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

Abstract

Coupling tissue-type plasminogen activator (tPA) to carrier red blood cells (RBC) prolongs its intravascular life span and permits its use for thromboprophylaxis. Here, we studied the susceptibility of RBC/tPA to PA inhibitors including plasminogen activator inhibitor-1 (PAI-1) that constrain its activity and may reduce the duration of its effect. Despite lesser spatial and diffusional limitations, soluble tPA was far less effective than RBC/tPA in dissolving clots formed in vitro from blood of wildtype (WT) mice (40 versus 80% lysis at equal doses of tPA). Furthermore, after i.v. injection, soluble tPA lost activity faster in transgenic mice expressing a high level of PAI-1 than in WT mice, whereas the activity of RBC/tPA was unaffected. PAI-1 inactivated soluble tPA at equimolar ratios in vitro, but it had no effect on the amidolytic or fibrinolytic activity of RBC/tPA. RBC/ tPA was also more resistant than soluble tPA to in vitro inhibition by other serpins (␣ 2 -macroglobulin and ␣ 1 -antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125 I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.

Enzymatic Antioxidants: Next Phase of Pharmacological Effort to Fight Oxidative Stress?

“…These data approve the possibility and efficiency of use of the components of vascular cell glycocalyx for targeted delivery of medical substances [43]. Moreover, glycocalyx of erythrocytes may protect the tissue plasminogen activator (tPA) bounded with it against inhibition interaction with inhibitor of plasminogen activator of type 1 (PAI-1), destroying the local electrostatic interactions among them, not disturbing the binding of tPA with fibrin and plasminogen [44]. Such stabilization by glycocalyx makes it possible to offer biotinilated components (tPA and erythrocytes), bounded via streptavidin, for thrombosis prophylaxis in patients with high risk of cerebral or vascular lesion [45,46].…”

Influence of the glycocalyx and plasma membrane composition on amphiphilic gold nanoparticle association with erythrocytes

“…55,56 When sialic acids were preferentially stripped, MUS : OT interactions increased significantly (2.2-fold) compared to unaltered cells treated with AuNPs, suggesting that the interaction of the anionic particles with the RBC membrane is substantially inhibited by these negatively-charged sugars (Fig. 4a).…”

“…Notably, even with the glycocalyx stripped, the electron micrographs showed that all NPs found were apparently membrane-associated and none were found in the cytosol of cells. When erythrocytes were treated with trypsin and chymotrypsin to remove membrane glycoproteins (chiefly of the glycophorin family [55][56][57] ), there was no significant difference in AuNP-membrane interaction following this treatment (Fig. 4b).…”

Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle

“…51 One possible explanation for this surprising result is that coupling to RBCs renders tPA less susceptible to plasma tPA inhibitors, 52 including the most physiologically relevant plasminogen activator inhibitor 1 (PAI-1). Further studies affirm the greater fibrinolytic potency of bound tPA compared with soluble tPA in mouse blood, and indicate that coupling to RBCs protects tPA against physiological and pathological concentrations of PAI-1, 53 and inhibition by other serpins (α 2 -macroglobulin and α 1 -antitrypsin). As the interaction of tPA and PAI-1 may be stabilized through salt bridges formed between cationic amino acid in tPA and anionic residues in PAI-1, 54 the authors propose that the protection is due to charge-mediated masking of vulnerable sites on tPA molecules by the negatively charged components of glycocalyxon on the surface of RBCs.…”