tPA variant tPA‐A296–299 Prevents impairment of cerebral autoregulation and necrosis of hippocampal neurons after stroke by inhibiting upregulation of ET‐1

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The primary drug treatment for ischemic stroke is tissue‐type plasminogen activator (tPA) (Lapchak, 2002). However, tPA is also neurotoxic (Choi, 1992). In part because it exacerbates uncoupling of cerebral blood flow (CBF) and metabolism that occurs post stroke by augmenting pre‐existing hyperactivity of N‐methyl‐D‐aspartate receptors (NMDA‐Rs), we propose that tPA is neurotoxic. Glutamatergic hyperactivity occurs in animal models of stroke even without administration of exogenous tPA (Lipton, 1999), and NMDA‐R antagonists are protective (Armstead, 2002a; Armstead, 2002b). Binding of glutamate to NMDA‐Rs elicits cerebrovasodilation, which couples local metabolism to CBF, but fosters excess excitoxicity (Armstead, 2002b; Faraci & Heistad, 1998). Cerebral autoregulation is a homeostatic mechanism by which the neurovascular unit (NVU) regulates CBF across a range of blood pressures. NMDA‐R activation contributes to impairment of cerebral autoregulation after traumatic brain injury (TBI; Faraci & Heistad, 1998). NMDA‐R driven NVU‐mediated impairment of autoregulation following TBI is linked to Glasgow Coma Scale (GCS), with greater autoregulatory impairment associated with worse GCS (Freeman, Udomphorn, Armstead, Fisk, & Vavilala, 2008). We speculate that these NVU functions link cerebral hemodynamics with cognitive function.
A critical barrier to identifying mechanisms of stroke in humans is the reliance upon rodent models that, because of the small size of the subjects, have the disadvantage of not permitting repeated measurements of systemic physiological variables and CBF. In contrast, by virtue of having a gyrencepahalic brain that contains substantial white matter similar to humans, which is more sensitive to ischemic damage than gray matter, pigs offer a key advantage. This study is based on prior findings in pigs after TBI or stroke in which, by activating NMDA‐Rs (Armstead, 2002a; Armstead, 2002b; Armstead et al., 2010b; Armstead et al., 2011a; Armstead, Kiessling, Riley, Cines, & Higazi, 2011b), we have shown that wild type (wt)‐ tPA impairs cerebral autoregulation. Although the NMDA‐R antagonist MK 801 protects against cerebral dysregulation after stroke and TBI (Armstead 2002a; Armstead, Riley, Kiessling, Cines, & Higazi, 2011b), its toxicity limits use in humans, indicating the need for novel approaches.
Moreover, the mechanism underlying the transition of NMDA‐R activation from vital to neurotoxic effects (Ankarcrona et al., 1995; Collingridge & Lester, 1989; Nakanishi & Masu, 1994) is uncertain. Toxic levels of glutamate that develop in the setting of stroke decrease cAMP (Ginty et al., 1993; Park et al., 2008; Shaywitz & Greenberg, 1999). Stroke is also associated with elevation of phosphorylated JNK mitogen‐activated protein kinase (MAPK) and of the spasmogen endothelin‐1 (ET‐1; Faraci & Heistad, 1998), both of which have been implicated in the impairment of cerebral autoregulation (Armstead, 1999; Armstead et al., 2011a; Freeman et al., 2008). However, the relationship between phosphorylated JNK, ET‐1 levels, and outcome after stroke is unknown.
Based on our prior finding that the “docking site” in wt‐tPA is required to bind and activate NMDA‐Rs (Nassar et al., 2010), we constructed a variant (tPA‐K296A/H297A/R298A/R299A [tPA‐A296–299]) that prevents the enzyme from binding to NMDA‐Rs, but retains fibrinolytic activity. Recent data show that, when given in a therapeutically relevant timeframe after stroke in pigs, tPA‐A296–299 prevents acute impairment of cerebral autoregulation by increasing intracellular cAMP and blocking upregulation of phosphorylated JNK (Armstead, Riley, Yarovoi, Higazi, & Cines, 2016). We hypothesize that activation of NMDA‐Rs by wt‐tPA after stroke leads to upregulation of JNK followed by an increase in ET‐1, imparing cerebral autoregulation and leading to tissue injury. As a corollary, we tested the hypothesis that a tPA variant that limits NMDA‐R activation, but retains fibrinolytic activity, would improve outcome by preventing ET‐1 upregulation, thereby preserving cerebrovascular autoreulation and tissue viability.

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