2Center of Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, ul. Kosygina 4, 117977 Moscow, Russia; fax: (495) 938-2533
3Physical Faculty, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-0126
* To whom correspondence should be addressed.
Received August 21, 2009
A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis–Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.
KEY WORDS: thromboplastin, tissue factor, immobilization, kinetics of factor VIIa–thromboplastin complex, spatial dynamics of blood coagulation