In vivo murine studies demonstrate that neutrophil activation by anti-NAP2 antibodies contributes to vaccine-induced immune thrombocytopenia and thrombosis (VITT)

Background: VITT involves thrombocytopenia and thrombosis post-initial anti-SARS-CoV-2 adenoviral vaccination. Most patients are found to have platelet-activating antibodies to the chemokine, platelet factor 4 (PF4) in the absence of heparin. VITT antibodies differ from those in heparin-induced thrombocytopenia (HIT), in which PF4 is bound to heparin. Distinct epitope sites on PF4 for VITT and HIT antibodies were defined (PMID34233346). We noted that the VITT antigenic site is conserved in mouse (m) PF4, and in the platelet-specific chemokine neutrophil-activating peptide 2 (NAP2), both human and mouse. We observed that VITT antibodies bind strongly to NAP2. In an active patient with VITT, we found that VITT antibodies circulate as immune complexes containing either PF4 or NAP2. Importantly, VITT antibodies plus NAP2 activates platelets. Aim(s): We tested in a passive-immunization murine model with VITT antibodies the ability to induce neutrophil-endothelial activation as an indicator of a prothrombotic state and identify the chemokines involved. Method(s): We studied two systems: A femoral vein and a cremaster venule model, using confocal intravital imaging and labeled neutrophils. VITT antibodies were infused into mice transgenic for FcgammaRIIA and lacking PF4 (FcgammaRIIA+/mPF4-/-). Result(s): This led to an immediately reduced neutrophil rolling by ~80% (14-3 m/sec) (Figure 1A,B). Subsequent infusion of PF4 slowed neutrophil rolling by another ~80% (3-0.6 m/sec). In contrast, VITT antibodies did not slow neutrophil speed in FcgammaRIIA+/ mPF4-/-/ mNAP2-/-mice (Figure 1C). Conclusion(s): These data suggest that both NAP2 and PF4 contribute to thrombosis in VITT and may explain the pathogenesis of VITT in patients with no detectable anti-PF4 antibodies. VITT may be prothrombotic because it involves co-activation of neutrophils via NAP2 by way of CXCR2 and FcgammaRIIA. Targeting NAP2 pathobiology may enhance understanding of the pathogenesis of VITT and lead to new therapeutics.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3“-galloyl-4”,6“-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh, inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. [Formula presented] Disclosures: No relevant conflicts of interest to declare.