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Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines substituted, and a subsequent APC cleavage diminished. Since the thrombotic potential of high plasma levels of FVIII has been described, we aimed to generate new factor VIII molecules where the APC cleavage was only modulated. The ultimate goal being to increase in vivo the FVIIIa half-life. Among the two APC cleavage sites, the sequence around the site 562 was the most conserved between species. This region was therefore chosen to be modified with a prior verification that the amino-acids to be modified were not described in any hemophilic phenotype. Subsequently, the six following mutations were realized in a BDD-FVIII cDNA: Q561N, G563A, N564D, N564A, N564Q and I566M as well as the controls R336I, R562K and R336I+R562K. The constructs were transiently expressed in BHK cells to determine the specific activities of the corresponding molecules. The mutant specific activities, as determined by one- and two-stage clotting assays, ranged from 40 to 94 % of the wild-type except for G563A that was almost inactive. CHO clones expressing FVIII molecules were obtained. The mutants and control FVIII molecules were produced, partially purified on heparin column and further analyzed. The comparative specific activities in a two-stage clotting assay were the following (+/− SD; n=6): BDD-FVIII 100 %, Q561N 105 % +/− 45, G563A 6 % +/− 6, N564D 50 % +/− 23, N564A 45 % +/− 21, N564Q 80 % +/− 26, I566M 100 % +/− 41. The one-stage clotting assay gave identical results than the two-stage assay for each mutant. The mutants were then activated by thrombin (1:1) and the occurrence of FVIII activity was monitored. A 20- to 25-fold increase in FVIII activity was measured within 2 minutes following the addition of thrombin for all mutants. The mutants, except for the inactive G563A, were then analyzed for their resistance to APC by three different assays: an APC resistance kit (Coatest, Chromogenix), an in vitro assay that measured APC sensitivity of FVIIIa and an immunoblot assay that visualized the cleavage efficiency of the A2 fragment. These three assays confirmed the APC resistance of the previously published R336I, R562K and R336I+R562K, as compared with wild-type FVIII. They also revealed that the mutants N564D, N564A and I566M behaved similarly to the wild-type FVIII whereas Q561N and N564Q mutants were partially resistant to APC. The APC resistance ratio were the following 2.3 +/− 0.3 for BDD-FVIII, 2.1 +/− 0.4 for N564Q, 1.7 +/− 0,1 for Q561N, 1.6 +/− 0.3 for R562K and 1.3 +/− 0.3 for R336I+R562K. The N564Q and Q561N mutants exhibited a profile intermediate between wild type FVIII and R562K regarding of the loss of FVIIIa activity that was confirmed on the immunoblot profile. In conclusion we have generated new factor VIII molecules that retained their full procoagulant function while possessing a reduced sensitivity to APC cleavage.