The glycocalyx protects erythrocyte-bound tissue-type plasminogen activator from enzymatic inhibition.
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 wild-type (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 (alpha2-macroglobulin and alpha1-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 125I-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.
Ganguly
Kumkum
K
Institute for Environmental Medicine, University of Pennsylvania Medical Center, 3620 Hamilton Walk, Philadelphia, PA 19104-6068, USA.
Murciano
Juan-Carlos
JC
Westrick
Randal
R
Leferovich
John
J
Cines
Douglas B
DB
Muzykantov
Vladimir R
VR
D000818:Animals; D001777:Blood Coagulation / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism*; D005342:Fibrinolysis / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D019276:Glycocalyx / Q000502:physiology*; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D017395:Plasminogen Activator Inhibitor 1 / Q000502:physiology; D015843:Serpins / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D007457:Iodine Radioisotopes; D017395:Plasminogen Activator Inhibitor 1; D015843:Serpins; D010959:Tissue Plasminogen Activator; D005947:Glucose