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Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke, but prevention of thrombus formation with an antithrombotic increases hemorrhagic risk. The RE–LY trial comparing warfarin with the direct thrombin inhibitor, dabigatran, in non–valvular AF patients demonstrated significant reductions in thromboembolic stroke and intracerebral hemorrhage with dabigatran. Focal ischemia is also associated with acute loss of β1 integrin from brain microvessels. We have shown that interaction of β1 integrin with the extracellular matrix stabilizes expression of the tight junction protein claudin–5 and maintains permeability barrier integrity, and that interfering with this interaction increases permeability. Since thrombin has been implicated in vascular injury during ischemia, we hypothesized that dabigatran could decrease the risk of intracerebral hemorrhage by direct inhibition of thrombin–induced permeability via protection of β1 integrin.
Primary murine brain endothelial cells (mBEC) were exposed to murine thrombin (0–10 U/ml) for 1 h prior to measuring permeability to 4–kDa FITC dextran. Expression of β1 integrin and claudin–5 following thrombin exposure was measured by flow cytometry.
Thrombin increased mBEC permeability in a concentration–dependent manner (Papp for control: 2.98 × 10–6 ± 0.87 × 10–6 cm/s, for 10 U/ml thrombin 4.37 × 10–6 ± 0.39 × 10–6 cm/s, n = 6, 2p = 0.0109). Thrombin (up to 20 U/ml) did not cause endothelial cell death. Pre–treatment of mBEC with dabigatran (24 h, 500 nM) completely abrogated the effect of 10 U/ml thrombin on permeability. These results were confirmed in the immortalized bEND.3 mouse brain endothelial cell line. Expression of β1 integrin and claudin–5 were not affected by thrombin treatment, suggesting that thrombin increases mBEC permeability by direct modification of other tight junction protein(s) or via a transcellular mechanism.
Dabigatran blocks thrombin–induced brain endothelial cell permeability in vitro. Future studies will explore the mechanisms by which thrombin leads to increased microvessel permeability and by which dabigatran promotes microvessel integrity.
Poster Sessions, Expo North
Saturday, March 09, 2013, 3:45 p.m.–4:30 p.m.
Session Title: Basic Science Underpinnings of Vascular Disease
Abstract Category: 33. Vascular Medicine: Basic
Presentation Number: 1166–162
- 2013 American College of Cardiology Foundation