The control of platelet function and coagulation is a fine balance between activation and inhibitory
mechanisms. Platelets become rapidly activated by multiple receptors agonists and have a central role in
thrombosis in acute coronary syndromes, and other disease states. Similarly, naturally occurring
anticoagulants are critical in preventing fibrin generation and thrombosis. Fivemembers of theprotein disulfide
isomerase (PDI) family of enzymes, PDI, ERp57, ERp5, ERp72 and ERp46 potentiate activation of ¿IIb¿3 and
thrombosis.We discovered atransmembrane
member of the PDI family found to inhibit activation of ¿IIb¿3 and
member of the PDI family in platelets, TMX1, which
thrombosis. TMX1
is the first
acts by a novel
mechanism of oxidizing thiols to disulfide bonds and is the last checkpoint inhibitor of the platelet activation
pathways that lead to conformational changes in ¿IIb¿3 and fibrinogen binding. The prothrombotic PDIs are
secreted from platelets and endothelial cells and support fibrin generation at the site of vascular injury. We
found that TMX1 is expressed on platelets and endothelial cells but,
negatively
procoagulant
in contras to the other PDIs, TMX1
regulates fibrin generation. One mechanism by which TMX1 inhibits coagulation is by limiting the
effect of endothelial cells and platelets. We propose to study
t
vascular TMX1 as a dual negative
regulator of platelets and coagulation by addressing the following Specific Aims. We will characterize 1. the
role of TMX1 in thrombus formation; 2. the mechanism of inhibition of ¿IIb¿3 activation by TMX1; 3. the effect
of TMX1 on the other platelet PDIs, and on other platelet surface substrates. A principal technique used will be
the laser-induced injury model of thrombosis. We will study the mechanism by which TMX1 negatively
regulates coagulation. To determine the underlying mechanisms by which TMX1 inhibits platelet function we
will integrate a platelet knockout mouse model with mass spectrometry-based identification of functional
cysteines. This proposal will determine the mechanisms by which TMX1 works, and how TMX1
counterbalances the PDI enzymes that support activation of ¿IIb¿3. Characterization of the negative regulatory
role of TMX1 will provide novel insight into how the network of PDI enzymes regulate thrombosis. Studies on
how TMX1 maintains the balance between thrombosis and hemostasis will elucidate optimal ways to promote
hemostasis and inhibit thrombosis and provide a basis for studying TMX1 in disease states.