Time courses of APC-mediated FVa inactivation.

(A) Time course of FVa inactivation in the FV R506Q/Y1702C doubly heterozygous propositus. The inactivation end-point patterns of normal (N) and R506Q (L) FVa are reported for comparison. In the normal control, the FV heavy chain (HC) is cleaved by APC at Arg506 and Arg306, resulting in a 30-kd immunoreactive fragment (HC_307-506). In the FV R506Q homozygote, cleavage of the FV heavy chain does not generate the HC_307-506fragment, and a 60/54-kd doublet (HC_307-679/709) appears instead. The propositus' FV shows an inactivation pattern indistinguishable from that of the R506Q FV homozygote, which indicates that the Y1702C FV is not expressed in plasma. HC_IIa is a shorted heavy chain of FVa, due to the prolonged exposure of FVa to the thrombin generated following clot formation and characterized by considerably lower cofactor activity. It suggests a potentially alternative method for FVa inactivation in FVR506Q individuals.50 51 Lane 1, FVa in the presence of phospsholipid vesicles before adding APC; lanes 2 to 4, FVa 5, 10, and 20 minutes after the addition of APC. Molecular weights (kd) are reported on the left. (B) Time course of FVa inactivation in family members carrying the FV Y1702C mutation as a single heterozygous defect. The normal FVa inactivation pattern (presence of the 30-kd band and absence of the 60/54-kd doublet) indicates that the Y1702C FV molecules do not contribute to the altered FVa inactivation pattern (propositus, Figure 4A). Lane 1, FVa in the presence of phospholipid vesicles before adding APC; lanes 2 to 5, FVa 3, 5, 10, and 20 minutes after the addition of APC.