Understanding Ectopically Expressed Factor VIII (F8) In Megakaryocytes: Implications for Optimum Platelet-Delivered F8 Activity for Gene Therapy

Abstract 2205

F8 ectopically expressed during megakaryopoiesis is stored in platelet (p) a-granules and released at sites of injury. This F8 is effective in hemostasis even in the presence of circulating inhibitors so that pF8 represents a novel strategy for therapy for patients with hemophilia A with inhibitors. However, cremaster laser injury studies showed that pF8 release from a-granules has a distinct temporal and spatial availability that leads to clot instability. We proposed that F8s, such as canine (c) B-domainless (B) F8, with greater specific activity than human (h) BF8, would prevent pF8 clot instability. We confirmed our thesis; however at the same time, pcBF8 levels were only ∼30% of phBF8 whether studied in transgenic mice models or by lentiviral/bone marrow transplantation (lenti/BMT) into F8^null mice. This was surprising because in baby hamster kidney cells, cBF8 was secreted into the media at three times greater than hBF8. This lower level in murine megakaryocytes (Megs) was not due to mRNA levels as shown by qRT-PCR analysis. Using cultured marrow from transgenic mice expressing hBF8 or cBF8, there was a marked decrease in relative number of Megs in both pF8s compared to wildtype (WT) Megs, with the pcBF8 cells showing a greater decrease (49 ± 5% for cBF8 Megs vs. 36 ± 8% for phBF8, p < 0.02). Cultured Megs from pcBF8 and phBF8 mice both showed increased numbers of small, low ploidy Megs, and this was again higher for pcBF8 Megs (58 ± 9% for cBF8 Megs vs. 32 ± 6% for phBF8, p < 0.02). TUNEL studies as an indication of apoptosis showed that Megs expressing either F8s showed significant increased apoptosis than WT Megs with pcBF8 showing 37 ± 22% vs. 19 ± 11% in phBF8. The above data show that pcBF8 is deleterious to Meg development and was supported by a retrospective analysis of lenti/BMT pF8 platelet counts where recipient mice platelets made up a higher % of recovered platelet counts (17% ± 3% for pcBF8 vs. 4 ± 1% for phBF8, p < 0.05). We then tested whether we can separate the greater specific activity affect of pcBF8 from its deleterious affect on megakaryopoiesis. Our group has previously shown that cBF8 may have increased stability because it is predominantly expressed as a single chain, likely involving an R1645H (RH) substitution at a conserved PACE/furin site in most F8 species. Lenti/BMT pF8 studies expressing phBF8RH showed that this pF8 was expressed at the same level in reconstituted mice as phBF8, but was more efficacious in several bleeding models, including near-normal hemostasis in the cremaster laser injury model in F8^null recipient mice, thus becoming the first lenti/BMT pF8-expressing F8^null mouse with near-normal hemostasis in a F8^null setting. Preliminary Meg count and apoptosis studies show that phBF8RH is no more deleterious to Megs than phBF8. Thus, our studies point out that F8 is deleterious to Megs with some species being more deleterious than others. This apoptosis limits F8 levels in Megs, and this deleterious effect can influence post-BMT outcome. We also present a model of how one can take advantage of a F8 variant that had a high specific activity while avoiding its low expression levels in Megs. Thus our studies provide important new insights into the biology of pF8 that may be important in developing this platelet-delivery strategy for the treatment of hemophilia A.