(A) Schematic diagram of transgene construct and insertion location. Construct of the 2bF8 cassette (from 5′ to 3′): the human GPIIb gene promoter (αIIb^pr), chimeric intron, hBDDFVIII, SV40 poly A, and neomycin resistance. Insertion of 2bF8 transgene was in the B1 band of chromosome 18. (B) PCR analysis showed that 2bF8 transgene was detected in 2bF8^trans and FVIII^null mice after receiving BMT from 2bF8^trans mouse and 3 weeks’ reconstitution. Lane 1, DNA marker; lane 2, WT; lane 3, FVIII^null; lane 4, 2bF8^trans; lane 5, FVIII^null following BMT (recipient); lane 6, H₂O; lane 7, 2bF8 vector. DNA was purified from peripheral blood, and transgene was amplified with primers P1 and P2 (left panel; see Table 1). Mouse FVIII exon 4 (mFVIII), which is not disrupted in FVIII^null mice, was amplified as an internal control (right panel).

(A–F) Localization of transgene protein expression was determined by immunofluorescent microscopy. Isolated platelets from FVIII^null (A–C) and 2bF8^trans mice (D–F) were immunostained for either human FVIII (hFVIII; A and D) or mouse VWF (B and E). The 2 images were merged in C and F, showing that in the platelets of 2bF8^trans mice (F) VWF and FVIII were colocalized (yellow). (G–K) Colocalization of human FVIII and mouse VWF was confirmed by electron microscopy. Isolated platelets from FVIII^null mice (G and H) and 2bF8^trans mice (I–K) were immunostained for human and mouse VWF. FVIII was probed with 5 nm colloidal gold and VWF with 10 nm colloidal gold; representative gold particles are indicated by arrows and arrowheads, respectively. The results show FVIII was stored together with VWF in platelet α-granules of 2bF8^trans mice (I–K). (L–Q) Confocal microscopy detected no human FVIII in mononuclear cells from transgenic mice. Isolated platelets and mononuclear cells from 2bF8^trans mice were immunostained for human FVIII. Nonspecific isotype-matched primary Ab was used for background staining. (R) Quantitative evaluation of FVIII:C levels in mouse mononuclear cell lysates. Platelets and mononuclear cells were isolated and lysed in 0.5% CHAPS. FVIII:C in lysates was determined by chromogenic assay. No FVIII:C was detected in mononuclear cell lysates from either 2bF8^trans or control mice (i.e., WT and FVIII^null mice). FVIII:C was detected in platelet lysates from 2bF8^trans mice. Scale bars: 8 μm (A–F and L–Q); 0.2 μm (G–K).

(A) Quantitative evaluation of FVIII:C levels in mouse plasma by chromogenic assay. No FVIII:C was detected in the plasma of FVIII^null and 2bF8^trans mice. (B) Platelet lysate added to dilutions of FVIII did not alter level of FVIII:C as measured by chromogenic assay. (C) Quantitative evaluation of FVIII:C levels in mouse platelet lysates. Platelets were isolated and lysed in 0.5% CHAPS. FVIII:C in lysates was quantitated by chromogenic assay. FVIII:C was detected in platelet lysates from 2bF8^trans mice and bone marrow recipients from 2bF8^tg+/– mice. Platelet FVIII:C was significantly increased in 2bF8^tg+/+ mice compared with 2bF8^tg+/– mice. FVIII:C was significantly decreased in 2bF8^transVWF^null mice. *P < 0.001. (D) Tail clip survival test assessing phenotypic correction of hemophilia A mice. All 2bF8^trans mice and their bone marrow recipients survived tail clipping. (E) Tail clip survival test assessing the beneficial effect of transgenic platelets. Different proportions of washed platelets were transfused into FVIII^null mice, which was followed by tail clipping. All FVIII^null mice that were infused with 2bF8^tg+/– platelets to a final level of 30% of total platelets survived tail clipping.

(A) Dose response to rhBDDFVIII infusion in FVIII^null mice. When rhBDDFVIII was infused into FVIII^null mice and tail clip was performed, all FVIII^null mice survived tail clipping when infused to a final level of 0.02 U/ml rhBDDFVIII. When rhBDDFVIII was infused at the same concentration (0.02 U/ml) into FVIII^null mice that had been pre-injected with mouse inhibitor plasma, no FVIII^null mice with 25 or 250 BU/ml survived tail clipping. (B) Dose response to FVIII inhibitory antibody infusion in 2bF8^trans mice. Different doses of inhibitor plasma from immunized FVIII^null mice were infused into 2bF8^tg+/– mice. All mice survived tail clipping when infused with less than 25 BU/ml inhibitory plasma, and 7 of 8 survived with a titer of 250 BU/ml. (C) Tail clip survival test on the recipients of spleen cell transplantation. Sublethally irradiated 2bF8^tg+/– or WT mice received spleen cells from immunized FVIII^null mice. Two weeks after transplantation, plasma was tested by inhibitor analysis and the tail clip survival test was performed. Transgenic 2bF8^trans mice demonstrated enhanced tail clip survival even in the presence of a titer greater than 5,000 BU/ml. (D) Tail clip survival test of immunized 2bF8^trans mice. Heterozygous 2bF8^tg+/– mice were immunized with rhBDDFVIII (with adjuvant) by i.p. injection once. Two weeks after immunization, plasma was used for inhibitor quantitation and the tail clip survival test was performed. All immunized 2bF8^trans mice survived tail clipping with a titer of 10–50 BU/ml.