Effect of ptfV chimeric variants on prothrombin time and thrombin generation in fV-depleted human plasma. (A) Sequence alignment of the a1-loop regions of ptfV, hfV, and hfVIII with 4 flanking residues to either side is shown (* indicates sulfation). (B) Prothrombin times were determined in fV-depleted human plasma before and after spiking in the ptfV chimeric variants (ptfV_hfVa1 and ptfV_hfVIIIa1) and ptfV and B-domainless hfV as controls. (C) Thrombin generation was measured in a purified system using human prothrombin, ptfXa, and ptfV or the 2 chimeric variants by the chromogenic assay. Inset shows early time points between 0 and 5 minutes used in calculating initial rates. mAu, milli–absorbance unit.