Fibrin Affinity of Erythrocyte-Coupled Tissue-Type Plasminogen Activators Endures Hemodynamic Forces and Enhances Fibrinolysis in Vivo

Abstract

Plasminogen activators (PAs; e.g., tissue-type, tPA) coupled to red blood cells (RBCs) display in vivo features useful for thromboprophylaxis: prolonged circulation, minimal extravasation, and preferential lysis of nascent versus preexisting clots. Yet, factors controlling the activity of RBC-bound PAs in vivo are not defined and may not mirror the profile of soluble PAs. We tested the role of RBC/PA binding to fibrin in fibrinolysis. RBC/ tPA and RBC/tPA variant with low fibrin affinity (rPA) bound to and lysed plasminogen-containing fibrin clots in vitro comparably. In contrast, when coinjected in mice with fibrin emboli lodging in pulmonary vasculature, only RBC/tPA accumulated in lungs, which resulted in a more extensive fibrinolysis versus RBC/rPA (p Ͻ 0.01). Reconciling this apparent divergence between in vitro and in vivo behaviors, RBC/tPA, but not RBC/rPA perfused over fibrin in vitro at physiological shear stress bound to fibrin clots and caused greater fibrinolysis versus RBC/rPA (p Ͻ 0.001). These results indicate that because of high fibrin affinity, RBC/tPA binding to clots endures hemodynamic stress, which enhances fibrinolysis. Behavior of RBC/PAs under hemodynamic pressure is an important predictor of their performance in vivo.

The Glycocalyx Protects Erythrocyte-Bound Tissue-Type Plasminogen Activator from Enzymatic Inhibition

“…RBC were isolated by centrifugation from fresh anticoagulated human and mouse blood. Approximately 6 ϫ 10 4 molecules of tPA were attached per RBC without loss of enzymatic activity or alterations in RBC biocompatibility as described previously (Muzykantov and Murciano, 1996;Murciano et al, 2003;Ganguly et al, 2005Ganguly et al, , 2006. Briefly tPA and RBCs (10% hematocrit) were biotinylated with 6-biotinylaminocaproic acid Nhydroxysuccinimide ester at 5-fold molar excess (b 5 tPA) or 10 M (b 10 RBC).…”

“…Unbound SA was removed by washing the cells three times followed by incubation with radiolabeled b 5 tPA. Unbound tPA was removed by washing, and cell-associated tPA was measured in a gamma counter (PerkinElmer Life and Analytical Sciences) (Murciano et al, 2003;Ganguly et al, 2005Ganguly et al, , 2006. The b 10 RBC/b 5 tPA conjugates are henceforth designated as RBC/tPA.…”

“…Injection of either preformed RBC/tPA (Murciano et al, 2003;Ganguly et al, 2005Ganguly et al, , 2006 or tPA derivatives targeted to complement receptor-1 on circulating RBC (Zaitsev et al, 2006) prevents subsequent formation of occlusive venous and arterial clots, with no overt harmful effects on the carrier RBC (Murciano et al, 2003;Ganguly et al, 2005, activation of coagulation (Murciano et al, 2003) or impaired postsurgical hemostasis (Zaitsev et al, 2006).…”

“…Previous studies showed that coupling tPA to red blood cells (RBC) produces long-circulating enzymatically active complexes, thus converting tPA from a therapeutic agent with considerable safety concerns into an effective and safe thromboprophylactic agent (Murciano et al, 2003;Ganguly et al, 2005Ganguly et al, , 2006. Injection of either preformed RBC/tPA (Murciano et al, 2003;Ganguly et al, 2005Ganguly et al, , 2006 or tPA derivatives targeted to complement receptor-1 on circulating RBC (Zaitsev et al, 2006) prevents subsequent formation of occlusive venous and arterial clots, with no overt harmful effects on the carrier RBC (Murciano et al, 2003;Ganguly et al, 2005, activation of coagulation (Murciano et al, 2003) or impaired postsurgical hemostasis (Zaitsev et al, 2006).…”

Targeting of a Mutant Plasminogen Activator to Circulating Red Blood Cells for Prophylactic Fibrinolysis

“…RBC/tPA circulate for many hours and incorporate into and rapidly dissolve newly formed, potentially occlusive clots from within (Murciano et al, 2003). Infusion of RBC/tPA in mice, rats, and pigs provides an effective short-term option to prevent thrombotic occlusion in diverse vascular systems, including the cerebral vasculature, without the hemorrhagic and CNS toxicity profile typically seen with free tPA (Murciano et al, 2003;Ganguly et al, 2005;Ganguly et al, 2006;Ganguly et al, 2007;Danielyan et al, 2008;Armstead et al, 2009).…”