anti-TF antibody (10 μg/ml) or an isotype-matched control antibody. Normal citrated plasma was added and then recalcified, and clotting was measured. Values depicted are those obtained 12 min after recalcification and represent means of results from triplicate wells ± SD.

FIG. 5.

Schematic of known interactions of GAS with the human coagulation system. Straight lines indicate pathways that contribute to coagulation; wavy lines indicate pathways that inhibit coagulation or contribute to fibrinolysis. (Interaction 1) M type 1 and 3 GAS stimulate TF production from endothelial cells (ECs) and monocytes (MOs) (this study). (Interaction 2) In experimental GAS necrotizing fasciitis induced by an M type 1 strain of GAS (23) and in humans with StrepTSS (22), Factor XII was decreased and the APTT was prolonged; however, the PT was normal and experimental animals displayed a hypercoagulable state (23). (Interaction 3) Streptokinase binds plasminogen and GAS M protein binds fibrinogen. This quaternary complex has potent plasminogen activator activity (15, 34). (Interaction 4) In a nonhuman primate model of M type 3 GAS necrotizing fasciitis and myonecrosis, animals had increased circulating levels of fibrin degradation products (FDP) (interaction 4a) and thrombin-antithrombin III (AT III) complexes (interaction 4b), indicating systemic activation of the coagulation system (28). (Interaction 5) Some strains of GAS, but notably not M type 1 or 3, bind protein S in plasma (30). (Interaction 6) Administration of TFPI in experimental animals with gram-negative bacteremia or in humans with sepsis has proven benefits; however, the efficacy of TFPI in StrepTSS remains to be determined.