Analysis
of the activation of plasma-derived hPro by prothrombinase.
Plasma-derived hPro (1.4 μM) was incubated in different mixtures
with PCPS vesicles (20 μM), and prothrombinase was assembled
with either wild-type rhfVa (panel A, 20 nM) or rhfVaNR→DE (panel B, 20 nM), as described in the Experimental
Section. At selected time intervals, aliquots of the reactions
were withdrawn and treated as described in the Experimental
Section. M represents the lane with the molecular
weight markers (from top to bottom): Mr 98 000, Mr 64 000, Mr 50 000, Mr 36 000, Mr 22 000. Lanes
1–17 represent samples from the reaction mixture before (0
min) the addition of fXa and 20, 40, 60, 80, 100, 120, 140, 160, 180,
200, 220, 240 s, 5, 6, 10, and 20 min, respectively, following the
addition of fXa. The hPro-derived fragments are shown as follows:
Pro (hPro, amino acid residues 1–579); P1 (prethrombin-1, amino
acid residues 156–579); F1•2-A (fragment 1•2-A
chain, amino acid residues 1–320); F1•2 (fragment 1•2,
amino acid residues 1–271); P2 (prethrombin-2, amino acid residues
272–579); B (B chain of α-thrombin, amino acid residues
321–579). F1 (fragment 1, amino acid residues 1−155);
F2 (fragment 2, amino acid residues 156−271).