Traumatic non-compressible hemorrhage is a leading cause of civilian and military mortality and its treatment requires massive transfusion of blood components, especially platelets. However, in austere civilian and battlefield locations, access to platelets is highly challenging due to limited supply and portability, high risk of bacterial contamination and short shelf-life. To resolve this, we have developed an I.V.-administrable ‘synthetic platelet’ nanoconstruct (SynthoPlate), that can mimic and amplify body’s natural hemostatic mechanisms specifically at the bleeding site while maintaining systemic safety. Previously we have reported the detailed biochemical and hemostatic characterization of SynthoPlate in a non-trauma tail-bleeding model in mice. Building on this, here we sought to evaluate the hemostatic ability of SynthoPlate in emergency administration within the ‘golden hour’ following traumatic hemorrhagic injury in the femoral artery, in a pig model. We first characterized the storage stability and post-sterilization biofunctionality of SynthoPlate in vitro. The nanoconstructs were then I.V.-administered to pigs and their systemic safety and biodistribution were characterized. Subsequently we demonstrated that, following femoral artery injury, bolus administration of SynthoPlate could reduce blood loss, stabilize blood pressure and significantly improve survival. Our results indicate substantial promise of SynthoPlate as a viable platelet surrogate for emergency management of traumatic bleeding.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Background Trauma‐associated hemorrhage and coagulopathy remain leading causes of mortality. Such coagulopathy often leads to a hyperfibrinolytic phenotype where hemostatic clots become unstable because of upregulated tissue plasminogen activator (tPA) activity. Tranexamic acid (TXA), a synthetic inhibitor of tPA, has emerged as a promising drug to mitigate fibrinolysis. TXA is US Food and Drug Administration‐approved for treating heavy menstrual and postpartum bleeding, and has shown promise in trauma treatment. However, emerging reports also implicate TXA for off‐target systemic coagulopathy, thromboembolic complications, and neuropathy. Objective We hypothesized that targeted delivery of TXA to traumatic injury site can enable its clot‐stabilizing action site‐selectively, to improve hemostasis and survival while avoiding off‐target effects. To test this, we used liposomes as a model delivery vehicle, decorated their surface with a fibrinogen‐mimetic peptide for anchorage to active platelets within trauma‐associated clots, and encapsulated TXA within them. Methods The TXA‐loaded trauma‐targeted nanovesicles (T‐tNVs) were evaluated in vitro in rat blood, and then in vivo in a liver trauma model in rats. TXA‐loaded control (untargeted) nanovesicles (TNVs), free TXA, or saline were studied as comparison groups. Results Our studies show that in vitro, the T‐tNVs could resist lysis in tPA‐spiked rat blood. In vivo, T‐tNVs maintained systemic safety, significantly reduced blood loss and improved survival in the rat liver hemorrhage model. Postmortem evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma‐targeted activity of the T‐tNVs. Conclusion Overall, the studies establish the potential of targeted TXA delivery for safe injury site‐selective enhancement and stabilization of hemostatic clots to improve survival in trauma.

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Severe hemorrhage associated with trauma, surgery, and congenital or drug-induced coagulopathies can be life-threatening and requires rapid hemostatic management via topical, intracavitary, or intravenous routes. For injuries that are not easily accessible externally, intravenous hemostatic approaches are needed. The clinical gold standard for this is transfusion of blood products, but due to donor dependence, specialized storage requirements, high risk of contamination, and short shelf life, blood product use faces significant challenges. Consequently, recent research efforts are being focused on designing biosynthetic intravenous hemostats, using intravenous nanoparticles and polymer systems. Here we report on the design and evaluation of thrombin-loaded injury-site-targeted lipid nanoparticles (t-TLNPs) that can specifically localize at an injury site via platelet-mimetic anchorage to the von Willebrand factor (vWF) and collagen and directly release thrombin via diffusion and phospholipase-triggered particle destabilization, which can locally augment fibrin generation from fibrinogen for hemostatic action. We evaluated t-TLNPs in vitro in human blood and plasma, where hemostatic defects were created by platelet depletion and anticoagulation. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM)based studies of clot viscoelasticity, and BioFlux-based real-time imaging of fibrin generation under simulated vascular flow conditions confirmed that t-TLNPs can restore fibrin in hemostatic dysfunction settings. Finally, the in vivo feasibility of t-TLNPs was tested by prophylactic administration in a tail-clip model and emergency administration in a liver-laceration model in mice with induced hemostatic defects. Treatment with t-TLNPs was able to significantly reduce bleeding in both models. Our studies demonstrate an intravenous nanomedicine continued...

Among blood components, platelets (PLTs) present the toughest logistic challenges in transfusion due to limited availability, difficult portability and storage, high contamination risks, and very short shelf life (approx. 5 days).

Non-compressible uncontrolled hemorrhage remains a major cause of mortality from traumatic injuries. Additionally, patients with congenital, disease-associated or drug-induced hemostatic dysfunctions, may often be at risk of excessive bleeding. Therefore, treatments that render rapid hemostasis are clinically significant in potentially saving lives. The clinical gold standard for this is the transfusion of whole blood (WB) or blood components (e.g. controlled ratios of platelets, RBCs, and plasma), as evidenced by several clinical studies (e.g. PROPPR, PROMMTT and PAMPer). However, the availability of such blood products is donor-dependent, their shelf-life is limited due to contamination risks, and, their portability and storage is often challenging. While extensive research efforts are currently being focused on addressing these challenges, e.g. using low titer Group O whole blood, cold-storage and freeze-drying of platelets and plasma, in vitro generation of platelets from iPSCs etc., a parallel research focus has emerged in designing biomaterials-based I.V.-administrable technologies (nanoparticles, polymers etc.) that can provide specific functional attributes of hemostasis while allowing donor-independent manufacturing, scale-up, and on-demand availability. Prominent examples of these are 'synthetic platelet' (SynthoPlate) nanoparticles that recapitulate platelet's binding interactions with von Willebrand Factor (vWF), collagen and active platelet integrin GPIIb-IIIa, flexible platelet-like particles (PLP) that bind fibrin to recapitulate platelet's biomechanical properties, fibrinogen function-mimicking nanoparticles that amplify the aggregation of active platelets, peptide-modified synthetic polymers (e.g. PolySTAT, HAPPI etc.) that render clot stabilization etc. In this framework, we present the design and evaluation of I.V.-administrable unique platelet-inspired nanoparticles that render injury site-targeted, enzyme-responsive direct delivery of thrombin, to site-specifically augment fibrin generation for hemostasis. Our design is inspired by platelets' crucial hemostatic mechanisms of : (i) rapidly accumulating at the injury site to form a plug and (ii) serving as a coagulation amplifier via presenting anionic phospholipids on the activated platelet surface to render tenase and prothrombinase factor assemblies leading to thrombin (FIIa) burst, which can then site-specifically convert fibrinogen to fibrin. Thrombin delivery to augment hemostasis is clinically well-accepted, as exemplified by products like Tisseel where thrombin and fibrinogen are co-delivered by syringe directly at wound site. Researchers have also studied thrombin-loaded topical dressings and topical administration of thrombin-loaded particles on wounds to mitigate bleeding, but these cannot be used intravenously. A recent interesting study has explored encapsulation of thrombin-loaded nanoparticles inside actual platelets with the idea of the particles being released (analogous to granule secretion) upon platelet activation, but this was only demonstrated in vitro because optimizing this complex strategy for consistent in vivo function may be challenging. Our approach circumvents these challenges by: (i) loading consistent amount of thrombin in I.V.-administrable lipid nanoparticles (LNPs), (ii) directly targeting the thrombin-loaded LNPs (TLNPs) to the injury site via specific binding to vWF and collagen, and (iii) releasing the loaded thrombin via particle destabilization by the action of injury site-specific enzyme phospholipase A2 for in situ fibrin production. We evaluated the TLNPs in vitro in human blood and plasma where hemostatic defects were created by platelet depletion and anticoagulant treatment. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM) based studies of clot characteristics and BioFlux microfluidics based real-time imaging of fibrin generation under simulated vascular flow conditions, confirmed the ability of TLNPs to restore fibrin generation in hemostatic dysfunction settings. Subsequently, the in vivo feasibility of these TLNPs was tested in a mouse tail-clip bleeding model where a combination of platelet depletion plus anticoagulant treatment was used to render significant hemostatic defect. TLNPs were able to effectively reduce tail-bleeding in mice. Figure 1 Figure 1. Disclosures Sen Gupta: Haima Therapeutics: Other: Co-founder, Patents & Royalties: US 9107845, US 9107963.