Membrane microparticles (MP) produced upon cell activation and/or damage possess coagulation activity, i.e. ability to accelerate blood clotting. They contain on their surface phosphatidylserine (PS), a substrate for assembling coagulation enzymatic complexes, and some of them tissue factor (TF), the initiator of clotting cascade reactions. In this study coagulation properties of MP derived from erythrocytes have been investigated. These MP were obtained from donor's erythrocytes activated with ionophore A23187 as well as from outdated erythrocyte concentrates for transfusion. MP were counted by flow cytometry. Coagulation activity of MP was examined by modified plasma recalcification assay. Involvement of PS and TF in this reaction was assessed using PS blocker lactadherin and anti-TF antibodies. TF activity in MP was measured by its ability to activate factor X in a chromogenic assay. Size of MP was evaluated by dynamic light scattering. Properties of erythrocyte MP were compared with previously characterized (using the same methodological approaches) MP derived from platelets and monocytic THP-1 cells, lacking and containing TF, respectively. Erythrocyte MP accelerated plasma clotting, but less actively than MP from platelets and MP from THP-1 cells, which demonstrated maximal activity. Lactadherin completely inhibited coagulation activity of all MP. Anti-TF antibodies did not affect clotting parameters in the presence of platelet and erythrocyte MP, but slowed clotting in the presence of MP from THP-1 cells. TF activity was not detected in erythrocyte and platelet MP, unlike MP from THP-1 cells expressing active TF. MP derived from erythrocytes were smaller than MP from platelets and THP-1 cells, with average diameter about 200 nm and 400 nm respectively. Thus, MP from erythrocyte possess less ability to accelerate plasma clotting in comparison with MP from platelet and THP-1 cells. The data obtained suggest that lesser coagulation activity of erythrocyte MP in comparison with MP from THP-1 cells is due to the absence of TF in erythrocyte MP (in contrast to MP from THP-1 cells) and to their smaller size, and in comparison with MP from platelets (which as erythrocyte MP do not express TF) is due to their smaller size only.
Membrannye mikrochastitsy (MCh), obrazuiushchiesia pri aktivatsii i/ili povrezhdenii kletok, obladaiut koaguliatsionnoĭ aktivnost'iu, to est' sposobnost'iu uskoriat' svertyvanie krovi. Oni soderzhat na svoeĭ poverkhnosti fosfatidilserin (FS) – substrat dlia sborki koaguliatsionnykh fermentnykh kompleksov, a nekotorye iz nikh tkanevoĭ faktor (TF) – initsiator reaktsiĭ svertyvaiushchego kaskada. V nastoiashcheĭ rabote issledovali koaguliatsionnye svoĭstva MCh éritrotsitov, kotorye poluchali iz éritrotsitov donorov, aktivirovannykh ionoforom A23187, a takzhe iz dlitel'no khraniashcheĭsia éritrotsitarnoĭ massy dlia perelivaniia. Kolichestvo MCh opredeliali s pomoshch'iu protochnoĭ tsitometrii, a ikh koaguliatsionnuiu aktivnost' s pomoshch'iu modifitsirovannogo testa rekal'tsifikatsii plazmy. Uchastie v étoĭ reaktsii FS i TF izuchali, ispol'zuia blokator FS, laktadgerin i anti-TF antitela. Aktivnost' TF v MCh otsenivali po sposobnosti aktivirovat' faktor X c pomoshch'iu khromogennogo testa. Razmer MCh izmeriali metodom dinamicheskogo svetorasseivaniia. Svoĭstva MCh éritrotsitov sravnivali s ranee okharakterizovannymi (s pomoshch'iu analogichnykh metodov) MCh trombotsitov i monotsitarnykh kletok THP-1, ne soderzhashchikh i soderzhashchikh TF, sootvetstvenno. MCh éritrotsitov uskoriali svertyvanie plazmy, odnako menee éffektivno, chem MCh trombotsitov i MCh kletok THP-1, kotorye demonstrirovali maksimal'nuiu aktivnost'. Laktadgerin polnost'iu podavlial koaguliatsionnuiu aktivnost' vsekh MCh. Anti-TF antitela ne vliiali na parametry svertyvaniia v prisutstvii MCh éritrotsitov i trombotsitov, no zamedliali svertyvanie v prisutstvii MCh kletok THP-1. V otlichie ot MCh kletok THP-1, ékspressiruiushchikh aktivnyĭ TF, aktivnost' TF ne obnaruzhena v MCh éritrotsitov i trombotsitov. MCh éritrotsitov byli mel'che MCh trombotsitov i kletok THP-1 – sredniĭ diametr okolo 200 nm i 400 nm, sootvetstvenno. Takim obrazom, MCh éritrotsitov obladaiut men'sheĭ sposobnost'iu uskoriat' svertyvanie plazmy po sravneniiu s MCh monotsitarnykh kletok THP-1 i trombotsitov. Poluchennye dannye ukazyvaiut na to, chto bolee nizkaia koaguliatsionnaia aktivnost' MCh éritrotsitov po sravneniiu s MCh kletok THP-1 obuslovlena otsutstviem v MCh éritrotsitov TF (v otlichie ot MCh kletok THP-1) i ikh bolee melkim razmerom, a po sravneniiu s MCh trombotsitov (ne soderzhashchimi TF, takzhe kak i MCh éritrotsitov) – tol'ko ikh bolee melkim razmerom.
Keywords: blood coagulation; erythrocytes; membrane microparticles; monocytes; platelets; tissue factor.