tPA‐mediated generation of plasmin is catalyzed by the proteoglycan NG2

Abstract

Paralysis resulting from spinal cord injury is devastating and persistent. One major reason for the inability of the body to heal this type of injury ensues from the local increase of glial cells leading to the formation of a glial scar, and the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the site of injury through which axons are unable to regenerate. Experimental approaches to overcome this problem have accordingly focused on reducing the inhibitory properties of CSPGs, for example by using chondroitinase to remove the sugar chains and reduce the CSPGs to their core protein constituents, although this step alone does not provide dramatic benefits as a monotherapy. Using in vitro and in vivo approaches, we describe here a potentially synergistic therapeutic opportunity based on tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen (plg) into the active protease plasmin. We show that tPA and plg both bind to the CSPG protein NG2, which functions as a scaffold to accelerate the tPA-driven conversion of plg to plasmin. The binding occurs via the tPA and plg kringle domains to domain 2 of the NG2 CSPG core protein, and is enhanced in some settings after chondroitinase-mediated removal of the NG2 proteoglycan side chains. Once generated, plasmin then degrades NG2, both in an in vitro setting using recombinant protein, and in vivo models of spinal cord injury. Our finding that the tPA and plg binding is in some instances more efficient after exposure of the NG2 proteoglycan to chondroitinase treatment suggests that a combined therapeutic approach employing both chondroitinase and the tPA/plasmin proteolytic system could be of significant benefit in promoting axonal regeneration through glial scars after spinal cord injury.

Annexin A2 Promotes Glioma Cell Invasion and Tumor Progression

“…To measure tPA activity, an amidolytic assay was used following an established protocol (Nolin et al, 2008). Duplicate protein samples (25 μl) were added to the reaction mixture containing plasminogen, S-2251 (a chromogenic plasmin substrate) and amiloride (an uPA inhibitor).…”

Impact of Tissue Plasminogen Activator on the Neurovascular Unit: From Clinical Data to Experimental Evidence

“…It is also interesting to note that NMDA-dependent nitric oxide production has been recently demonstrated to be dependent on tPA (Parathath et al, 2006), unveiling a possible role of tPA in the control of local cerebral perfusion. In contrast to its well-admitted exacerbating effect on neuronal excitotoxic necrosis (Nolin et al, 2008), tPA modulates apoptotic neuronal (Liot et al, 2006) and oligodendrocyte death independently of its proteolytic activity (Correa et al, 2011). Together with the previous observations that nonproteolytically active tPA can activate microglia after binding to annexin II (Siao and Tsirka, 2002), these antiapoptotic mechanisms of tPA strongly support the notion of a 'cytokine-like' or even 'neurotrophic-like' function of this molecule.…”

A nonfibrin macromolecular cofactor for tPA-mediated plasmin generation following cellular injury

“…15 Hence, fibrinindependent tPA-mediated plasmin generation sensitizes neurons to excitotoxicity-an insult that contributes to infarction during stroke, 17 traumatic brain injury, 18 and numerous other neurodegenerative paradigms. Whereas ␤-amyloid and NG2 are likely cofactors for plasmin generation during Alzheimer disease 19 and spinal cord injury, 20 respectively, the "fibrin-like" cofactor that promotes tPA-mediated plasmin formation within the brain during stroke and other acute cerebral injuries remains entirely unknown.…”