BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches.
METHODS: We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1.
RESULTS: We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity.
CONCLUSIONS: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.

Coagulation proteases, in addition to playing an essential role in blood coagulation, often influence diverse cellular functions by inducing specific signaling pathways via the activation of protease-activated receptors (PARs). PAR activation-induced cellular effects are known to be cell-specific as PARs are expressed selectively in specific cell types. However, a growing body of evidence indicates that coagulation protease-induced PAR activation in a specific cell type could affect cellular responses in other cell types via communicating through extracellular vesicles (EVs) as coagulation proteases induced-PAR signaling could promote the release of EVs in various cell types. EVs are membrane-enclosed nanosized vesicles that facilitate intercellular communication by transferring bioactive molecules, such as proteins, lipids, mRNAs, and microRNAs, from donor cells to recipient cells. Our recent findings established that factor VIIa (FVIIa) promotes the release of EVs from vascular endothelium via endothelial cell protein C receptor-dependent activation of PAR1-mediated biased signaling. FVIIa-released EVs exhibit procoagulant activity and cytoprotective responses both in vitro and in vivo model systems. This review discusses how factor VIIa and other coagulation proteases activate PAR-mediated cell signaling and trigger the release of EVs. The review specifically discusses how FVIIa-released EVs are enriched with phosphatidylserine and anti-inflammatory miRNAs and the impact of FVIIa-released EVs on hemostasis in therapeutic settings. The review also briefly highlights the therapeutic potential of FVIIa-released EVs in treating bleeding and inflammatory disorders, such as hemophilic arthropathy and sepsis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) has emerged as an important link between coagulation and the complications of obesity including metabolic dysfunction-associated steatotic liver disease (MASLD). PAR1 is expressed by various cells and cleaved by different proteases to generate unique tethered agonists that activate distinct signaling pathways. Mice expressing PAR1 with an R41Q mutation have disabled canonical thrombin-mediated signaling, whereas R46Q mice express PAR1 resistant to non-canonical signaling by activated protein C (APC).
METHODS: Mice with whole body and hepatocyte-selective PAR1 deficiency, as well as PAR1 R41Q and R46Q mice were fed a high fat diet to induce MASLD.
CONCLUSIONS: High fat diet (HFD)-fed R41Q mice displayed reduced hepatic steatosis and liver/body weight ratio. In contrast, HFD-fed R46Q mice displayed increased relative liver weight and hepatic steatosis alongside increased serum ALT activity. Surprisingly, despite the distinct impact of PAR1 mutations on steatosis, selective deletion of PAR1 in hepatocytes had no impact. To evaluate a viable PAR1-targeted approach, mice with HFD-induced obesity were treated with the allosteric PAR1 modulator NRD-21, which inhibits canonical PAR1 inflammatory signaling but promotes PAR1 protective, non-canonical anti-inflammatory signaling. NRD-21 treatment reduced plasma TNFα, serum ALT activity, hepatic steatosis, and insulin resistance (HOMA-IR), but increased plasma active GLP-1. The results suggest non-hepatocellular canonical PAR1 cleavage drives MASLD in obese mice and provide translational proof-of-concept that selective pharmacological modulation of PAR1 yields multiple metabolic benefits in experimental obesity.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

By the time a Drosophila egg is laid, both major body axes have already been defined and it contains all the nutrients needed to develop into a free-living larva in 24 h. By contrast, it takes almost a week to make an egg from a female germline stem cell, during the complex process of oogenesis. This review will discuss key symmetry-breaking steps in Drosophila oogenesis that lead to the polarisation of both body axes: the asymmetric divisions of the germline stem cells; the selection of the oocyte from the 16-cell germline cyst; the positioning of the oocyte at the posterior of the cyst; Gurken signalling from the oocyte to polarise the anterior-posterior axis of the somatic follicle cell epithelium around the developing germline cyst; the signalling back from the posterior follicle cells to polarise the anterior-posterior axis of the oocyte; and the migration of the oocyte nucleus that specifies the dorsal-ventral axis. Since each event creates the preconditions for the next, I will focus on the mechanisms that drive these symmetry-breaking steps, how they are linked and the outstanding questions that remain to be answered.

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/-) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6.

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Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis.
We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition.
During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation.
Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Infection by Staphylococcus aureus is the leading cause of infective endocarditis (IE). Activation of platelets by this pathogen results in their aggregation and thrombus formation which are considered to be important steps in the development and pathogenesis of IE. Here, we show that a secreted cysteine protease, staphopain A, activates human platelets and induces their aggregation. The culture supernatant of a scpA mutant deficient in staphopain A production was reduced in its ability to trigger platelet aggregation. The platelet agonist activity of purified staphopain A was inhibited by staphostatin A, a specific inhibitor, thus implicating its protease activity in the agonism. In whole blood, using concentrations of staphopain A that were otherwise insufficient to induce platelet aggregation, increased binding to collagen and thrombus formation was observed. Using antagonists specific to protease-activated receptors 1 and 4, we demonstrate their role in mediating staphopain A induced platelet activation.

Studies spanning the last 3 decades have fundamentally altered our understanding of the interplay between the hemostatic and immune systems. A plethora of studies have revealed that there is bidirectional crosstalk between these two systems at multiple levels that likely evolved as a means to coordinate the host response to numerous challenges, including trauma, infection, and thermal or chemical injury. Such challenges require reestablishment of vascular integrity, the clearance of pathogens, and removal of cellular and external debris. Clearly, bidirectional coordination of hemostasis and immunity would be beneficial in such contexts. Many types of malignancies take advantage of the interplay between hemostasis and immunity, co-opting these mechanisms to promote tumorigenesis, the formation of a supportive stroma, and metastasis to distant organs. Three important \"bridges\" that mechanistically link the hemostatic system to immune functions that have been shown to play a key role in cancer biology include the platelet/fibrinogen axis, protease activated receptor-1 (PAR-1) and protease activated receptor-2 (PAR-2). These hemostatic system components have been shown to regulate a variety of immune functions that support tumorigenesis in the context of inflammation-driven malignancy, metastasis, and escape from adaptive antitumor immunity. Understanding the mechanisms coupling these bridges between hemostasis and immunity, as well as others, could provide novel targets for the prevention and treatment of a variety of cancers.