Fig. 6.
Fig. 6. Thrombus formation induced by S pyogenesimmobilized on different substrates. / (A) Schematic representation of the chimerical proteins used to complement ΔM5 streptococci. The IgA-binding region (indicated by “A”) of the M4 protein was replaced by the 2 B-repeats (indicated by “B”) from the M1 protein or the 4 B-repeats from the M5 protein. (Panel B) Different streptococcal strains (108 bacteria/mL) were immobilized on glass coverslips coated with human serum albumin (HSA) at 100 μg/mL. Strains pM5 and pM5ΔB are ΔM5 streptococci complemented with plasmids encoding M5 and M5ΔB, respectively; strains pM4, pM4/M5, and pM4/M1 are complemented, respectively, with plasmids encoding M4 or the chimerical proteins shown in panel A; M1 and M3 are wild-type strains expressing the corresponding M proteins. Blood was perfused at 340 s−1, and surface coverage by platelets and platelet thrombi was measured after 8 minutes on an area of 45 000 μm2. (C) Strains M5 or M5ΔB, or no bacteria in the control, were immobilized on coverslips coated with fibrinogen (Fg; 200 μg/mL) or fibronectin (Fn; 100 μg/mL). Blood from the same donor as in panel B was perfused with or without addition of the anti-FcγRIIA antibody IV.3 (20 μg/mL, incubated for 15 minutes). The total volume occupied by platelet thrombi on an area of 45 000 μm2 was measured after 10 minutes of perfusion at 1600 s−1. (D) Strain ΔΜ5, or no bacteria in the controls, was immobilized on coverslips coated with fibronectin (Fn; 100 μg/mL). Blood from the same donor as in panel B was perfused with or without addition of antibodies (100 μg/mL each) against α5 (BIIG2), β1 (P5D2), or αIIbβ3 (LJ-CP8). Surface coverage by platelets and thrombi was measured after 8 minutes of perfusion at 1000 s−1. The data are representative of 3 separate experiments. (E) Strain M5, or no bacteria in the control, was immobilized on extracellular matrix (ECM) deposited by endothelial cells. Other conditions are the same as those as described in the legend to panel C, with the exception that blood was perfused at the 2 indicated wall shear rates. Surface coverage was measured as described in the legend to panel B. Representative images of the surface are also shown.

Thrombus formation induced by S pyogenesimmobilized on different substrates.

(A) Schematic representation of the chimerical proteins used to complement ΔM5 streptococci. The IgA-binding region (indicated by “A”) of the M4 protein was replaced by the 2 B-repeats (indicated by “B”) from the M1 protein or the 4 B-repeats from the M5 protein. (Panel B) Different streptococcal strains (108 bacteria/mL) were immobilized on glass coverslips coated with human serum albumin (HSA) at 100 μg/mL. Strains pM5 and pM5ΔB are ΔM5 streptococci complemented with plasmids encoding M5 and M5ΔB, respectively; strains pM4, pM4/M5, and pM4/M1 are complemented, respectively, with plasmids encoding M4 or the chimerical proteins shown in panel A; M1 and M3 are wild-type strains expressing the corresponding M proteins. Blood was perfused at 340 s−1, and surface coverage by platelets and platelet thrombi was measured after 8 minutes on an area of 45 000 μm2. (C) Strains M5 or M5ΔB, or no bacteria in the control, were immobilized on coverslips coated with fibrinogen (Fg; 200 μg/mL) or fibronectin (Fn; 100 μg/mL). Blood from the same donor as in panel B was perfused with or without addition of the anti-FcγRIIA antibody IV.3 (20 μg/mL, incubated for 15 minutes). The total volume occupied by platelet thrombi on an area of 45 000 μm2 was measured after 10 minutes of perfusion at 1600 s−1. (D) Strain ΔΜ5, or no bacteria in the controls, was immobilized on coverslips coated with fibronectin (Fn; 100 μg/mL). Blood from the same donor as in panel B was perfused with or without addition of antibodies (100 μg/mL each) against α5 (BIIG2), β1 (P5D2), or αIIbβ3 (LJ-CP8). Surface coverage by platelets and thrombi was measured after 8 minutes of perfusion at 1000 s−1. The data are representative of 3 separate experiments. (E) Strain M5, or no bacteria in the control, was immobilized on extracellular matrix (ECM) deposited by endothelial cells. Other conditions are the same as those as described in the legend to panel C, with the exception that blood was perfused at the 2 indicated wall shear rates. Surface coverage was measured as described in the legend to panel B. Representative images of the surface are also shown.

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