Shiga-Toxin Induced Inflammation and Thrombosis

Platelet-rich thrombi in the kidney may contribute significantly to the clinical problems, but little is known about the factors responsible for the generation of these thrombi. Platelet function is not directly activated by Shiga toxin, indicating that other mechanisms must be involved. Shiga toxin binds to receptors on epithelial and endothelial cells leading to inhibition of protein synthesis and a stress which can result in local inflammatory responses. Monocytes and macrophages can also release inflammatory chemokines and certain ones are powerful co-activators of platelet function, such as SDF-1 (stromal cell-derived factor 1) and MDC (macrophage-derived chemokine). We will therefore test the hypothesis that chemokines contribute to the vascular and thrombotic problems of Shiga-toxin infection. Our proposed research has three main aims directed to the above hypothesis. First, endothelial (EC) and epithelial (EP) cells and then monocytes and macrophages will be exposed to Shiga toxin and the cell media tested for their ability to activate platelet function, as assessed by aggregation, adhesion and secretion. Second, we propose to identify the chemokines and other factors in the media which may be responsible for platelet stimulation. Then, we plan to analyze whether chemokine-treated platelets adhere to EC previously exposed to chemokines under varied shear-stress conditions. New quenched- and continuous-flow techniques developed in our laboratory will be employed to follow not just the rapid functional kinetics (less than 5 sec), but also platelet biochemical events. Third, we will examine the need for low levels of primary platelet agonists such as ADP, to enable Shiga-toxin effects on function, using new blocking reagents and treatment of platelets with apyrase. Finally, the role of proteases in the Shiga-toxin effects on platelet function will be examined.