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Abnormal clot structure has been identified in patients with thrombotic disorders. Anticoagulant therapy offers clear benefits for thrombosis prevention and treatment by reducing blood clot formation and size; nevertheless, there are limited data on the effects of different anticoagulants, where clotting is initiated with different triggers, on clot structure. Our aim was to investigate the effects of vitamin K antagonists and factor Xa inhibitors on clot structure. Clots from pooled plasma spiked with rivaroxaban, apixaban, or enoxaparin, as well as plasma from patients on warfarin, were compared to plasma without anticoagulation. The kinetic profile of polymerizing clots was obtained by turbidity, fiber density was determined by confocal microscopy, clot pore size was investigated by permeation, and fiber size was analyzed using scanning electron microscopy. Clotting agonist was either tissue factor or thrombin. Following clotting with tissue factor, all anticoagulated clots had a significantly increased lag time, with the exception of enoxaparin. Rivaroxaban additionally led to significantly less dense and more permeable clots, with thicker fibers. In contrast, turbidity analysis following initiation with thrombin showed few effects of anticoagulation, with only enoxaparin leading to a prolonged lag time. Enoxaparin clots made with thrombin were less dense and more permeable. Our results show that anticoagulants modulate clot structure particularly when induced by tissue factor, most likely due to reduction of thrombin generation. We propose that the effects of different anticoagulants could be assessed with a global clot structure measurement such as permeation or turbidity, providing information on clot phenotype.

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.

The Effect of Dimethylbiguanide on Thrombin Activity, FXIII Activation, Fibrin Polymerization, and Fibrin Clot Formation Kristina F. Standeven 1 , Robert A.S. Ariëns 1 , Paul Whitaker 1 , Alison E. Ashcroft 2 , John W. Weisel 3 and Peter J. Grant 1 1 Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds, U.K. 2 School of Biochemistry and Molecular Biology, University of Leeds, Leeds, U.K. 3 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Abstract The antihyperglycemic drug dimethylbiguanide (DMB, also known as metformin) reduces the risk of cardiovascular complications in type 2 diabetes, although the mechanism(s) involved are unclear. DMB reduces glycosylation-related protein cross-linking, a process similar to fibrin cross-linking catalyzed by activated factor XIII (FXIII). To investigate whether the cardioprotective effect of DMB could be related to effects on clot stabilization, we studied the effects of DMB on FXIII, thrombin activity, and cleavage of fibrin(ogen). Activity of purified and plasma FXIII was inhibited by DMB. Analysis by mass spectrometry and FXIII-coupled magnetic particles excluded binding of DMB to FXIII. Thrombin-induced cleavage of the activation peptide from FXIII was inhibited in a dose-dependent manner, as was fibrinopeptide cleavage from fibrinogen. Ancrod-induced cleavage of fibrinopeptide A was not affected. DMB prolonged clotting time of normal plasma. Fiber thickness and pore size of fibrin clots, measured by permeation experiments and visualized by scanning electron microscopy, decreased significantly with DMB. No interactions between DMB and the active site of thrombin were found. Turbidity experiments demonstrated that DMB changed polymerization and lateral aggregation of protofibrils. These results suggest that DMB interferes with FXIII activation and fibrin polymerization, but not only by binding to thrombin on a different location than the active site. In patients on DMB therapy, FXIII antigen and activity levels in vivo were reduced over a 12-week period. These findings indicate that part of the cardioprotective effect of DMB in patients with type 2 diabetes may be attributed to alterations in fibrin structure/function. Footnotes Address correspondence and reprint requests to Robert A.S. Ariëns, Unit of Molecular Vascular Medicine, University of Leeds, G-Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, U.K. E-mail: r.a.s.ariens{at}leeds.ac.uk . Received for publication 19 December 2000 and accepted in revised form 22 October 2001. ANCOVA, analysis of covariance; DMB, dimethylbiguanide; FXIII, factor XIII; HPLC, high-performance liquid chromatography; MI, myocardial infarction; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; UKPDS, U.K. Prospective Diabetes Study.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent low-grade inflammation and a markedly increased risk of cardiovascular diseases (CVD). Leukocytes play an important role not only in host defense but are also increasingly recognized as key contributors to haemostasis and thromboinflammatory processes. In DM, chronic hyperglycaemia, oxidative stress and inflammation lead to leukocyte dysfunction, including enhanced cell activation, impaired mitochondrial function, and dysregulated interactions with platelets and endothelial cells. These alterations promote the thromboinflammatory state that contributes to vascular complications in DM. Thus, the modulation of oxidative stress and inflammation are important. Polyphenols are a class of plant secondary metabolites widely studied for their antioxidant and anti-inflammatory properties. This review comprehensively explores leukocyte dysfunction in DM, its contribution to thromboinflammation, and the mechanistic role of polyphenols in modulating these processes. The evidence presented suggests that polyphenols may contribute to the modulation of thromboinflammatory pathways. Further research in this area is required to enhance our understanding of thromboinflammation in DM and to translate these findings into effective adjunctive strategies, alongside standard pharmacological therapies to reduce CVD risk in individuals with DM.

Background Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. Methods Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. Results Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. Conclusions In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and appliedmethods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolizationwithout affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Cardiovascular diseases (CVDs) are a significant health burden worldwide. One of the key pathological processes underlying CVD is thrombosis–the formation of a blood clot (thrombus) within the blood vessel. Thrombus composition typically includes fibrin, platelets, red blood cells, leukocytes, and neutrophil extracellular traps (NETs). Polyphenols, a diverse group of naturally occurring compounds abundant in plant-based foods, have shown potential cardiovascular protective properties. This review discusses and summarizes the effects of polyphenols on the endothelium, platelet function and activity, and blood coagulation, and how this may potentially contribute to attenuated thrombus formation. The available evidence discussed in this review suggests that polyphenols may confer cardiovascular benefits not only through antioxidant and anti-inflammatory actions, but also by directly modulating thrombosis-related mechanisms. Nevertheless, in vivo studies remain limited, and the lack of standardized procedures contributes to discrepancies among reported results. Moreover, differences in compound structure, absorption and bioavailability should be considered when interpreting findings and their potential application as part of preventative strategies. The evidence presented in this review suggests that polyphenols may offer benefits towards lowering thrombosis risk and reducing recurrence among patients with thrombosis, although additional studies are required to further explore their mechanistic effects.