Platelet-Inspired Intravenous Nanomedicine for Injury-Targeted Direct Delivery of Thrombin to Augment Hemostasis in Coagulopathies.

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 approach for injury-site-targeted direct delivery of thrombin to augment hemostasis.

Platelet-mimicking procoagulant nanoparticles augment hemostasis in animal models of 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.

Bioinspired artificial platelets: past, present and future.

Platelets are anucleate blood cells produced from megakaryocytes predominantly in the bone marrow and released into blood circulation at a healthy count of 150,000-400,00 per µL and circulation lifespan of 7-9 days. Platelets are the first responders at the site of vascular injury and bleeding, and participate in clot formation via injury site-specific primary mechanisms of adhesion, activation and aggregation to form a platelet plug, as well as secondary mechanisms of augmenting coagulation via thrombin amplification and fibrin generation. Platelets also secrete various granule contents that enhance these mechanisms for clot growth and stability. The resultant clot seals the injury site to stanch bleeding, a process termed as hemostasis. Due to this critical role, a reduction in platelet count or dysregulation in platelet function is associated with bleeding risks and hemorrhagic complications. These scenarios are often treated by prophylactic or emergency transfusion of platelets. However, platelet transfusions face significant challenges due to limited donor availability, difficult portability and storage, high bacterial contamination risks, and very short shelf life (~5-7 days). These are currently being addressed by a robust volume of research involving reduced temperature storage and pathogen reduction processes on donor platelets to improve shelf-life and reduce contamination, as well as bioreactor-based approaches to generate donor-independent platelets from stem cells in vitro. In parallel, a complementary research field has emerged that involves the design of artificial platelets utilizing biosynthetic particle constructs that functionally emulate various hemostatic mechanisms of platelets. Here, we provide a comprehensive review of the history and the current state-of-the-art artificial platelet approaches, along with discussing the translational opportunities and challenges.

Bioinspired artificial platelets for transfusion applications in traumatic hemorrhage.

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). Robust research efforts are being directed to develop biologic PLTs in vitro as well as design biosynthetic and artificial PLT technologies that can potentially resolve these challenges to allow adequate availability and timely transfusion to improve survival in trauma.

Trauma-targeted delivery of tranexamic acid improves hemostasis and survival in rat liver hemorrhage model.

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.

Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve 'golden hour' survival in a porcine model of traumatic arterial hemorrhage.

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.