Angiotensin II (AngII) contributes to the pathogenesis of hypertension and other cardiovascular diseases. AngII induces a pro-oxidative, pro-inflammatory, and pro-thrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, it remains unclear whether and how the blood cell recruitment is linked to the production of reactive oxygen species (ROS). In this study, we addressed the contributions of AngII type-1 receptors (AT1r), and gp91phox to the recruitment of leukocytes and platelets and ROS production in venules during chronic (2 wks) infusion of AngII in wild type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle post-capillary venules. In WT mice, AngII infusion induced a time-dependent increase in the adhesion of leukocytes and platelets and enhanced ROS production in venules. These changes in blood cell adhesion and ROS production were not observed in AT1r−/− mice, in AT1r−/− bone marrow chimeras (blood cells deficient in AT1r), gp91phox−/−, gp91phox−/− chimeras (blood cells or endothelial cells deficient in gp91phox) and in WT mice rendered granulocytopenic via i.p injection of anti-mouse Gr-1 antibody. Thrombocytopenic WT mice (platelets depleted by i.p. injection of rabbit anti-mouse thrombocyte antiserum) responded similar to WT mice. These findings implicate leukocyte-associated AT1r and gp91phox in the induction of the pro-oxidative, proinflammatory and prothrombogenic phenotype assumed by microvessels that are chronically exposed to elevated AngII.

Patients with inflammatory bowel disease (IBD) are susceptible to microvascular thrombosis and thromboembolism. The increased incidence of thrombosis is accompanied by enhanced coagulation and abnormalities in platelet function. Clinical studies have also revealed alterations in platelet activation, enhanced platelet-leukocyte interaction, and elevated plasma levels of prothrombotic cytokines, such as IL-6. This study was directed towards determining whether: 1) experimental colitis, induced by 6 days of dextran sodium sulfate (DSS) ingestion, is associated with platelet activation and the formation of platelet-leukocyte aggregates (PLAs), 2) IL-6 deficiency alters these responses to DSS colitis, and 3) the platelet abnormalities observed in DSS mice can be recapitulated by chronic infusion of murine recombinant IL-6. Flow cytometry was used to characterize platelet function in heparin-anticoagulated whole blood. Platelets were identified by characteristic light scattering and membrane expression of CD41. Platelet activation was monitored using the expression of an activation epitope of GPIIb/IIIa integrin (with JON/A antibody). The combination of CD41, CD45.2, Gr-1, F4/80 and isotype control antibodies were used to detect and quantify aggregates of leukocytes, neutrophils and monocytes with platelets in control, wild type (WT) colitic, IL-6 -/- colitic, and WT mice implanted with IL-6 loaded Alzet osmotic minipumps (for 6 days). Our results indicate that DSS colitis is associated with increased numbers of activated platelets and the formation of aggregates of leukocytes (PLA), neutrophils (PNA) and monocytes (PMA) with platelets. These platelet responses to experimental colitis were largely undetected in IL-6 -/- mice. Chronic infusion (at a rate that yielded plasma IL-6 levels similar to those detected in DSS colitic mice) of IL-6 recapitulated the increased platelet activation and formation of PLA, PNA, and PMA observed in DSS-colitic mice. Collectively, these findings show that the altered platelet function detected in human IBD can be reproduced in an animal model of colonic inflammation and that interleukin-6 plays a critical role in the genesis of these platelet abnormalities in the setting of experimental IBD.