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.

Background Assays measuring thrombin generation (TG) in plasma increasingly gained attention in the field of thrombosis and hemostasis. Adaptation of the method enabled the measurement of TG in whole blood (WB). Despite their potential, TG assays did not reach the stage of universal clinical application, partly because of the absence of normal ranges. Our study aimed to accurately determine normal ranges and interindividual variability of TG and correlate results with coagulation factor levels, sex, and oral contraceptive usage. Methods The study protocol was evaluated by the local medical ethical board. In total, 129 healthy volunteers gave full informed consent. Normal ranges of TG in platelet-poor plasma (PPP), platelet-rich plasma (PRP), and WB were determined according to CLSI guidelines. Results Our study is the first to measure normal ranges of TG in PPP, PRP, and WB in a large healthy cohort. Significant correlations were found between TG in plasma and WB. Interindividual variability of TG in WB was comparable to that of plasma. Oral contraceptive use increased TG in PPP, PRP, and WB. The inhibitory effect of thrombomodulin on TG was significantly lower in females than in males. This effect was more pronounced upon oral contraceptive use. Primary clotting factor determinants for TG parameters depended on the tissue factor concentration, but were similar in WB and plasma. Conclusions Establishing normal ranges for TG brings us 1 step closer to clinical use. Good correlations between plasma and WB (including clotting factor determinants for TG) suggest that WB TG can be reliably used in clinic.

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.