Role of Thiol Isomerase ERp5 in Thrombus Formation

Abstract

Abstract 370Protein disulfide isomerase is required for thrombus formation in various in vivo models of thrombosis. Another member of the thiol isomerase family, endoplasmic reticulum protein 5 (ERp5), is released from activated platelets and co-immunoprecipitates with beta 3 integrin (Jordan et al, 2005). We further investigated the association of ERp5 with the platelet fibrinogen receptor alpha IIb beta 3 and the significance of ERp5 release in thrombus formation in vivo. Recombinant purified ERp5 was labeled with Alexa 488 and used in direct binding assays to CHO cells expressing wild type (WT) alpha IIb beta 3, CHO cells expressing mutant alpha IIb beta 3 (containing an Asp119Tyr substitution in the beta 3 subunit) and to control CHO cells. The mutant alpha IIb beta 3 does not bind fibrinogen. ERp5 bound to CHO cells expressing wild type (WT) alpha IIb beta 3 in a dose-dependent manner but did not bind to CHO cells expressing mutant alpha IIb beta 3 or to control CHO cells. The relative increase in the geomean of Alexa 488-labeled ERp5 binding to 0.5 ×106 WT alpha IIb beta 3 CHO cells over that bound to control CHO cells was 20, 45 and 85% for ERp5 concentrations of 80, 160 and 400 nM respectively. Binding of ERp5 (160 nM) to WT alpha IIb beta 3 expressing CHO cells was further increased by 75% when the integrin was activated with 2 mM Mn2+ compared to non-activated WT alpha IIb beta 3 CHO cells. A role for ERp5 in thrombus formation was studied in the laser injury model of thrombosis in mouse cremaster arterioles using a rabbit polyclonal anti-ERp5 antibody, immunoaffinity purified against recombinant ERp5. This antibody detected ERp5 in the releasate of thrombin-activated mouse platelets in vitro by Western blot and on the surface of thrombin-activated mouse platelets by flow cytometry. Dylight 649-labeled anti-CD42b was infused into the mouse circulation to detect platelet accumulation and Alexa 488-labeled anti-ERp5 antibody at 0.05 ug/g, a dose that does not inhibit thrombus formation, was infused to detect ERp5. The fluorescent anti-ERp5 signal detected at the thrombus site was compared to the signal produced by a non-specific IgG labeled with Alexa 488 infused into a control mouse. Anti-ERp5 fluorescence was detected in the thrombus with kinetics that followed platelet accumulation whereas there was minimal signal from the control IgG. We examined whether higher doses of anti-ERp5 affect thrombus formation. Platelet and fibrin accumulation were detected using fluorescently labeled anti-CD42b antibody and monoclonal anti-fibrin-specific antibody respectively before or after the injection of unlabeled anti-ERp5 antibody or pre-immune IgG at 2.5 ug/g. Platelet and fibrin accumulation, expressed as area under the curve of the median integrated fluorescence over time, was obtained from 14 thrombi in 6 mice formed before infusion of antibody, 18 thrombi in 2 mice formed after infusion of control IgG and 29 thrombi in 3 mice formed after infusion of anti-ERp5. Anti-ERp5 infusion caused a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (p<0.01). There was no difference in platelet and fibrin accumulation before infusion of antibody and after infusion of control antibody. These results provide evidence for a role of a second thiol isomerase, ERp5, in thrombus formation, a function which may be mediated through its association with alpha IIb beta 3. Disclosures:No relevant conflicts of interest to declare.