Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates in vitro blood coagulation similarly to thromboplastin on fibroblasts

Abstract

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.

Hemostasis and thrombosis beyond biochemistry: roles of geometry, flow and diffusion

“…Such kind of systems was developed by several research groups and included models in non-stirred plasma with immobilized activator [13,15,59]. In the experimental system that was developed by our group, coagulation was initiated either by TF-bearing cells, glass edge [13], or TF immobilized to a plastic surface [60]. After activation on the surface, coagulation propagated freely into the bulk of non-stirred plasma, and fibrin clot growth was observed by light scattering.…”

Predicting prothrombotic tendencies in sepsis using spatial clot growth dynamics

“…Plasma clotting was activated with surface-immobilized tissue factor (TF) [24] and propagated into the bulk of the plasma (Fig. 1a).…”

Thrombin inhibitors based on single-stranded DNA aptamers

“…Then 6 ml of 1 mol/l CaCl 2 was added, and mixture was placed into thin flat plastic chamber of experimental device at 378C. Coagulation was triggered in a nonstirred plasma sample by a surface of activator covered with immobilized tissue factor [42]. Clot started to grow from the activator surface into the bulk of plasma.…”