Dataset Information

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.

SUBMITTER: Noubouossie DF

PROVIDER: S-EPMC7059516 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Noubouossie Denis F DF Henderson Michael W MW Mooberry Micah M Ilich Anton A Ellsworth Patrick P Piegore Mark M Skinner Sarah C SC Pawlinski Rafal R Welsby Ian I Renné Thomas T Hoffman Maureane M Monroe Dougald M DM Key Nigel S NS

Blood 20200301 10

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 asse ...[more]

PMID: 31971571

Similar Datasets

Project description:Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply mandates optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate, a difference that has been attributed to variable rates of energy usage and metabolism in red blood cells. Specific metabolite abundances are heritable traits; however, the link between heritability of energy metabolism and red blood cell storage profiles is unclear. Herein we performed a comprehensive metabolomics and proteomics study of red blood cells from 18 mono- and di-zygotic twin pairs to measure heritability and identify correlations with ATP and other molecular indices of energy metabolism. Without using affinity-based hemoglobin depletion, our work afforded the deepest multi-omic characterization of red blood cell membranes to date (1280 membrane proteins and 330 metabolites), with 119 membrane protein and 148 metabolite concentrations found to be over 30% heritable. We demonstrate a high degree of heritability in the concentration of energy metabolism metabolites, especially glycolytic metabolites. In addition to being heritable, proteins and metabolites involved in glycolysis and redox metabolism are highly correlated, suggesting that crucial energy metabolism pathways are inherited en bloc at distinct levels. We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of these proteins together. This can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic diseases.

Project description:The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), ?-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas ?-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure