Thrombosis and Bleeding in Patients with Vaccine-Induced Immune Thrombotic Thrombocytopenia: A Systematic Review of Published Cases.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic reaction to COVID-19 (coronavirus disease 2019) adenoviral vector vaccines. Its distinct bleeding and thrombotic patterns compared with other platelet consumptive disorders remain unclear. METHODS: We performed a systematic review of the literature (PubMed and Embase) up to July 31, 2022, including case reports and case series providing nonaggregate data of VITT patients. Accurate VITT diagnosis required fulfillment of the following criteria: (1) endorsement by the authors, (2) consistent vaccine type and timing, (3) presence of thrombocytopenia and thrombosis, (4) detection of anti-platelet factor 4 antibodies. Data are presented as frequencies with 95% confidence intervals (CIs) calculated with the exact binomial method. RESULTS: We retrieved 143 eligible studies, describing 366 patients. Of 647 thrombotic events, 53% (95% CI: 49-56) were venous thromboses at unusual sites and 30% (95% CI: 27-34) were cerebral venous sinus thromboses (CVSTs). The ratio of venous-to-arterial events was 4.1. Thromboses in most sites were associated with at least another thrombotic event, with the exception of CVST and CNS arterial thrombosis (isolated in 49 and 39% of cases, respectively). Bleeding occurred in 36% (95% CI: 31-41) of patients; 68% (95% CI: 59-75) of bleeding events were intracranial hemorrhages (ICHs). Overall mortality was 24% (95% CI: 19-29), and 77% (95% CI: 58-90) in patients with isolated CVST complicated by ICH. CONCLUSION: VITT displays a venous-to-arterial thrombosis ratio comparable to heparin-induced thrombocytopenia. However, VITT is characterized by a higher prevalence of CVST and ICH, which contribute to the increased bleeding frequency and mortality.

Proteolytic Activities of Enterovirus 2A Do Not Depend on Its Interaction with SETD3.

Enterovirus 2Apro is a protease that proteolytically processes the viral polyprotein and cleaves several host proteins to antagonize host responses during enteroviral infection. Recently, the host protein actin histidine methyltransferase SET domain containing 3 (SETD3) was identified to interact with 2Apro and to be essential for virus replication. The role of SETD3 and its interaction with 2Apro remain unclear. In this study, we investigated the potential involvement of SETD3 in several functions of 2Apro. For this, we introduced the 2Apro from coxsackievirus B3 (CVB3) in a mutant of encephalomyocarditis virus (EMCV) containing an inactivated Leader protein (EMCV-Lzn) that is unable to shut down host mRNA translation, to trigger nucleocytoplasmic transport disorder (NCTD), and to suppress stress granule (SG) formation and type I interferon (IFN) induction. Both in wt HeLa cells and in HeLa SETD3 knockout (SETD3KO) cells, the virus containing active 2Apro (EMCV-2Apro) efficiently cleaved eukaryotic translation initiation factor 4 gamma (eIF4G) to shut off host mRNA translation, cleaved nucleoporins to trigger NCTD, and actively suppressed SG formation and IFN gene transcription, arguing against a role of SETD3 in these 2Apro-mediated functions. Surprisingly, we observed that the catalytic activity of enteroviral 2A is not crucial for triggering NCTD, as a virus containing an inactive 2Apro (EMCV-2Am) induced NCTD in both wt and SETD3KO cells, albeit delayed, challenging the idea that the NCTD critically depends on nucleoporin cleavage by this protease. Taken together, our results do not support a role of SETD3 in the proteolytic activities of enterovirus 2Apro.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.