Platelet-Localized FXI Promotes a Glycoprotein Ib Alpha Dependent Feedback Loop in Arterial Hypertension and Vascular Inflammation

Backgroud: Vascular inflammation is a hallmark of atherosclerosis and arterial and venous thromboembolic disease. Arterial hypertension is a highly significant risk factor for death and cardiovascular disease. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring leukocyte recruitment and reactive oxygen species production within the vessel wall. Interactions of platelets, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating thrombosis. Platelets provide a pro-coagulant surface for amplified thrombin generation in hemostasis and thrombosis, and play important roles in vascular inflammation by preserving vascular integrity. They also promote leukocyte recruitment in wire injury and angiogenesis models through glycoprotein Ib alpha (GPIbalpha) interacting with the integrin alphaMbeta2 (CD11b/CD18 or Mac-1) on leukocytes and regulate monocyte and neutrophil activation. How platelets, coagulation factors, leukocytes and the vessel wall cooperate to promote vascular inflammation in arterial hypertension is unclear.

Objective: The aim of this work was to explore the roles of TF, FXI, FXII, thrombin and platelet GPIbalpha on inflammatory monocyte-driven vascular dysfunction and arterial hypertension in ATII infused mice and rats as well as in 5/6 nephrectomized (5/6Nx) rats.

Methods: FXII^-/-, FXI^-/-, and hIL-4R/Ibalpha mice and 5/6NX rats were used for this study. Mice and rats where treated with ATII (1 mg×kg^-1 ×d^-1 for 7 days) using osmotic minipumps. Blood pressure was recorded using tail cuff measurement and telemetry carotid implants. To assess vasodilator properties, isolated aortic segments were mounted to force transducers in organ chambers to test their response to acetylcholine (ACh) and glyceryl trinitrate (GTN). ROS production was quantified within the aorta using dihydroethidium-derived fluorescence. Thrombin generation in platelet rich plasma (PRP) was measured using calibrated automated thrombography.

Results: ATII induces an upregulation of tissue factor, thrombin-dependent endothelial cell VCAM-1 expression and integrin alpha4- and platelet-dependent leukocyte adhesion to arterial conductance vessels. Depletion of platelets using an anti-GPIbalpha antibody as well as injection of anti-Mac-1 directed against the CD11b/CD18 integrin on leukocytes similarly prevented leukocyte rolling and adhesion. Reduced vascular recruitment of leukocytes was paralleled by diminished vascular ROS production assessed by the superoxide-sensitive dye dihydroethidium. ATII-induced vascular dysfunction unexpectedly involved the activation of FXI but not FXII. Moreover, inhibition of FXI synthesis attenuates blood pressure increase in response to ATII. The platelet FXI receptor GPIbalpha supports the upregulation of thrombin feedback activation in ATII-treated mice. Blockade of TF during ATII administration attenuated both endothelial dysfunction (acetylcholine concentration-relaxation curves) and smooth muscle dysfunction as demonstrated by improved vascular relaxation in response to the endothelium independent vasodilator glyceryl trinitrate and reduced ROS within the vessel wall. Five/6Nx rats had endothelial dysfunction that was prevented by FXI Antisense oligonucleotide. Thrombin-induced thrombin generation was increased in PRP of 5/6 Nx rats and markedly diminished by FXI Antisense oligonucleotide, mimicking the results from ATII-infused mice.

Conclusion: Our results reveal a critical role of platelet GPIbalpha to promote localized thrombin amplification and a FXI-thrombin feedback loop in ATII-induced vascular inflammation. Importantly, pharmacologic inhibition of FXI synthesis is sufficient to prevent thrombin propagation on platelets, to reduce vessel wall leukocyte infiltration, and to diminish ATII-induced endothelial dysfunction and arterial hypertension in mice and rats.Targeting FXI could be a novel therapeutic possibility to interrupt this heterotypic cellular coagulation-inflammatory circuit.