M protein from Streptococcus pyogenes induces tissue factor expression and pro-coagulant activity in human monocytes

Abstract

Invasive infections caused by the important pathogen Streptococcus pyogenes are often associated with disturbed blood coagulation in the human host, and may in severe cases develop into the life-threatening condition disseminated intravascular coagulation. In this study, the addition of M1 protein to human blood or purified peripheral blood mononuclear cells led to a dose-dependent increase of pro-coagulant activity, which was mediated by an upregulation of tissue factor on monocytes. Analysis of the resulting clots by transmission electron microscopy revealed that the cells were covered with a fibrin network that seemed to originate from the cell surface. Taken together, the results imply an important role for M proteins in the induction of haemostatic disorders in invasive streptococcal infectious diseases.

Fig. 1.

Pro-coagulant activity of human blood in response to bacterial proteins. (a) Human blood was incubated overnight with M proteins of different serotypes (M1, M3, M5 and M49), protein H (pH), protein A (pA), protein G (pG), protein L (pL), PAB or LPS at a final dilution of 1 μg ml⁻¹. Samples were then washed, and 100 μl of cell suspension was added to 100 μl of pre-warmed, fresh human citrated plasma, recalcified with 100 μl CaCl₂. The time to form a clot was subsequently determined in a coagulometer and normalized to buffer-treated cells. (b) Human blood was treated with M1 protein (1 μg ml⁻¹), the corresponding concentration of the control preparation, or buffer alone. After an overnight incubation, samples were washed and analysed as above. The values express clotting times relative to the clotting time evoked by buffer-treated cells. Values below 1 indicate increased pro-coagulant activity. Bars represent means and sd from three different donors. Asterisks indicate statistically significant differences as compared to buffer-treated cells, if not otherwise indicated: *, P<0.05; **, P<0.01; ***, P<0.001.

Fig. 2.

M1 protein induces pro-coagulant activity in human blood. Human whole blood was treated with different concentrations of M1 protein. After 24 h incubation at 37 °C, the samples were washed twice in order to remove M1 protein and plasma components. Then 100 μl fresh, untreated, citrated human plasma was pre-incubated with 100 μl CaCl₂ at 37 °C for 60 s in a coagulometer. Washed blood cells (100 μl) were added and the time to form a clot was determined. LPS (100 ng ml⁻¹) served as a positive control. All samples were analysed in duplicates. The figure shows the mean±sd of three individual experiments. **, P<0.01; ***, P<0.001.

Fig. 3.

M1 protein induces pro-coagulant activity in human PBMCs. PBMCs were isolated from human blood and incubated with M1 protein at different concentrations or 100 ng LPS ml⁻¹ for 24 h at 37 °C. Cell suspensions (100 μl) were then added to 100 μl of pre-warmed citrated human plasma, recalcified with 100 μl CaCl₂, and the time to form a clot was measured. Values represent the mean±sd of three separate experiments, each done in duplicates. *, P<0.05 and **, P<0.01.

Fig. 4.

Pro-coagulant activity induced by M1 protein fragments. Human PBMCs (1×10⁶ ml⁻¹) were incubated with M1 protein (5 μg ml⁻¹) (130 nM), fragments A-S or S-C3 at equimolar concentrations (2.0 and 1.7 μg ml⁻¹, respectively), LPS (100 ng ml⁻¹) or buffer alone for 20 h at 37 °C, and the ability of the cell suspensions to induce clot formation was thereafter determined in a coagulometer. The figure shows the mean±sd of three individual experiments. Asterisks indicate statistically significant differences relative to buffer control cells: *, P<0.05; **, P<0.01. Fragments A-S and S-C3 are schematically depicted at the top.

Fig. 5.

Monocytes treated with M1 protein form a fibrin network on the cell surface. PBMCs untreated (b, c) or stimulated with M1 protein (1 μg ml⁻¹) (d, e) or fragment S-C3 (f, g) were incubated with human plasma in the absence (b, d, f) or presence (c, e, g) of Ca²⁺. Pelleted cells (b, d, f) and clots (c, e, g) were fixed, thin-sectioned, and examined by transmission electron microscopy as described in Methods. Monocytes that were not incubated with plasma served as control (a). Bar, 2 μm.