Vascular Proteomics Reveals Critical Roles of ADAMTS18 in Stroke Associated with Imbalanced Level of Von Willebrand Factor Versus ADAMTS13

Background and Purpose-Stroke is the second leading cause of death worldwide. Acute ischemic stroke (AIS) is frequently induced by internal carotid artery thrombosis. Clinically, most arterial thrombus formation is initiated by platelets adhering to the damaged vessel wall. The adherent platelets become activated and bind with fibrinogen and von Willebrand factor (vWF), allowing formation of a platelet aggregate or mural thrombus. Identifying new mechanism involved in this pathogenesis is critical for understanding the molecular basis of thrombus formation and ultimately developing new therapeutic interventions for stroke. The ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) family comprises 19 secreted metalloproteinases that cleave extracellular matrix components and have diverse functions in numerous disease and physiological contexts. Several ADAMTS genes (ADAMTS -7,-8, 12, and 13) have been shown to be involved in cardiovascular disease and pediatric stroke. ADAMTS18 remains "orphan" protease. We have previously shown that ADAMTS18 deficiency causes abnormal remodeling of the common carotid artery (CCA) and an increase in carotid thrombosis and size of cerebral infarct in mice. Thus, the stroke-prone Adamts18 KO mice could be valuable for identification of molecular signatures of accelerated carotid thrombosis related to stroke. One of the major steps yet to be taken by the field is the widespread use of proteomics to uncover the critical molecular events and signaling pathway involved in ADAMTS gene-related stroke.

Methods and Results-Here, we carried out the first proteomics comparison of protein expression patterns in CCA and abdominal artery (AA) between Adamts18 knockout (KO) mice and their wild-type (WT) littermates. A total of 1654 proteins were identified in four separate CCA samples (0.4-1 mg/wet weight each), and 1810 proteins were detected in four separate AA samples (2.3-3.6 mg/wet weight each). The abundances of 154 proteins in CCA and 55 proteins in AA were significantly different between KO and WT mice. Ingenuity^® Pathway Analysis revealed that ADAMTS18 deficiency predisposes mice to stroke by altering the abundance of many proteins related to cytoskeleton, prothrombotic activity, coagulation, angiogenesis, oxidative homeostasis, and energy metabolism. Western blotting and qRT-PCR analyses further revealed the imbalanced level of von Willebrand factor and ADAMTS13, the pivotal pathogenic factors of ischemic stroke, in CCA of KO mice.

Conclusion and Significance-Results of this first vascular proteomic study of Adamts18 KO mice establish a substantial new resource for investigation of how ADAMTS gene affects cellular phenotypes of stroke. These new findings contribute to inform novel prevention strategies and direct therapeutic development for stroke.