Engineered biomimetic platelet membrane-coated nanoparticles as platelet decoys to reduce coagulopathy and improve outcomes in sepsis

Abstract Introduction: Platelets (plt) play crucial roles not only in hemostasis but also in immune defense. Thrombocytopenia is common in sepsis and strongly correlates with poor outcomes. We previously engineered plt-membrane coated nanoparticles (PNP) that protect against murine systemic MRSA infection. This project aims to identify mechanisms by which PNP act as plt decoys to protect the host in sepsis, paving the way for translational therapeutic studies. Methods: Plt membrane was derived by freeze-thawing. Poly(lactic-co-glycolic acid) core, prepared by acetone/water nanoprecipitation, was added to plt membrane and sonicated to create PNP that retain surface characteristics of plt. Effects of PNP (600µg/mL) on plt aggregation in response to soluble agonists adenosine diphosphate (ADP; 5µg/mL), arachidonic acid (AA; 125µg/mL), thrombin (0.01U/mL), and a toxin (20µg/mL) were assessed by light transmission aggregometry. To assess PNP effects on plt killing of bacteria, plts were infected with Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), or methicillin-resistant Staphylococcus aureus (MRSA) (5x106 colony forming unit; CFU) ± PNP (60µg) for 1h at 37°C, followed by CFU enumeration. SA and MRSA cultures were incubated with PNP (1mg/mL) in human plasma for 24h at 37°C. Biofilm was stained with crystal violet and quantified at 595nm. To determine whether PNP block coagulase-induced consumptive coagulopathy, blood was infected with vehicle or MRSA (2x105CFU/mL) for 3h at 37°C. Plasma was isolated and re-calcified with 8.3mM CaCl2 to initiate fibrin formation, measured as change in turbidity at 405 nm. PNP effects on coagulation were assessed by incubating plasma with thrombin (0.01U/mL) or lipopolysaccharide (LPS; 200µg/mL) ± PNP (800µg/mL), followed by measuring fibrin generation. To determine PNP effects on coagulation enzyme activity, factors (F) XIIa (50nM), XIa (1nM), and Xa (5nM) were incubated with PNP (80µg/mL) for 30min at 4°C, followed by ultracentrifugation to pellet PNP. Residual enzyme activity was determined using enzyme-specific chromogenic substrates to measure rate of hydrolysis at 405nm. To determine whether PNP protect FXIa from inhibition by protease nexin-II (PN-II), PNP-bound FXIa activity was measured after incubation with PN-II (1µg/mL). PNP effects on thrombosis were assessed by treating wildtype (WT) mice intravenously (i.v) with vehicle or PNP (40mg/kg) for 15min, followed by i.v thromboplastin infusion to induce systemic thrombosis. Blood and organs were harvested at 1min for hematological and histological analyses. Circulating thrombin anti-thrombin (TAT) levels were measured by ELISA. PNP effects on sepsis-induced coagulopathy were studied by infecting WT mice i.v with PA(1x106CFU), followed by immediate treatment with vehicle or PNP (40mg/kg; i.v). At 16h post infection, blood and organs were harvested for hematological and histological analyses and TAT ELISA. Results: PNP reduced plt aggregation induced by ADP, AA, thrombin, and a toxin by 50%, while enhancing plt killing of both Gram- (by 40%) and Gram+ bacteria (by 50%; eliminating SA and MRSA growth). PNP reduced SA and MRSA biofilm formation by 75%, and blocked MRSA-induced consumptive coagulopathy in whole blood, suggesting that PNP may protect plt during infection by absorbing pro-coagulant proteins and bacterial toxins so that plt are less activated, survive longer, and have greater antibacterial activity. PNP interfered with thrombin- and LPS-induced plasma clotting, suggesting that PNP also interact with components of the coagulation cascade. We found that PNP selectively bound FXIIa and FXIa but not FXa. Unlike activated plt, PNP did not enhance FXIIa and FXIa enzymatic activity or protect FXIa from inhibition by PN-II. Murine thromboplastin infusion induced thrombocytopenia, leukopenia, and increased TAT levels, which were alleviated by PNP treatment. PA-infected mice treated with PNP exhibited reduced sepsis severity and lower plasma TAT levels, suggesting that PNP exerted antithrombotic effects that improved sepsis outcomes. Conclusion: We engineered PNP as plt decoys to harness and deploy biomimetic natural host cell membranes to counteract processes that drive severe pathology due to bacterial infection. Our findings suggest a conserved mechanism of PNP action targetting core thrombotic pathways in sepsis that may be effective against many different microbes independent of their antimicrobial susceptibility.