INTRODUCTION:A freeze-dried, platelet-derived hemostatic agent (FPH) was developed for acute hemorrhage. The canine product (cFPH) was developed for use in preclinical models supporting human product (hFPH) investigations. MATERIALS AND METHODS:A carotid artery bypass graft (CABG) study in dogs compared 3 dosages of cFPH to canine liquid stored platelets (cLSP) and vehicle (VEH) control groups. Histopathological analysis and blood loss assessments were completed. A separate ex-vivo synthetic graft study assessed thrombogenicity via blood from human and canine donors that was combined with species-specific FPH or apheresis platelets. Characterization of cFPH and hFPH included thrombin generation, total thrombus formation, and scanning electron microscopy. RESULTS:Blood loss was reduced in CABG dogs receiving standard of care (cLSP) or cFPH treatment compared to VEH control; a cFPH dose effect signal was observed. Further, cFPH dosing up to 5 × 109 cells/kg was not associated with increased mortality or occlusion of the anastomosis sites, and histopathologic evidence of off-target thrombosis was not detected. When passed through a synthetic graft (ex vivo), whole blood combined with species-specific FPH did not result in thrombosis beyond that of whole blood control. In vitro testing and imaging of cFPH and FPH were comparable. CONCLUSIONS:A single dose of cFPH or cLSP reduced blood loss in a pilot surgical study and was well tolerated with no related adverse events. Further, the hemostatic activity and characteristics of cFPH are comparable to that of hFPH, suggesting that research findings from the canine product are likely to inform the development of the human product.

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.

BACKGROUND:A safe and efficacious hemostatic product with a long shelf-life is needed to reduce mortality from hemorrhage due to trauma and improve surgical outcomes for persons with platelet deficiency or dysfunction. Thrombosomes, a trehalose-stabilized, leukoreduced, pooled blood group-O freeze-dried platelet-derived hemostatic (FPH) with a 3-year shelf-life, may satisfy this need. OBJECTIVES:To characterize the mechanism of action of FPH. METHODS:FPH's ability to adhere to collagen, aggregate with and without platelets, and form clots was evaluated in vitro. Nonobese diabetic-severe combined immunodeficiency mouse models were used to assess circulation persistence and hemostatic efficacy. RESULTS:FPH displays the morphology and surface proteins of activated platelets. FPH adheres to collagen, aggregates, and promotes clots, producing an insoluble fibrin mesh. FPH is rapidly cleared from circulation, has hemostatic efficacy comparable to apheresis platelets in a murine tail-cut, and acts in a dose-dependent manner. CONCLUSION:FPH is a first-in-class investigational treatment and shows strong potential as a hemostatic agent that is capable of binding exposed collagen, coaggregating with endogenous platelets, and promoting the coagulation cascade. These properties may be exploited to treat active platelet-related or diffuse vascular bleeding. FPH has the potential to fulfill a large unmet patient need as an acute hemostatic treatment in severe bleeding, such as surgery and trauma.

BACKGROUND:The current global pandemic has created unprecedented challenges in the blood supply network. Given the recent shortages, there must be a civilian plan for massively bleeding patients when there are no blood products on the shelf. Recognizing that the time to death in bleeding patients is less than 2 h, timely resupply from unaffected locations is not possible. One solution is to transfuse emergency untested whole blood (EUWB), similar to the extensive military experience fine-tuned over the last 19 years. While this concept is anathema in current civilian transfusion practice, it seems prudent to have a vetted plan in place.METHODS AND MATERIALS:During the early stages of the 2020 global pandemic, a multidisciplinary and international group of clinicians with broad experience in transfusion medicine communicated routinely. The result is a planning document that provides both background information and a high-level guide on how to emergently deliver EUWB for patients who would otherwise die of hemorrhage.RESULTS AND CONCLUSIONS:Similar plans have been utilized in remote locations, both on the battlefield and in civilian practice. The proposed recommendations are designed to provide high-level guidance for experienced blood bankers, transfusion experts, clinicians, and health authorities. Like with all emergency preparedness, it is always better to have a well-thought-out and trained plan in place, rather than trying to develop a hasty plan in the midst of a disaster. We need to prevent the potential for empty shelves and bleeding patients dying for lack of blood.

Controlling hemorrhage has been a focus of survival since man recognized that the loss of blood led to death. Papyri from 1600 BCE describe methods for hemorrhage control including; direct pressure, ligature and the use of sutures. Multiple studies have demonstrated the survival advantage of early transfusion of whole blood or red cells and plasma. The added survival impact of early transfusion of platelets was recently reported in a substudy of the prospective Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial. Early transfusion of platelets demonstrated a statistically significant survival benefit at 24 h and 30 days post-injury. [1] Platelet availability is limited due to the short shelf life (5-7 days) and storage requirements (room temperature with constant agitation). Providing platelets or platelet derived products for prehospital treatment and to rural and some urban hospitals is an unmet medical need. The interest in novel and alternative platelet products has grown over the past decade and the status of novel platelet products is presented herein. Development, approval, and distribution of hemostatically effective approved platelet products for prehospital use and routine stockage in rural and urban centers could significantly increase survival rates in bleeding patients.

BACKGROUND:Hemorrhage causes significant morbidity and mortality in people aged <65 years. A lyophilized platelet-derived hemostatic agent (Thrombosomes) demonstrated hemostatic efficacy in animal models. We report the results of the first safety trial of autologous Thrombosomes given to normal subjects. STUDY DESIGN AND METHODS:Ten subjects received autologous Thrombosomes prepared from their apheresis platelets, and five control subjects received a buffer solution. There were five cohorts, with three subjects per cohort (two in the Thrombosomes group and one in the control group). Doses escalated from 1/1,000 to 1/10 of a proposed efficacious dose. Cohorts 4 and 5 received the highest dose, but in Cohort 5, one-half the dose was infused 2 hours apart. Cohorts 1 through 3 were monitored for 42 days, Cohorts 4 and 5 were monitored for 60 days using hematology, coagulation, and chemistry assays and antibody testing. RESULTS:There were no serious adverse events (AEs) and no subject withdrawals. There were eight treatment-related AEs (TRAEs) in 5 of 15 subjects (33%) (four in the Thrombosomes group and one in the control group). Of four subjects receiving the highest doses, three had TRAEs. One had elevated D-dimer, prothrombin fragment 1 + 2, and white blood cell count (subject had concurrent upper respiratory tract infection); one had T-wave inversions in precordial leads V2 and V3 without elevated troponin or symptoms; and one had a platelet autoantibody without change in platelet count. All subjects' TRAEs resolved by Day 21. CONCLUSION:There were no serious AEs in this small study. Thrombosomes were considered safe at the doses assessed. Future, larger trials will be needed to further assess safety and efficacy.

INTRODUCTION:Lyophilization may facilitate production of a safe, portable, easily storable, and transportable source of platelets for bleeding patients. The objective of this study was to examine the impact of lyophilized human and porcine platelets in a swine liver injury model of nonsurgical hemorrhage.METHODS:Anesthetized pigs (40 kg) had a controlled 35% total blood volume bleed from the right jugular vein followed by cooling to 35°C and resuscitation with Ringer's lactate to achieve a 3:1 blood withdrawal resuscitation. Through a midline laparotomy, the liver was injured with two standardized 5 × 5-cm grids with lacerations 1 cm apart and 0.5 cm deep. After 2 min of uncontrolled hemorrhage, the animals were treated with placebo (n = 5), lyophilized human (n = 5, HP), or swine platelets (n = 5, SP). At 15 min, shed blood was calculated. The animals then underwent abdominal closure. At 48 h, the animals were killed for histopathologic evaluation of the lung, kidney, and heart.RESULTS:Intraoperative blood loss at 15 min was significantly higher in the HP arm (SP: 4.9 ± 2.9 mL/kg, HP: 12.3 ± 4.7 mL/kg, and control: 6.1 ± 2.5 mL/kg; P = 0.013). Mortality at 48 h was 20% in all three arms, due to uncontrolled intra-abdominal bleeding. At the time the animals were killed, SP animals had a significantly higher hematocrit (SP: 22.0% ± 3.0%, HP: 15.1% ± 4.9%, and control: 13.9% ± 0.6%; P = 0.026). No significant difference was found in platelet count (SP: 319.3 ± 62.1 × 10(3)/µL, HP:361.5 ± 133.6 × 10(3)/µL, and control: 242.7 ± 42.5 × 10(3)/µL; P = 0.259). Histopathology of kidneys, lungs, and heart demonstrated no evidence of thromboembolic complications.CONCLUSION:In this swine model of liver injury, human lyophilized platelets increased intraoperative blood loss. With the use of species-specific lyophilized platelets, however, this effect was abolished, with a decrease in blood loss at 48 h after injury.

BackgroundUncontrolled hemorrhage is responsible for ∼80% of the potentially survivable deaths in combat and over 40% of early mortality in the under 65 age group in the United States. Providing an easily used infusible hemostatic agent to first responders could significantly reduce these fatalities. We report on an infusible lyophilized platelet‐derived hemostatic agent stabilized with trehalose and polysucrose prior to and during lyophilization.Study Design and MethodsCharacterization included determining the particle population size range, surface marker expression GPIb, GPIIbIIIa, and Annexin V binding. Function was assessed by aggregation, thromboelastography, and thrombin generation. Pharmacokinetics, biodistribution, and immunogenicity established using Indium111 labeled Thrombosomes in healthy New Zealand white rabbits (NZWRs), efficacy in thrombocytopenic NZWR, and safety in NZWRs, canines, and nonhuman primates.ResultsThrombosomes retained GPIIbIIIa expression (98.71% ± 0.18 of the rehydrated particles), a reduced expression of GPIb (47.77% ± 6.65), and Annexin V binding (86.05% ± 2.65). Aggregation to all agonists except thrombin in buffer (78.15% ± 2.5) was <50%. Thrombin generation and thromboelastography results demonstrated a concentration gradient that was consistent from lot to lot. There were no observed adverse events in any safety study and blood loss was reduced by >80% in the thrombocytopenic ear bleed model.ConclusionOur in vitro characterization studies in conjunction with preclinical animal safety and efficacy studies demonstrated lot consistency in manufacturing, maintenance of hemostatic functions of Thrombosomes, safety at high dose concentrations, and the potential to provide an effective hemostatic agent at the site of injury.

Thrombosomes® (a freeze dried human platelet derived hemostatic agent) was developed by Cellphire Inc. to provide an infusible hemostat for the treatment of uncontrolled hemorrhage in military and civilian settings at all treatment levels. Thrombosomes® have been shown to adhere to damaged vascular tissue, recruit endogenous platelets from the circulation, form a stable clot and reduce bleeding in a thrombocytopenic animal model. Cellphire recently received FDA approval for a first in human exploratory IND clinical trial. This sub-therapeutic dose SAFETY trial is a result of applying a transitional medicine approach as much as possible to all phases of product development. One of the key elements of that process has been characterization of the product and transitioning from discovery type experiments and documentation to GLP and cGMP compliant methods. Successful transition requires a full understanding of the technology, the material being lyophilized, and the preclinical and clinical studies that will be needed for FDA approval. Early adoption of GLP compliant practices could accelerate development of promising therapeutics by reducing the number of experiments needed to validate a finding. A systematic approach, applying the translational medicine process, from discovery to human use for a freeze dried therapeutic will be offered. Data used in support if the exploratory IND will be used to illustrate the impact of using this approach. Source of funding: None declared. Conflict of interest: None declared. [email protected]