This preclinical evaluation of a prohemostatic agent involved patients who received aspirin and clopidogrel before coronary artery stenting, the use of a humanized animal model to assess the hemostatic properties of patient platelets, as well as microfluidic assays to measure platelet reactivity. We demonstrate that our investigational product can bypass the effects of dual antiplatelet therapy (DAPT) by generating thrombin at sites of vascular injury, thereby restoring the hemostatic properties of patient platelets. Importantly, its effects could be reversed upon administration of a thrombin inhibitor. Thus, this product offers a titratable and reversible strategy for the management of defective hemostasis associated with DAPT.

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; 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.

BACKGROUND Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. METHODS Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. RESULTS Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. CONCLUSION Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.