Background: Hematological complications associated with prothrombotic events with extrapulmonary manifestations have been demonstrated in autopsies of patients affected by coronavirus disease 2019 (COVID-19). Based on the close relationship of coagulation and immune response, we hypothesized that hypercoagulability in COVID-19 could result from the activation of tissue factor (F3) and subsequent alterations in Activated Protein C (APC) signaling (Figure 1).

Aims: We aimed to identify changes in the expression of APC signaling network in liver, peripheral blood and nasal epithelium of COVID-19 patients that may contribute to local and systemic disarrangement of hemostasis.

Methods: For the expression of PROC and receptor genes public single-cell-RNA-sequencing datasets were analyzed from COVID-19 patients and healthy individuals, using the toolkit Scanpy 1.7.2 in Phyton.

Results: The key compounds of Protein C (PC) activation and signaling; PROCR, F2R, THBD, S1PR1 and PROC were downregulated in COVID-19 patients; a greater expression of F3 in all COVID-19 tissues analyzed and upregulation of AGTR1, NFKB1, PTPN1, THBS1, PTGS2, PLAU, SERPINE1 and F5 pro-inflammatory and procoagulant genes in the liver of COVID-19 patients compared to control (Figure 2B, E and G). The hepatocyte PROC expression was changed in COVID-19 patients from hepatocyte 4 ADH1B+ PCK1+ in healthy liver (Figure 2F) to hepatocyte 3 CYP2A6+ in the liver of COVID-19 patients (Figure 2A). The ACE2 expression was increased in all COVID-19 tissues (Figure 2B, E and G) overlapping the PROC expression in the epithelium (Figure 2D) and liver tissues (Figure 2A). There was a co-expression of ACE2, PROC, PROS1, RHOA, and RAC1 in ciliated cells of COVID-19 patients (Figure 2C-D).

Conclusion(s): The results provide evidence indicating a deficient synthesis and activation of PC and its receptors in COVID-19 patients that might contribute to a pronounced hypercoagulable state in response to endothelial COVID-19- related injury. Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Figure 1. APC signaling in the endothelial cell.

When thrombin binds to TM on endothelial cell surfaces, PC is converted to its active form -APC- and EPCR is a necessary factor for optimal PC activation. Furthermore, the colocalization of PAR-1, EPCR in caveolin-1-rich membrane microdomains specialized in the cell membrane is essential for PAR-1-mediated cytoprotective APC signaling. The anti-inflammatory and anticoagulant actions of APC occur mainly through PAR-1, which, when activated, propagates pro-inflammatory and anti-inflammatory responses, according to the protease that cleaved it. PAR1 is sensitive to proteolysis at its N-terminal exodomain by multiple plasma proteases, including thrombin, APC, plasmin, factor FVIIa, Factor Xa, and matrix metalloproteases. Cleavage of PAR-1 at the R41 / 46 sites at the N-terminal exodimine by APC initiates signaling, promoting structural reorganization of the receptor and coupling to the G protein complex -GRK-5- to phosphorylate the intracellular region of PAR-1 and recruitment of β-arrestin 2, which serves as a framework for cytoprotective signaling. This cascade initiates a series of intracellular signaling pathways, including an upregulation of SPHK1 activity and therefore S1P production, leading to activation of signaling through its S1PR1 receptor, which stimulates a marked increase in Rac1 activation to perform a protective barrier function by stabilizing the endothelial cytoskeleton and inhibiting RhoA signaling. The Tie2 receptor transmembrane has also been shown to be able to initiate downstream signaling pathways and maintain vascular integrity through direct APC binding, which results in increased ZO-1 phosphorylation of Akt via PI3K and ERK inhibition, contributing to increasing the integrity of the barrier. Protein S cofactor-linked APC can intervene at various points during the systemic response to infection. It exerts an antithrombotic effect by inactivating factors Va and VIIIa, limiting thrombin generation. Finally, PAR-3 cleavage by APC can initiate PAR1-dependent cytoprotective signaling in endothelial cells in the presence of EPCR. TM: Thrombomodulin; PC: Protein C; EPCR: Endothelial protein receptor; PAR-1: protease-activated receptor 1; SPHK1: sphingosine kinase – 1; S1P: sphingosine-1-phosphate; Tie2: transmembrane receptor tyrosine kinase; PAR-3: protease-activated receptor 3.

Figure 2. Expression of genes involved in APC signaling in Liver, Nasal epithelium and Peripheral mononuclear cells of COVID-19 patients compared to healthy individuals based on single-cell transcriptomics.

A. Matrix plot showing expression of genes related to APC signaling according to cell type in the liver of patients infected with SARS-CoV-2. B. Matrix plot showing expression of genes related to APC signaling according to cell type in the liver of patients infected with SARS-CoV-2. C. UMAP embedding of Nasal epithelial cells grouped into 18 clusters. D. Visualization of APC-related genes expression in Nasal epithelial cells UMAP embedding. E. Matrix plot showing expression of genes related to APC signaling in patients infected with SARS-CoV-2 vs uninfected individuals. F. Matrix plot showing expression of genes related to APC signaling according to cell type in a healthy liver. G. Matrix plot showing expression of genes related to APC signaling in patients with COVID-19 vs healthy individuals. The graphics were generated using the Scanpy toolkit in phyton.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/hypercoagulability-in-covid-19-and-its-putative-association-with-the-downregulation-of-protein-c-signalling/