Session: 301. Platelets and Megakaryocytes: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Bleeding and Clotting, Translational Research, Platelet disorders, Diseases
Methods: We generated a novel humanized HPS-1 model with a 16-base pair duplication (C57BL/6J-Hps1em16mal; ΔHps116dupB6) utilizing genomic engineering to knock-in the most common HPS1 mutation, c.1472_1487dup16-bp (HPS1 16bpdup) found in HPS1 patients. Platelet function and the effects of FPH were assessed by electron microscopy, lumi-aggregometry, flow cytometry mepacrine uptake, and in mixed FPH: murine platelet agglutination. Traditional tail bleed transection assays and total blood loss were used to assess bleeding before and after infusion of FPH at 1.6x109 particles/kg relative to saline control. Student’s t-test was used to compare differences between mean treatment groups.
Results: The ΔHps116dupB6 model had hypopigmentation of the tail and ears and prolonged bleeding times, typical of other genetic mouse models of HPS. This model also had a pre-disposition to lung fibrosis, with foamy and enlarged Type II alveolar cells. Electron microscopy revealed absent dense granules in ΔHps116dup compared to background C57BL/6J wild-type (WT) mice. In response to 10 µg/mL collagen, the WT platelets aggregated 69% and released 1.36 nmoles of ATP, while the ΔHps116dup platelets aggregated 23% and released 0.00 nmoles ATP. In response to 80 µM PAR4-AP (which activates the murine thrombin receptor), WT platelets aggregated 71% and released 1.57 nmoles ATP, while the ΔHps116dupB6 platelets aggregated 59% and released 0.00 nmoles ATP. There was also significant reduction in the mean fluorescence of mepacrine uptake into platelets from ΔHps116dupB6 mice compared to WT (1520±108.0 vs. 3417±377.6, p<0.0001).
FPH decreased the shed blood volume relative to saline (0.204±0.226 vs. 0.651±0.191 mL, p=0.0008) as well as the bleeding time of the ΔHps116dupB6 mice (initial stop 2.86± 1.08 vs. 19.46±1.31 min, p= <0.0001; final stop 12.73±3.26 vs. 19.46±1.312 min, p= <0.0001). In vitro, FPH co-aggregated with ΔHps116dup mouse platelets in the presence of collagen and induced expression of the active form of murine GPIIB/IIIa as judged by antibody Jon/A staining.
Conclusions: Our novel ΔHps116dupB6 mouse recapitulated the major clinical phenotypes of HPS-1 patients. The bleeding phenotype in ΔHps116dupB6 was significantly corrected by infusion of the investigational product, FPH. The ability of FPH to cease bleeding was due, at least in part, to co-aggregation at the site of exposed collagen and expression of activated murine GPIIB/IIIa. Additional translational and clinical studies are required to ascertain if FPH can cease bleeding in Type 1 HPS patients.
Disclosures: Moskowitz: Cellphire: Current Employment, Current equity holder in private company. Booth: Cellphire: Current Employment, Current equity holder in private company. Brown: Cellphire: Current Employment. Gahl: Cellphire: Research Funding. Malicdan: Cellphire: Research Funding.
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