Thrombin and Protease-Activated Receptors
Thrombin is a serine protease that plays a critical role in coagulation in distal microvessels. In addition, thrombin has pleiotropic extravascular effects. It can induce protection at low doses but act as a neurotoxin at high doses, killing cells via the protease-activated receptors (PAR). Using a protease activatable cell-penetrating probe, the Lyden Laboratory has shown directly that thrombin protease activity participates in damaging the neurovascular unit. The lab did this by comparing quantified vascular leakage and cell death markers with the quantity of thrombin activation (probe fluorescence) in the parenchyma after middle cerebral artery occlusion (MCAo). The Lyden Lab blocked thrombin with a direct thrombin inhibitor, argatroban, and greatly reduced ischemic edema and tissue damage at realistic therapeutic time window (delay dose three hours after reperfusion).
The Lyden Laboratory also exacerbated tissue damage by infusing thrombin during ischemia. The results suggested a critical role for thrombin in mediating brain injury in focal ischemia. The lab hypothesizes that thrombin partially mediates edema and cell death during stroke via activity at the PAR-1 receptor. The Lyden Laboratory has made significant progress in studying this mechanism of neuroprotection post-MCAo with different activated protein C (APC) mutants selective for PAR-1 receptors (3K3A-APC mutant in Phase I clinical trial). The lab has made significant progress in understanding the role of PAR-1 by using lentivirus-mediated shRNA knockdown of PAR-1 in rats and by studying the effect of stroke in aged PAR-1, PAR-3 and PAR-4 knockout animals. In addition, we have developed conditional knockout animal models to further investigate the function of thrombin and thrombin receptors in the central nervous system.
Therapeutic hypothermia is the most powerful neuroprotectant ever documented in stroke models. The Lyden Laboratory showed the protective effect of a single degree Celsius (C) in our quantal bioassay model. However, this preclinical benefit has failed to translate into clinical trial success. Based on our parallel observations of astrocyte protection of neurons, members of the Lyden Lab asked whether cooling could disrupt the astrocyte-mediated protection of neurons. In fact, we showed that hypothermia interferes with the astrocytes, in a graded, temperature-dependent manner (Lyden et al. JCBFM2018). We then showed that ultra-fast cooling to 33°C for a short time was more powerful in the middle cerebral artery occlusion model than were longer cooling periods. These exciting and novel data, if confirmed, suggest a reason for the failures of therapeutic hypothermia in some clinical trials. Moreover, our data suggests testable hypotheses about the effects of temperature in modulating neurovascular unit protection during ischemia.