Figure 2
Effect of the interaction between VWF and FH on FH cofactor activity, ADAMTS13-mediated VWF proteolysis, and VWF:RCo assay. (A-B) Cofactor activity of FH for CFI. FH (A, 0-32 nM; B, 13 nM), FI (17 nM), and C3b (8 nM) were incubated in the presence of VWF (A, 80 nM; B, 0-80 nM) or human serum albumin (HSA) (A, 80 nM) for 30 minutes at 37°C. The proteolysis of C3b into iC3b was analyzed by 10% SDS–polyacrylamide gel electrophoresis under reducing conditions using a goat anti-human C3 antibody. The presence of VWF enhanced FH cofactor activity resulting in increased cleavage of the C3b α′ chain into the α 65- and α 43-kDa fragments. (C) Effect of FH on ADAMTS13-mediated VWF proteolysis. Kinetics of digestion of VWF (10 nM) in the presence or absence of FH (645 nM) by recombinant ADAMTS13 (10 nM) in Tris-buffered saline. VWF digestion was carried out without shear stress and without denaturing agent. At selected times, the reaction was stopped by EDTA. The residual VWF was measured by a sandwich ELISA using a monoclonal antibody against the N-terminal site of VWF for capture and an anti–C-terminal antibody for detection. Plain curves represent the data fitted to the 1-phase exponential decay (r2 ≥ 0.87). Data depict means ± standard deviation of 3 independent experiments. (D) Distribution of VWF multimers. The samples recovered after 12 hours of incubation were separated by an SDS–2% agarose gel, transferred to a nitrocellulose membrane, and revealed using a polyclonal anti-VWF IgG coupled to horseradish peroxidase. The figure depicts the migration profile of VWF (10 nM) incubated alone, in the presence of ADAMTS13 (10 nM) with or without FH (645 nM). (E) Effect of FH on VWF:RCo assay. VWF (4 nM) incubated with purified FH (344 nM, 30 minutes at 37°C) and added on formol-fixed platelets in the presence of ristocetin. VWF:RCo was assessed using a commercial kit (Siemens) on an APAC4004 platelet aggregometer (Elitech). Percentage of VWF-mediated platelet aggregation was compared with normal human plasma. FI, complement factor I; RCo, ristocetin cofactor.

Effect of the interaction between VWF and FH on FH cofactor activity, ADAMTS13-mediated VWF proteolysis, and VWF:RCo assay. (A-B) Cofactor activity of FH for CFI. FH (A, 0-32 nM; B, 13 nM), FI (17 nM), and C3b (8 nM) were incubated in the presence of VWF (A, 80 nM; B, 0-80 nM) or human serum albumin (HSA) (A, 80 nM) for 30 minutes at 37°C. The proteolysis of C3b into iC3b was analyzed by 10% SDS–polyacrylamide gel electrophoresis under reducing conditions using a goat anti-human C3 antibody. The presence of VWF enhanced FH cofactor activity resulting in increased cleavage of the C3b α′ chain into the α 65- and α 43-kDa fragments. (C) Effect of FH on ADAMTS13-mediated VWF proteolysis. Kinetics of digestion of VWF (10 nM) in the presence or absence of FH (645 nM) by recombinant ADAMTS13 (10 nM) in Tris-buffered saline. VWF digestion was carried out without shear stress and without denaturing agent. At selected times, the reaction was stopped by EDTA. The residual VWF was measured by a sandwich ELISA using a monoclonal antibody against the N-terminal site of VWF for capture and an anti–C-terminal antibody for detection. Plain curves represent the data fitted to the 1-phase exponential decay (r2 ≥ 0.87). Data depict means ± standard deviation of 3 independent experiments. (D) Distribution of VWF multimers. The samples recovered after 12 hours of incubation were separated by an SDS–2% agarose gel, transferred to a nitrocellulose membrane, and revealed using a polyclonal anti-VWF IgG coupled to horseradish peroxidase. The figure depicts the migration profile of VWF (10 nM) incubated alone, in the presence of ADAMTS13 (10 nM) with or without FH (645 nM). (E) Effect of FH on VWF:RCo assay. VWF (4 nM) incubated with purified FH (344 nM, 30 minutes at 37°C) and added on formol-fixed platelets in the presence of ristocetin. VWF:RCo was assessed using a commercial kit (Siemens) on an APAC4004 platelet aggregometer (Elitech). Percentage of VWF-mediated platelet aggregation was compared with normal human plasma. FI, complement factor I; RCo, ristocetin cofactor.

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