Coupling Tissue Type Plasminogen Activator to Carrier Erythrocyte Protects Against Plasma Inhibitors.

Conjugating tissue plasminogen activator (tPA) to carrier red blood cells (RBC) restricts its permeation into tissues and pre-existing hemostatic clots, minimizing side effects, and prolongs its circulation, which permits it to be incorporated within nascent clot which it lyses from inside. We now report that RBC/tPA dissolves clots formed from mouse blood more effectively than free tPA, while they were equipotent against clots formed in vitro from PAI-1 KO mouse blood. To test whether tPA acquires resistance to plasma inhibitors when conjugated to RBC, we compared the activity of RBC/tPA vs free tPA in the presence of purified PAI-1, α₂ macroglobulin (α₂M) and α₁anti-trypsin (α₁AT). At equimolar concentrations, PAI-1 completely inhibited the activity of soluble tPA in vitro, whereas RBC/tPA retained 100% and 75% of its amidolytic and fibrinolytic activity, respectively. RBC/tPA, but not free tPA, was also resistant to equimolar concentrations of α₂M and α₁AT. However, tPA coupled to RBC pre-incubated with a mixture of hyaluronidase, heparinase and neuraminidase was as susceptible to inactivation by PAI-1, α₂M and α₁AT as free tPA. Further, tPA coupled to glycocalyx-stripped RBC bound two-fold more ¹²⁵I-labeled PAI-1 than tPA coupled to naïve RBC. We conclude that the RBC glycocalyx protects tPA from inactivation by PA inhibitors, likely by steric hindrance. This unexpected benefit may enhance the utility of RBC/tPA in thromboprophylaxis and suggests a previous under-appreciated role for the glycocalyx in modulating PA activity on the vasculature and other cell surfaces.