Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways

Abstract

Storage lesion–induced, red cell–derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV–induced coagulation activation, the ability of storage lesion–induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV–induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Microvesicle Formation Induced by Oxidative Stress in Human Erythrocytes

“…RBC-derived EVs were shown to express phosphatidylserine and cell-specific band 3 epitopes on the surface, as well as to contain enzymes involved in redox homeostasis (glutathione S-transferase, ubiquitin, thioredoxin, peroxiredoxin-1, peroxiredoxin-2) and complement-inhibiting proteins CD55 and CD59. The EV effects in health, disease, and blood transfusion are a matter of continued investigation [ 3 , 15 , 54 , 55 , 56 , 57 , 58 ]. However, it has already been defined that MVs could be captured by other circulated blood cells and by endothelial cells; thereby, EVs are involved in coagulation promotion, inflammation, immune modulation, endothelial dysfunction, and vasodilatation impairment, as well as having vasoactive properties potentially altering oxygen delivery homeostasis [ 17 ].…”

Extracellular Vesicles Derived From Platelets, Red Blood Cells, and Monocyte-Like Cells Differ Regarding Their Ability to Induce Factor XII-Dependent Thrombin Generation

“…The importance of FXI was as well reported by Rubin et al (2013), who showed that rbcEVs at concentration of ≥ 5 × 10 9 /L induced thrombin generation despite the presence of CTI. Very recently, EVs from red blood cell units have been suggested to initiate the intrinsic pathway by two mechanisms leading to FIX activation, (i) the normal FXIIa-FXI-FIX pathway and (ii) the activation of kallikrein that in turn directly activates FIX (Noubouossie et al, 2020). Notably, the authors of this study described that heating at 60 • C or treatment with trypsin abolished the ability of rbcEVs to induce thrombin generation, suggesting that EV-associated proteins were responsible for the activation of FXII and kallikrein.…”

“…In our study, protease treatment did not affect the thrombogenicity of rbcEVs or pEVs. It should be noted, however, that the rbcEVs used in the studies by Rubin et al (2010); Gao et al (2013), and Noubouossie et al (2020) were derived from red blood cell units that had been stored for 42-45 days, which is likely associated with oxidative changes and storage lesion (D'Alessandro et al, 2015;Almizraq et al, 2018). This enhanced thrombogenicity upon prolonged storage of red blood cell units may also be the cause of a higher incidence of deep vein thrombosis after transfusion of aged blood units (Koch et al, 2019).…”

Novel anti-thrombotic mechanisms mediated by Mas receptor as result of balanced activities between the kallikrein/kinin and the renin-angiotensin systems

“…The subsequent cleavage of FXI by FXIIa triggers a proteolytic cascade of coagulation factors, leading to thrombin generation, which promotes thrombus formation by activating platelets and generating fibrin. Recently, plasma kallikrein has also been shown to activate FIX independent of FXIa, another pathway by which contact activation may influence thrombin formation [ 21 , 22 ]. In addition to activation in pathologic states, the kallikrein/kinin system is physiologically activated by prolylcarboxypeptidase (PRCP), an endothelial cell membrane serine protease that activates prekallikrein independent of FXII [ [23] , [24] , [25] ].…”