In this project we will further investigate two key observations made in the past funding period: 1) P-selectininduces the formation of pro-coagulant microparticles (MPs) from leukocytes, and 2) activation of the plateletmetalloproteinase ADAM17 (TACE) leads to platelet clearance.In the first aim, we will study the cell biology of MP production induced by IPS or a recombinant P-selectinmolecule (P-sel-lg), in vitro and in vivo. The working hypothesis is that MPs are released from the plasmamembrane through a budding process that employs cellular machinery used for the formation of multivesicularendosomes and for retrovirus budding. We will study the importance of ubiquitination and the cellularcytoskeleton in this process. The effect of shear stress on MP production and the adhesion moleculesinvolved in MP recruitment to thrombi will be examined together with Project 3. With Project 2, we willcharacterize MPs produced in mice over-expressing soluble P-selectin (deltaCT-P-sel mice) for their cytoskeletonand surface proteins. MP generation will be studied in cytoskeleton-mutant mice (filamin chimeras, gelsolin -/-).The role of the cytoplasmic domain of tissue factor (TF) in TF targeting to MPs and in the regulation of its procoagulantactivity will be studied in mice expressing TF lacking the CT domain (deltaCT-TF mice).In the second aim, we propose to study the role of TACE in platelet biology. We hypothesize that the signalingpathways that regulate the proteolytic activity of TACE are important for both platelet function in hemostasisand in the regulation of platelet clearance. We will examine the intracellular processing and the sub-cellularlocalization of TACE upon platelet activation or mitochondrial injury. It is our hypothesis that, in platelets,TACE translocation to the plasma membrane coincides with its activation. We will address the signalingpathways from mitochondrial injury to TACE activity with a focus on AMP-activated protein kinase (AMPK), ametabolic stress-sensing kinase, and on p38 MAP kinase. In in vitro and in vivo models of arterial and venousthrombosis, we will determine the role of TACE and the kinases involved in its activation in thrombusformation. For these studies we will use genetically modified mice and inhibitors of the activating kinases.In the third aim, we will address problems directly relevant to transfusion biology: how are damaged(mitochondrial injury or aging) platelets cleared from circulation, and how to improve the efficacy of platelettransfusions by adding TACE inhibitors to platelet concentrates and/or co-infusing P-sel-lg. We will test thehypotheses that TACE regulates platelet clearance by shedding of GPIba and/or clustering of GPIb-V-IXcomplexes, and that P-selectin, by producing pro-coagulant MPs, would enhance hemostasis inthrombocytopenic mice.
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