Neuroprotection and vasculoprotection using genetically targeted protease-ligands

Area of Science:

• Neuroscience
• Vascular Biology
• Biochemistry

Background:

• Thrombin and activated protein C (APC) are coagulation factors impacting brain via protease-activated receptors (PARs).
• Wild-type (WT) proteases affect cell survival; therapeutic APC forms are in development.
• Engineered proteases aim to separate procoagulant/anticoagulant from cytoprotective effects.

Purpose of the Study:

• To investigate the differential effects of engineered thrombin and APC on neuronal survival and vascular integrity in a rodent stroke model.
• To determine if cytoprotective and coagulation effects of these proteases can be dissociated.

Main Methods:

• Middle cerebral artery occlusion (MCAo) model in rodents (2-hour and 4-hour durations).
• Administration of WT murine APC, mutant APCs (5A-APC, 3K3A-APC), engineered thrombin, and a GpIIb/IIIa inhibitor (GR144053).
• Assessment of neuronal degeneration and vascular disruption post-reperfusion.

Main Results:

• WT APC and 5A-APC reduced neuronal death after 2-hour MCAo.
• 5A-APC protected neurons during 4-hour MCAo, but both WT APC and 5A-APC exacerbated vascular disruption.
• Human APC mutants reduced delayed neuronal injury, yet high doses worsened vascular leakage.
• Mutated thrombin worsened vascular damage without affecting neurons; GR144053 had no significant effect.

Conclusions:

• Engineered proteases demonstrate differential effects on neurons and vasculature in stroke models.
• Murine 3K3A-APC and 5A-APC protected neurons but worsened vascular leakage at high doses.
• Cytotoxic effects of plasma proteases may be separable from coagulation effects; dose and timing are critical.