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. Author manuscript; available in PMC: 2013 Apr 24.
Published in final edited form as: Biochemistry. 2012 Apr 10;51(16):3451–3459. doi: 10.1021/bi300070z

Figure 2. Thrombin Cleavage of the Single Swap P3-P3′ FVIII mutants.

Figure 2

WT and the 372(P3-P3′)740 and 740(P3-P3′)372 FVIII variants (100 nM) were reacted with thrombin over a 25 min time course and samples were subjected to Western blotting using C5, R8B12, and 2D2 monoclonal antibodies for detection of A1, A2 and A3-C1-C2 subunits, respectively, as described in “Experimental Procedures.” (A) Representative blots for rates of A1 subunit generation (top) for reactions using 0.75 nM thrombin for WT and 740(P3-P3′)372 and 0.075 nM thrombin for 372(P3-P3′)740; (B) A2 subunit generation (center) using 0.75 nM thrombin for WT and 740(P3-P3′)372 and 0.075 nM thrombin for 372(P3-P3′)740; and (C) A3-C1-C2 subunit generation (bottom) using 0.13 nM thrombin for WT and variants. SC refers to single chain and LC to light chain.