Date Published: January 25, 2017
Publisher: Public Library of Science
Author(s): William D. Whetstone, Breset Walker, Alpa Trivedi, Sangmi Lee, Linda J. Noble-Haeusslein, Jung-Yu C. Hsu, Michael G. Fehlings.
Thrombin-induced secondary injury is mediated through its receptor, protease activated receptor-1 (PAR-1), by “biased agonism.” Activated protein C (APC) acts through the same PAR-1 receptor but functions as an anti-coagulant and anti-inflammatory protein, which counteracts many of the effects of thrombin. Although the working mechanism of PAR-1 is becoming clear, the functional role of PAR-1 and its correlation with APC in the injured spinal cord remains to be elucidated. Here we investigated if PAR-1 and APC are determinants of long-term functional recovery after a spinal cord contusive injury using PAR-1 null and wild-type mice. We found that neutrophil infiltration and disruption of the blood-spinal cord barrier were significantly reduced in spinal cord injured PAR-1 null mice relative to the wild-type group. Both locomotor recovery and ability to descend an inclined grid were significantly improved in the PAR-1 null group 42 days after injury and this improvement was associated with greater long-term sparing of white matter and a reduction in glial scarring. Wild-type mice treated with APC acutely after injury showed a similar level of improved locomotor recovery to that of PAR-1 null mice. However, improvement of APC-treated PAR-1 null mice was indistinguishable from that of vehicle-treated PAR-1 null mice, suggesting that APC acts through PAR-1. Collectively, our findings define a detrimental role of thrombin-activated PAR-1 in wound healing and further validate APC, also acting through the PAR-1 by biased agonism, as a promising therapeutic target for spinal cord injury.
Spinal cord injury results in direct vascular damage, followed by an inflammatory response and a cascade of events that further disrupt the blood-spinal cord barrier. One of the earliest factors produced following injury is the serine protease thrombin [1, 2], which is a 36 kDa protein comprised of two chains, A and B, that are linked by a disulfide bond . Thrombin primarily contributes to coagulation by the conversion of fibrinogen to fibrin and the activation of platelets and several coagulation factors [4–6]. It is also known to mediate inflammation and endothelial permeability [7, 8].
All procedures involving animals were approved by the UCSF Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. PAR-1 null mice were a generous gift from Dr. Sean Coughlin (Cardiovascular Research Institute, University of California, San Francisco) and bred on a C57Bl/6 background. Adult PAR-1 null mice have been extensively characterized and demonstrate normal physiology [35, 36]. All efforts were made to minimize animal suffering.
The thrombin receptor PAR-1 is critical to a number of wound healing events after spinal cord injury. We demonstrate that spinal cord-injured PAR-1 null mice display reduced leukocyte infiltration, vascular barrier disruption, glial scar formation, and improved white matter sparing and locomotor recovery. We further find that injured wild-type mice treated with rAPC, presumably acting through the same PAR-1 receptor, improves neurological recovery to a similar degree to that of the injured PAR-1 null mice, suggesting that APC is acting through PAR-1 by the property of biased agonism. Together, these findings suggest that the PAR-1 receptor is an effective target for therapeutic intervention after spinal cord injury.
In conclusion, we have demonstrated a detrimental role for thrombin-activated PAR-1 in wound healing and locomotor recovery after spinal cord injury. Administration of APC counteracts thrombin-PAR-1-induced adverse effects and provides protection. APC has been shown to exert beneficial effects in a spinal cord compression model in the rat  as well as in spinal cord ischemia in the rat and rabbit [77, 99]. Together with the current study, APC has now been independently validated in three species using different models of spinal cord injury. Understanding the mechanisms underlying the efficacy of APC provides opportunity for its further refinement with the goal of optimizing long-term neurological outcomes and translating this effort to human clinical trials.