Recent research has proposed several host factors required for SARS-CoV-2 infection and involved in the inflammatory response. Among these, members of the human serpin family and PAR2 have been suggested to play a relevant role. As it has been shown that one of the multiple activities of protease inhibitor SerpinB3 is the activation of PAR2, we have modulated the expression of these two molecules on both human bronchial and hepatic cells and assessed cell surface Spike binding and SARS-CoV-2 infectivity. Our findings indicate that both SerpinB3 and PAR2 play a pivotal role in viral infection and downregulate the expression of interferon-γ, a cytokine with a well-known antiviral effect. These results underscore the potential of the SerpinB3-PAR2 axis as a target for antiviral therapy and provide support for addressing serpins as targets for this purpose.

A T cell-inflamed tumor microenvironment is predictive of better prognosis and clinical response to immunotherapy. Proteinase-activated receptor 2 (PAR2), a member of G-protein coupled receptors is involved in inflammatory process and the progression of various cancers. However, the role of PAR2 in modulating the tumor microenvironment remains unclear. Here, we found that PAR2 high-expression was associated with a favorable prognosis in patients with colorectal cancer. Intriguingly, PAR2 expression in human colorectal cancer was mainly confined to tumor cells and was significantly associated with CD8+ T cell infiltration. Tumor-intrinsic PAR2 deficiency blunted antitumor immune responses to promote tumor growth and attenuated the therapeutic efficacy of anti-PD1 in a mouse model of colon cancer. Tumors with downregulated PAR2 showed decreased CD8+ T cell infiltration and impaired effector function. Mechanistically, PAR2 activation in tumor cells induced CXCL9 and CXCL10 production via PI3K/AKT/mTOR signaling, thereby enhancing CD8+ T cell recruitment in the tumor microenvironment. In addition, PAR2 was essential for dendritic cell activation and differentiation towards conventional type 1 subset. PAR2 deficiency in dendritic cells markedly impaired their ability to prime CD8+ T cells and control tumor growth in vivo. Thus, our findings identify new roles for PAR2 in promoting antitumor immunity and provide a promising target to improve immunotherapy efficacy in colorectal cancer.

Colorectal cancer (CRC), the third most prevalent cancer globally, exhibits a notable association with venous thromboembolism (VTE), significantly impacting patient morbidity and mortality. We delve into the complex pathogenesis of cancer-associated thrombosis (CAT) in CRC, highlighting the interplay of clinical risk factors and tumor-specific mechanisms. Our comprehensive review synthesizes the current understanding of CRC's pro-thrombotic tendencies, examining both general clinical factors (e.g., age, gender, obesity, prior VTE history) and tumor-specific aspects (e.g., tumor location, stage, targeted therapies). Key findings illustrate how CRC cells themselves actively contribute to coagulation cascade activation through various procoagulant elements such as tissue factor, cancer procoagulant, and extracellular vesicles. We also explore how CRC influences host cells to adopt a procoagulant phenotype, thereby exacerbating thrombotic risks. This review underscores the role of genetic mutations in CRC (e.g., KRAS, p53) in modulating coagulation-related protein expression and thrombosis risks. An in-depth understanding of the genetic landscape specific to CRC subtypes is essential for developing targeted anticoagulation strategies and could significantly advance thrombosis prevention while improving the overall management of patients with CRC. This highlights the urgent need for precision in addressing CAT within clinical settings.

The coagulation system is a mechanism for wound healing after injury, but it also participates in host early immune defense. The coagulation factor Ⅶ (FⅦ) can initiate extrinsic pathway and play an important role in the coagulation process. However, studies of the immune function of FVII are scarce, especially in fish. In this study, we cloned and characterized an FⅦ-like gene from large yellow croaker (Larimichthys crocea) (LcFⅦL). The open reading frame of LcFⅦL consists of 1437 base pairs and encodes 478 amino acid residues. LcFⅦL contains conserved domains that are present in other vertebrate FⅦs or FⅦLs, including a prepropeptide, a gamma-carboxy glutamic acid domain, two epidermal growth factor-like domains, and a serine protease domain. LcFⅦL was highly expressed in the liver and brain, but its expression was low in the other tested tissues. At the cellular level, LcFⅦL was highly expressed in macrophages, and its expression was induced by exposure to Pseudomonas plecoglossicida. We produced the recombinant LcFⅦL light chain (rLcFⅦL-LC), and found that it had obvious antibacterial effects against Gram-positive bacteria but low against Gram-negative bacteria. The rLcFⅦL-LC promoted the proliferation of macrophages. It also significantly induced the expression of proinflammatory factor (IL-1β and IL-6) and increased reactive oxygen species activity in large yellow croaker macrophages, while inhibited the expression of anti-inflammatory factor (TGF-β), suggesting that rLcFⅦL-LC may promote polarization of macrophages towards the M1 type. Taken together, these findings provide insight into the function of fish FⅦ, and advance our understanding of the role of the coagulation system in host defense.

Human saliva contains extracellular vesicles (EVs). These EVs expose extrinsic tenase complexes of tissue factor (TF) and activated factor VII (FVIIa), and trigger blood coagulation. Here, we show that EVs exposing extrinsic tenase complexes are also present in saliva of persons with severe hemophilia A, that is, persons with FVIII deficiency. Addition of these salivary EVs to autologous FVIII-deficient blood results in FXa generation, thereby compensating for the lack of FXa generation via intrinsic tenase (FVIIIa/FIXa) complexes. Consistently, in our retrospective analysis of persons with severe hemophilia A who do not receive prophylactic FVIII substitution, oropharyngeal mucosal bleedings are infrequent and self-limited. Conversely, in saliva of persons with severe FVII deficiency, in whom oropharyngeal bleedings are prevalent, functional extrinsic tenase complexes are absent, because EVs lack FVII. Saliva of persons with severe FVII deficiency is unable to restore blood coagulation, which is because of the absence of FVII in both their saliva and blood. Picomolar levels of recombinant FVIIa can restore the coagulant potential of saliva of persons with FVII deficiency. Taken together, our findings may explain the paucity of oropharyngeal bleedings in persons with hemophilia A as well as the occurrence of such bleedings in persons with severe FVII deficiency.

Although hypercoagulability is commonly associated with malignancies, whether coagulation factors directly affect tumor cell proliferation remains unclear. Herein, by performing single-cell RNA sequencing (scRNA-seq) of the prostate tumor microenvironment (TME) of mouse models of castration-resistant prostate cancer (CRPC), we report that immunosuppressive neutrophils (PMN-MDSCs) are a key extra-hepatic source of coagulation factor X (FX). FX activation within the TME enhances androgen-independent tumor growth by activating the protease-activated receptor 2 (PAR2) and the phosphorylation of ERK1/2 in tumor cells. Genetic and pharmacological inhibition of factor Xa (FXa) antagonizes the oncogenic activity of PMN-MDSCs, reduces tumor progression, and synergizes with enzalutamide therapy. Intriguingly, F10high PMN-MDSCs express the surface marker CD84 and CD84 ligation enhances F10 expression. Elevated levels of FX, CD84, and PAR2 in prostate tumors associate with worse survival in CRPC patients. This study provides evidence that FXa directly promotes cancer and highlights additional targets for PMN-MDSCs for cancer therapies.

Excess fibrotic remodeling causes cardiac dysfunction in ischemic heart disease, driven by MAP (mitogen-activated protein) kinase-dependent TGF-ß1 (transforming growth factor-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is not completely understood.

Mice with permanent ligation of the left anterior descending artery were used to model nonreperfused MI and analyzed by single-cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-FVIIa (activated factor VII)-integrin ß1-PAR2 (protease-activated receptor 2) signaling complex by utilizing genetic mouse models and pharmacological intervention.

Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared with controls. Proximity ligation assays of monocytic cells demonstrated that colocalization of FVIIa with integrin ß1 was diminished in monocyte/macrophage FVII-deficient mice after MI. Compared with controls, F7fl/fl CX3CR1 (CX3C motif chemokine receptor 1)Cre mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells. Single-cell mRNA sequencing of CD45 (cluster of differentiation 45)+ cells 3 and 7 days after MI uncovered a trajectory from recruited monocytes to inflammatory TF+/TREM (triggered receptor expressed on myeloid cells) 1+ macrophages requiring F7. As early as 7 days after MI, macrophage F7 deletion led to an expansion of reparative Olfml 3 (olfactomedin-like protein 3)+ macrophages and, conversely, to a reduction of TF+/TREM1+ macrophages, which were also reduced in PAR2R38E mice. Short-term treatment from days 1 to 5 after nonreperfused MI with a monoclonal antibody inhibiting the macrophage TF-FVIIa-PAR2 signaling complex without anticoagulant activity improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI.

Extravascular TF-FVIIa-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the malignant tumors with the worst prognosis in head and neck cancers. The transformation from normal tissue through low-grade and high-grade intraepithelial neoplasia to cancerous tissue in HSCC is typically viewed as a progressive pathological sequence typical of tumorigenesis. Nonetheless, the alterations in diverse cell clusters within the tissue microenvironment (TME) throughout tumorigenesis and their impact on the development of HSCC are yet to be fully understood.

We employed single-cell RNA sequencing and TCR/BCR sequencing to sequence 60,854 cells from nine tissue samples representing different stages during the progression of HSCC. This allowed us to construct dynamic transcriptomic maps of cells in diverse TME across various disease stages, and experimentally validated the key molecules within it.

We delineated the heterogeneity among tumor cells, immune cells (including T cells, B cells, and myeloid cells), and stromal cells (such as fibroblasts and endothelial cells) during the tumorigenesis of HSCC. We uncovered the alterations in function and state of distinct cell clusters at different stages of tumor development and identified specific clusters closely associated with the tumorigenesis of HSCC. Consequently, we discovered molecules like MAGEA3 and MMP3, pivotal for the diagnosis and treatment of HSCC.

Our research sheds light on the dynamic alterations within the TME during the tumorigenesis of HSCC, which will help to understand its mechanism of canceration, identify early diagnostic markers, and discover new therapeutic targets.

Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses.

Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart.

We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in β-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of β-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice.

These data provide insights into a TF-FVIIa signaling axis through PAR2-β-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

Imbalances in proteolytic activity have been linked to the development of inflammatory bowel diseases (IBD) and experimental colitis. Proteases in the intestine play important roles in maintaining homeostasis, but exposure of mucosal tissues to excess proteolytic activity can promote pathology through protease-activated receptors (PARs). Previous research implicates microbial proteases in IBD, but the underlying pathways and specific interactions between microbes and PARs remain unclear. In this study, we investigated the role of microbial proteolytic activation of the external domain of PAR2 in intestinal injury using mice expressing PAR2 with a mutated N-terminal external domain that is resistant to canonical activation by proteolytic cleavage. Our findings demonstrate the key role of proteolytic cleavage of the PAR2 external domain in promoting intestinal permeability and inflammation during colitis. In wild-type mice expressing protease-sensitive PAR2, excessive inflammation leads to the expansion of bacterial taxa that cleave the external domain of PAR2, exacerbating colitis severity. In contrast, mice expressing mutated protease-resistant PAR2 exhibit attenuated colitis severity and do not experience the same proteolytic bacterial expansion. Colonization of wild-type mice with proteolytic PAR2-activating Enterococcus and Staphylococcus worsens colitis severity. Our study identifies a previously unknown interaction between proteolytic bacterial communities, which are shaped by inflammation, and the external domain of PAR2 in colitis. The findings should encourage new therapeutic developments for IBD by targeting excessive PAR2 cleavage by bacterial proteases.