ABSTRACT Stroke in small brain vessels in subcortical white matter (WM) regions account for 25% of all strokes. It leads to vascular cognitive impairment and dementia (VCID), and is the second leading cause of dementia overall. Despite such clinical importance, the pathophysiology of ischemic WM injury (WMI) and VCID is still poorly understood. Moreover, there is no yet an approved therapy for prevention and/or treatment of WM strokes and VCID. Here, we propose collaborative studies between the Zlokovic and Griffin labs on activated protein C (APC) pathways in the WM, and to evaluate therapeutic potential of APC-based therapies for ischemic WMI using a model of vasoconstriction of small brain vessels in the WM. Our previous studies using models of large artery infracts, brain trauma and neurodegeneration led to discovery of vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective, and anti-inflammatory activities of APC and its cytoprotective-selective mutants. In 2019, these findings have been translated into successfully completed phase 2 trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective-selective APC analog with >90% loss of anticoagulant activity. However, whether activation of APC pathways in the WM is beneficial or not during ischemic WMI, remains unknown. Our goals include: 1) providing proof of concept for hypothesized mechanisms for protective activities of APC in the WM; and 2) characterizing novel protease activated receptor 1 (PAR1)-related P1-47 and PAR3-related P3-42 APC-mimetic peptides, and 3) testing improved 3rd generation APC R-46-selective biologics for treating and preventing ischemic WMI and WM stroke. Our pilot data support our hypotheses that: i) APC will be beneficial for ischemic WMI via PAR1 cleavage at Arg46 to protect WM fiber tracts, oligodendrocytes and BBB from ischemic WMI (AIM 1); ii) APC-mimetic peptides derived from PAR1 and PAR3 sequences (e.g,, P1-47 and P3-42, (i.e., the tethered PAR agonists created by APC cleavages) exhibit synergistic biased agonism, and will elicit -arrestin 2-dependent cytoprotective signaling in brain endothelium and oligodendrocytes in vitro and in vivo after WM stroke (AIM 2); and iii) E56K-APC and D180E-APC newly engineered APC mutants have enhanced ability to cleave PAR1 at Arg46 and will provide improved APC biologics for WM stroke therapy (AIM 3). To address our hypotheses, we will use i) WM model of stroke; ii) new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 point mutations, and -arrestin 2-/- and G12-/- mice; iii) new APC-mimetic PAR1- and PAR3-related peptides with the respective PAR1 and PAR3 tethered-ligand amino acid sequences; iv) new APC R46-cleavage site selective biologics; v) in vivo mutiparametric longitudinal MRI of WM lesion volume, BBB integrity, blood flow, structural and connectivity changes, and tract-tracing based connectomics for circuit level analysis; vi) behavior tests; vii) immunohistology, neuropathology; and vii...