Obesity is a prevalent prothrombotic risk factor marked by enhanced fibrin formation and suppressed fibrinolysis. Fibrin both promotes thrombotic events and drives obesity pathophysiology, but a lack of essential analytical tools has left fibrinolytic mechanisms affected by obesity poorly defined. Using a plasmin-specific fluorogenic substrate, we developed a plasmin generation (PG) assay for mouse plasma that is sensitive to tissue plasminogen activator, α2-antiplasmin, active plasminogen activator inhibitor (PAI-1), and fibrin formation, but not fibrin crosslinking. Compared with plasmas from mice fed a control diet, plasmas from mice fed a high-fat diet (HFD) showed delayed PG and reduced PG velocity. Concurrent to impaired PG, HFD also enhanced thrombin generation (TG). The collective impact of abnormal TG and PG in HFD-fed mice produced normal fibrin formation kinetics but delayed fibrinolysis. Functional and proteomic analyses determined that delayed PG in HFD-fed mice was not due to altered levels of plasminogen, α2-antiplasmin, or fibrinogen. Changes in PG were also not explained by elevated PAI-1 because active PAI-1 concentrations required to inhibit the PG assay were 100-fold higher than circulating concentrations in mice. HFD-fed mice had increased circulating thrombomodulin, and inhibiting thrombomodulin or thrombin-activatable fibrinolysis inhibitor (TAFI) normalized PG, revealing a thrombomodulin- and TAFI-dependent antifibrinolytic mechanism. Integrating kinetic parameters to calculate the metric of TG/PG ratio revealed a quantifiable net shift toward a prothrombotic phenotype in HFD-fed mice. Integrating TG and PG measurements may define a prothrombotic risk factor in diet-induced obesity.

Objective: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor α2bβ ₃ and platelet-derived serotonin. Conclusions: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently crosslinks Von Willebrand Factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study, we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C-domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C-domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. Significance The role of VWF in platelet-dependent thrombus formation is explained.