In vivo, platelet adhesion/aggregation on activated endothelium may proceed via upregulation of endothelial cell (EC) molecules adhesive for platelets, especially 1v23 (the vitronectin receptor), ICAM-1, P-selectin, and ultra large von Willebrand factor (vWF) strings, accompanied and followed by tissue factor (TF)-driven mural platelet thrombosis in arteries or arterioles. The necessary EC activation may arise via several routes, including oxidized low density lipoproteins, bacterial toxins, and/or cytokines. The present work will test the hypothesis and paradigm that upregulation of these adhesion molecules permits the initiation of platelet adhesion/aggregation on endothelium, a process which is then amplified by both EC-associated TF and blood- borne TF. Blood-borne TF exists in the form of microparticles derived from leukocytes, platelets, and ECs. Upregulation of the EC-associated type of TF may take place by a variety of mechanisms, studied here: a) increases in TF mRNA stability and/or decreases in TFPI mRNA stability, b) enhancement of phosphatidylserine on the EC membrane outer leaflet, and c) reduction in the regulatory ability of EC-associated TFPI. We further hypothesize that mural platelet thrombi, in turn, slow blood flow sufficiently to allow activated endothelium to assume an even more procoagulant phenotype. A prime example of this paradigm is the epidemic childhood hemolytic uremic syndrome (HUS), in which food-borne, Shiga toxin (STX)-producing E. coli lead to a systemic inflammatory and prothrombotic state, with complications which can include renal failure, seizures, coma, and death. In an in vitro model of HUS we have demonstrated that STX significantly augments functional TF on monolayers of human glomerular ECs (HGECs), and markedly enhances platelet strings (associated with ultra large vWF strings) and small aggregate formation on these monolayers.
Specific Aim 1 is to determine the mechanism(s) of TF augmentation observed with exposure of activated HGECs to STX.
Specific Aim 2 is to demonstrate that STX augments platelet adhesion/aggregation in flowing blood on activated HGECs via upregulation of the above adhesion molecules, but also via upregulation of the TF pathway. These studies should elucidate the potential of therapeutic interventions in HUS directed at blocking adhesion molecules and blocking EC surface and/or blood-borne TF.
We will test the hypothesis that the presence of certain molecules on vascular endothelium (the blood vessel wall) which are adhesive for blood platelets are what initiates platelet thrombus formation on the inflamed blood vessel wall. This thrombus formation is then amplified by the clotting activity known as tissue factor, expressed on the surface of this same inflamed endothelium. A prime application of this hypothesis, we believe, is the epidemic childhood hemolytic uremic syndrome (HUS), in which food- borne toxin-producing E. coli leads to a disease state with complications including kidney failure, seizures, coma, and death. Our studies promise to clarify the potential of treatments for HUS directed at blocking the above adhesive molecules and vessel wall tissue factor.
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