Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19.

BACKGROUND: COVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. OBJECTIVES: From the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. METHODS: We utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. RESULTS: Compared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. CONCLUSION: We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

NLRP3-Induced NETosis: A Potential Therapeutic Target for Ischemic Thrombotic Diseases?

Ischemic thrombotic disease, characterized by the formation of obstructive blood clots within arteries or veins, is a condition associated with life-threatening events, such as stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism. The conventional therapeutic strategy relies on treatments with anticoagulants that unfortunately pose an inherent risk of bleeding complications. These anticoagulants primarily target clotting factors, often overlooking upstream events, including the release of neutrophil extracellular traps (NETs). Neutrophils are integral components of the innate immune system, traditionally known for their role in combating pathogens through NET formation. Emerging evidence has now revealed that NETs contribute to a prothrombotic milieu by promoting platelet activation, increasing thrombin generation, and providing a scaffold for clot formation. Additionally, NET components enhance clot stability and resistance to fibrinolysis. Clinical and preclinical studies have underscored the mechanistic involvement of NETs in the pathogenesis of thrombotic complications, since the clots obtained from patients and experimental models consistently exhibit the presence of NETs. Given these insights, the inhibition of NETs or NET formation is emerging as a promising therapeutic approach for ischemic thrombotic diseases. Recent investigations also implicate a role for the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as a mediator of NETosis and thrombosis, suggesting that NLRP3 inhibition may also hold potential for mitigating thrombotic events. Therefore, future preclinical and clinical studies aimed at identifying and validating NLRP3 inhibition as a novel therapeutic intervention for thrombotic disorders are imperative.

A pH-tuned chitosan-PLGA nanocarrier for fluconazole delivery reduces toxicity and improves efficacy against resistant Candida.

Fluconazole (FLZ) is a broad-spectrum antifungal used against Candida infections. Candida auris displays resistance to FLZ. Drug nanocarriers composed of natural (chitosan, C) or synthetic polymers (polylactide co-glycolide, PLGA) show improved drug characteristics, efficacy and reduction in toxicity. Here, C-PLGA nanoparticles (110 nm) were synthesized by coacervation method and loaded with FLZ, achieving ~8-wt% drug loading. The nanoformulation displayed pH-tuned slow sustained drug release (83 %) up to 5 d, at pH 4, while 34 % release occurred at pH 7.0. Fluorescent-tagged C-PLGA-NPs were localized on the Candida cell wall/membrane as seen by confocal microscopy. This resulted in ~1.9-fold reduced efflux of R6G dye as compared to bare drug treatment in Candida albicans and resistant C. auris. The nanoformulation showed a significant 16- and 64-fold (p < 0.0001) enhanced antifungal activity (MIC 5 and 2.5 µg/ml) against C. albicans and C. auris, respectively, as compared to FLZ. The nanoformulation showed highly effective antifungal activity in-vivo against C. albicans and C. auris. Moreover, the nephrotoxicity and hepatotoxicity was negligible. Thus, PLGA NPs-mediated fluconazole delivery can contribute to increased drug efficacy and to reduce the problem of fungal resistance.

Design and synthesis of a new topical agent for halting blood loss rapidly: A multimodal chitosan-gelatin xerogel composite loaded with silica nanoparticles and calcium.

Uncontrolled hemorrhage often causes death during traumatic injuries and halting exsanguination topically is a challenge. Here, an efficient multimodal topical hemostat was developed by (i) ionically crosslinking chitosan and gelatin with sodium tripolyphosphate for (ii) fabricating a robust, highly porous xerogel by lyophilization having 86.7 % porosity, by micro-CT and large pores ∼30 µm by SEM (iii) incorporating 0.5 mg synthesized silica nanoparticles (SiNPs, 120 nm size, -22 mV charge) and 2.5 mM calcium in xerogel composite that was confirmed by FTIR analysis with peaks at 3372, 986 and 788 cm-1, respectively. XPS analysis displayed the presence of SiNPs (Si2p peak for silicon) and calcium (Ca2p1, Ca2p3 transition peaks) in the composite. Interestingly, in silico percolation simulation for composite revealed interlinked 800 µm long-conduits predicting excellent absorption capacity and validated experimentally (640 % of composite dry weight). The composite achieved >16-fold improved blood clotting in vitro than commercial Celox and Gauze through multimodal interaction of its components with RBCs and platelets. The composite displayed good platelet activation and thrombin generation activities. It displayed high compressive strength (2.45 MPa) and withstood pressure during application. Moreover, xerogel composite showed high biocompatibility. In vivo application of xerogel composite to lethal femoral artery injury in rats achieved hemostasis (2.5 min) significantly faster than commercial Celox (3.3 min) and Gauze (4.6 min) and was easily removed from the wound. The gamma irradiated composite was stable till 1.5 yr. Therefore, the xerogel composite has potential for application as a rapid topical hemostatic agent.