Licking the wound with procoagulant vesicles

In this issue of Blood, Thaler and colleagues define the role of salivary procoagulant extracellular vesicles (EVs) by demonstrating the differences in samples obtained from patients with hemophilia A vs patients with factor VII (FVII) deficiency (also known as hypoconvertinemia).¹ 

It is over 3 decades since the early reports of “procoagulant microparticles,” where the observed clotting activity was attributed to the presence of tissue factor (TF).² Understandably, the predominant interest in these vesicles was their appearance and activity within the blood. These EVs explained how an otherwise insoluble protein could retain functionality while being released and circulating in the bloodstream. Although initially the concentrations of TF in blood were thought to be too low to be detectable, concerted effort has produced advances in measurement of TF activity in EVs.³ A recent multicenter study has highlighted the discrepancies in quantifying TF-EVs,⁴ and it has been recommended that isolating EVs from plasma and measuring TF activity using a functional assay produces a more accurate means of quantification. It is conceivable that heterogeneous TF-EV populations may promote differential activities and, therefore, functions.

The recognition of the potential influence of TF-EVs on physiological processes, together with the need to better characterize the vesicles, the mechanisms of formation, and interactions with cells from various tissues, was highlighted a decade ago.⁵ Additionally, advancements in the understanding of regulation and interactions of TF and the decoding of TF-mediated signaling⁶ have supported the concept that TF-EVs play important physiological roles. The surrounding environment also influences and alters the nature of the signals,⁷ suggesting that there are differential roles for TF-EVs within and outside the blood system.

In this article, Thaler et al demonstrate the strength of the interaction of the recruited FVIIa on the TF-EVs. The data clearly show that the TF-EVs are present in a form that retains FVIIa-dependent tenase activity. The functional interaction of TF-EVs, released into body fluids, supports the physiological importance of these EVs in these fluids. One of the authors (Rienk Nieuwland) pointed out in a seminar that when we cut our finger, the finger goes in the mouth, and that “animals tend to lick their wounds”; hence, the title for this commentary.

A number of contrasting features between the sites, extent, and tendency of bleeding in hemophilia A and hypoconvertinemia have previously been detailed.^8,9 Although rare, FVII deficiency can cause serious or fatal bleeds in vital tissue such as the brain and gastrointestinal tract. Given the presence of fluids associated with these organs, this further highlights the importance of TF in fluids other than blood and raises the notion that TF-EVs have diverse hemostatic functions that can influence physiological processes. Additionally, as the authors point out, understanding the symptoms associated specifically with these (and other) bleeding phenotypes enables the clinical staff to advise patient as to what they may safely do and what should be avoided, as well as likely remedial actions upon incidental and accidental bleeds. In this study, through stepwise examination of the properties of saliva samples from groups of patients with the hemophilia A and FVII deficiency, the role of TF-EVs in salivary-induced activation of coagulation was examined and found to be different. For completeness the study was carried out with whole blood, plasma, and then using chromogenic substrates. Addition of the EVs from saliva of persons with severe hemophilia A, that is, persons with FVIII deficiency, to autologous FVII-deficient blood results in FXa generation, compensating for the lack of FXa generation via intrinsic tenase (FVIIIa/FIXa) complexes. This finding explains why oropharyngeal mucosal bleedings are infrequent and self-limiting in this patient population, even those not receiving prophylactic FVIII substitution. Conversely, in saliva from persons with severe FVII deficiency, in whom oropharyngeal bleedings are frequent, functional extrinsic tenase complexes are absent, as their EVs lack FVII. These studies also highlight the previously reported similarities in bleeding patterns between FVII deficiency with combined vascular-coagulation defect, although hemophilia A has symptoms that are typical, with a prevalence of soft-tissue hemorrhages.⁹ The advent of multiomics together with the superior analytical algorithms is likely to accelerate the understanding of the condition through cataloging of compounding risk factors and aid the avoidance and possibly even treatment of bleeding disorders.

However, the understanding of the molecular interactions between the proteins (and other components) of the EVs with cells or tissues has shown that EVs are fast-acting mediators during response to injury, as well as the initiation of repair processes. Examinations of the modifications of these proteins contributes to the exploration of the role of TF-EVs during pathological conditions in the blood and extravascular fluids. This study used specific inhibitors, inactivated factors, and antibodies as tools to discern important properties of these EVs, as carriers of FVIIa. Interestingly, the robust association of FVIIa with the salivary EVs does not appear to be common to all TF-EV types and has been proposed to arise from affinity maturation of TF on incorporation into EVs and not merely due to the presence of phosphatidylserine.¹⁰ Consequently, examination of TF-EVs in different fluids and on mucosal surfaces may reveal differentially produced TF-EVs with varied functions.

On reading the article by Thaler et al, one quickly finds that the rather modest title of the article heralds a wealth of interesting scientific and clinical information certain to generate many more studies on TF-EVs.

Conflict-of-interest disclosure: C.E. declares no competing financial interests.