Arterial thrombosis contributes to some of the most frequent causes of mortality globally, such as myocardial infarction and stroke. Platelets are essential mediators of physiological haemostasis and pathological thrombosis. Platelet activation is controlled by a multitude of signalling pathways. Upon activation, platelets shed platelet-derived extracellular vesicles (pEVs). In this Special Issue: Extracellular Vesicles, Moon et al. investigate the impact of various platelet agonists (thrombin, ADP, collagen) on the proteome of pEVs. The study demonstrates that pEVs exhibit an agonist-dependent altered proteome compared to their parent cells, with significant variations in proteins related to coagulation, complement, and platelet activation. The study observes the rapid generation of pEVs following agonist stimulation with specific proteome alterations that underscore an active packaging process. This commentary highlights the implications of their findings and discusses the role of pEV cargo in cardiovascular disease with potential novel therapeutic and diagnostic opportunities.

Background This study investigated the anticoagulant properties of sulfated chitosan derived from the internal bone of the spineless cuttlefish Sepiella inermis. Chitosan, a biopolymer, is used in various biomedical applications including anticoagulation. Sulfation of chitosan enhances its biological activity, making it a potential therapeutic agent. This study explored the efficacy of sulfated chitosan in preventing blood clot formation to provide a novel anticoagulant alternative. Objectives This study aimed to synthesize and characterize the anticoagulant properties of sulfated chitosan extracted from the internal bone of the spineless cuttlefish S. inermis using Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM), and X-Ray Diffraction (XRD) and evaluate the anticoagulant properties of sulfated chitosan extracted from the internal bone of spineless cuttlefish S. inermis. Materials and methods Chitin and chitosan were extracted from the cuttlebone of a specimen of S. inermis, and sulfated chitosan was synthesized by sulfation of chitosan. Sulfated chitosan was subsequently used to evaluate its anticoagulant properties using tests such as activated partial thromboplastin time (APTT) and prothrombin time (PT). Characteristic investigations were conducted, including FTIR, FESEM, and XRD analyses. Results The results of this study suggested the possibility of using S. inermis internal bone as an unconventional source of natural anticoagulant that can be combined with biomedical applications. Anticoagulant activity measured using APTT and PT showed that sulfated chitosan was a strong anticoagulant. Conclusion We examined the anticoagulant activity of S. inermis extract using thrombin and activated partial thromboplastin times. Our results demonstrated the heparin-like anticoagulant action of the extracted sulfated chitosan, suggesting that it may be a great alternative anticoagulant treatment.

This study presents a thrombin-loaded cationized chitosan (TCCS) sponge with highly effective hemostatic and antibacterial activity. The TCCS sponge, prepared using a multistep method, features a porous structure, favorable mechanical properties, excellent water absorption ability, and shape recovery triggered by water or blood. The TCCS sponge exhibited strong antibacterial activity against Methicillin-resistant Staphylococcus aureus and Escherichia coli. Additionally, it demonstrated enhanced procoagulant and hemostatic efficacy in rat tail amputation and rat liver perforation wound models compared to commercial hemostats. Furthermore, the sponge exhibited favorable biocompatibility and biosafety. These findings suggest that the TCCS sponge has substantial potential for practical applications in managing severe hemorrhages and bacterial infections.

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.

BACKGROUND: Congenital factor V (FV) deficiency is a rare clotting disorder affecting ∼1 in 1,000,000, with bleeding severity that ranges broadly for poorly understood reasons.
OBJECTIVE: To help understand the molecular basis of the observed phenotype in FV deficient patients, the genetics and biochemistry causing a patient\'s FV deficiency were evaluated.
RESULTS: A 71-year-old female, who had serious life-long bleeding upon provocation and profound menorrhagia that lead to hysterectomy, was found to have 3% of normal plasma FV antigen with normal electrophoretic mobility. Platelet FV was similarly low, although the banding pattern was less fragmented than normal. Plasma clotting activity was <1% of normal. Familial inheritance and DNA sequence analysis from peripheral blood leukocytes were consistent with novel compound heterozygosity with missense mutations in exon XVII, Leu1821 to Ser (L1821S) and exon XXV, Gly2192 to Cys (G2192C). The respective single-mutation variants were expressed and purified. Explaining why the antigen level and activity were inequivalent, thrombin activation of recombinant (r) FV/L1821S was impaired, and rFV/G2192C was unable to bind to a procoagulant phospholipid membrane.
CONCLUSIONS: These findings are consistent with the observed phenotype, highlighting the importance of understanding FV biochemical function to rationalize clinical bleeding severity when the circulating antigen level is discordant.

BACKGROUND: Acute lung injury (ALI) following pneumonia involves uncontrolled inflammation and tissue injury, leading to high mortality. We previously confirmed the significantly increased cargo content and extracellular vesicle (EV) production in thrombin-preconditioned human mesenchymal stromal cells (thMSCs) compared to those in naïve and other preconditioning methods. This study aimed to investigate the therapeutic efficacy of EVs derived from thMSCs in protecting against inflammation and tissue injury in an Escherichia coli (E. coli)-induced ALI mouse model.
METHODS: In vitro, RAW 264.7 cells were stimulated with 0.1 µg/mL liposaccharides (LPS) for 1 h, then were treated with either PBS (LPS Ctrl) or 5 × 107 particles of thMSC-EVs (LPS + thMSC-EVs) for 24 h. Cells and media were harvested for flow cytometry and ELISA. In vivo, ICR mice were anesthetized, intubated, administered 2 × 107 CFU/100 µl of E. coli. 50 min after, mice were then either administered 50 µL saline (ECS) or 1 × 109 particles/50 µL of thMSC-EVs (EME). Three days later, the therapeutic efficacy of thMSC-EVs was assessed using extracted lung tissue, bronchoalveolar lavage fluid (BALF), and in vivo computed tomography scans. One-way analysis of variance with post-hoc TUKEY test was used to compare the experimental groups statistically.
RESULTS: In vitro, IL-1β, CCL-2, and MMP-9 levels were significantly lower in the LPS + thMSC-EVs group than in the LPS Ctrl group. The percentages of M1 macrophages in the normal control, LPS Ctrl, and LPS + thMSC-EV groups were 12.5, 98.4, and 65.9%, respectively. In vivo, the EME group exhibited significantly lower histological scores for alveolar congestion, hemorrhage, wall thickening, and leukocyte infiltration than the ECS group. The wet-dry ratio for the lungs was significantly lower in the EME group than in the ECS group. The BALF levels of CCL2, TNF-a, and IL-6 were significantly lower in the EME group than in the ECS group. In vivo CT analysis revealed a significantly lower percentage of damaged lungs in the EME group than in the ECS group.
CONCLUSIONS: Intratracheal thMSC-EVs administration significantly reduced E. coli-induced inflammation and lung tissue damage. Overall, these results suggest therapeutically enhanced thMSC-EVs as a novel promising therapeutic option for ARDS/ALI.

BACKGROUND: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown.
METHODS: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury.
RESULTS: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors.
CONCLUSIONS: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.

Surgery remains the standard treatment for spinal metastasis. However, uncontrolled intraoperative bleeding poses a significant challenge for adequate surgical resection and compromises surgical outcomes. In this study, we develop a thrombin (Thr)-loaded nanorobot-hydrogel hybrid superstructure by incorporating nanorobots into regenerated silk fibroin nanofibril hydrogels. This superstructure with superior thixotropic properties is injected percutaneously and dispersed into the spinal metastasis of hepatocellular carcinoma (HCC) with easy bleeding characteristics, before spinal surgery in a mouse model. Under near-infrared irradiation, the self-motile nanorobots penetrate into the deep spinal tumor, releasing Thr in a controlled manner. Thr-induced thrombosis effectively blocks the tumor vasculature and reduces bleeding, inhibiting tumor growth and postoperative recurrence with Au nanorod-mediated photothermal therapy. Our minimally invasive treatment platform provides a novel preoperative therapeutic strategy for HCC spinal metastasis effectively controlling intraoperative bleeding and tumor growth, with potentially reduced surgical complications and enhanced operative outcomes.

COVID-19 patients experience a complex interplay involving ACE2, thrombin, D-dimer, and lipid profile, yet its full understanding remains elusive. ACE2, a pivotal regulator of the renin-angiotensin system and the primary receptor for SARS-CoV-2 undergoes downregulation upon viral binding, potentially leading to severe cases with acute respiratory distress syndrome (ARDS). A specific ACE2 gene polymorphism (rs2285666) may be associated with COVID-19 susceptibility, with the A allele potentially increasing infection risk. COVID-19 disease progression is linked to coagulation abnormalities, but the exact connection with thrombin and D-dimer remains uncertain. A study examining coagulation parameters in COVID-19 patients admitted to Al-Diwania Educational Hospital from February to May 2022 found that thrombin and D-dimer levels were directly related to disease severity. Severe cases exhibited significantly altered coagulation function compared to mild and recovered cases, with notably higher D-dimer levels and elevated thrombin serum concentrations. Moreover, dyslipidemia, particularly low HDL cholesterol, is a prevalent comorbidity in COVID-19 patients and may be linked to worse outcomes. In conclusion, COVID-19 is associated with a prothrombotic state and dysregulation of the renin-angiotensin system due to ACE2 downregulation following viral binding. The intricate interplay between ACE2, thrombin, D-dimer, and lipid profile necessitates further investigation. The multifaceted nature of the disease demands continued research to unravel its pathogenesis and identify potential therapeutic targets.

Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.