A Freeze-Dried Platelet-Derived Hemostat Decreases Bleeding in a Novel Murine Model of Type 1 Hermansky-Pudlak Syndrome

Introduction: Hermansky-Pudlak Syndrome (HPS) is a rare autosomal recessive, multi-genotype, vesicle trafficking disorder clinically presenting as oculocutaneous albinism, platelet delta granule deficiency with bleeding from birth and premature mortality from pulmonary fibrosis in three of the 11 genetic subtypes. Animal models that recapitulate all disease aspects are lacking. Platelet transfusions to treat bleeds are avoided because of alloantibody formation. A Freeze-Dried Platelet-Derived Hemostatic (FPH) is under clinical development to treat severe surgical bleeding (NCT05771831). Here, we developed a murine model of HPS-1 that recapitulates key clinical sequelae and assessed the ability of FPH to cease bleeding after surgical injury.

Methods: We generated a novel humanized HPS-1 model with a 16-base pair duplication (C57BL/6J-Hps1em16mal; ΔHps1^16dupB6) 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 ΔHps1^16dupB6 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 ΔHps1^16dup 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 ΔHps1^16dup 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 ΔHps1^16dupB6 platelets aggregated 59% and released 0.00 nmoles ATP. There was also significant reduction in the mean fluorescence of mepacrine uptake into platelets from ΔHps1^16dupB6 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 ΔHps1^16dupB6 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 ΔHps1^16dup 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 ΔHps1^16dupB6 mouse recapitulated the major clinical phenotypes of HPS-1 patients. The bleeding phenotype in ΔHps1^16dupB6 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.

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.