Abstract/Summary

Thiol isomerases are endoplasmic reticulum (ER) resident proteins that modulate the formation, reduction and isomerisation of disulphide bonds, which are often rate-limiting reactions during protein folding. Recent evidence suggests that this family of proteins are surface presented in a number of different cell types, where they are important in cell remodelling and recognition. Previous research has identified thiol isomerases within platelet surfaces, including PDI, ERp5, ERp57 and ERp72, which play pivotal roles in the regulation of platelet function. ERp44 is a thiol protein with one hydrophobic N-terminal thioredoxin domain containing a CRFS motif, followed by two thioredoxin inactive domains arranged in a clover-leaf-like structure. It also acts as a multifunctional chaperone protein that regulates redox homeostasis and modulates thiol-mediated quality control, and calcium and KDEL-receptor signalling. Most functionally important platelet thiol isomerases have been identified to be present on the surface of platelets and their levels increase upon platelet activation. Also, the inhibition of the catalytic activity of these enzymes leads to diminished platelet function and thrombosis. These properties raise the possibility that ERp44 may also contribute to platelet regulation. Here, we investigated the expression and localisation of ERp44 in platelets and evaluated its role in the regulation of platelet function using affinity-purified function blocking anti-ERp44 antibodies in response to different platelet agonists. In addition, we have focused my attention on understanding how ERp44 is tightly associated with different surface substrate proteins in human platelets, using mass spectrometry and co-immunoprecipitation techniques. The expression of ERp44 in human platelets and mouse platelets, as well as in the human megakaryocytic cell line (MEG-01), was confirmed using western blot. The location, levels and subcellular associations of ERp44 with different organelles were therefore examined. Immunofluorescence microscopy (IFM) of permeabilised platelets revealed ERp44 to be dispersed throughout the cytoplasm in a punctuated arrangement. Following activation, ERp44 partially translocated to a ring-like staining pattern towards the plasma membrane in confocal images and colocalised with proteins known to be present in the membrane of dense tubular system. Subcellular fraction of platelets by sucrose density gradient centrifugation revealed ERp44 to be principally located in low-density fractions, consistent with interactions with the plasma membrane, and suggested that, like other platelet thiol isomerases, this enzyme is not located in α-granules. Indeed, cell surface localisation of ERp44 was detected on resting platelets by flow cytometry and found to increase following stimulation with 1U/mL thrombin. Platelets treated with anti-ERp44 AP antibodies inhibited a range of platelet activities, such as aggregation, fibrinogen binding to integrin αIIbβ3, degranulation, intracellular calcium mobilisation and integrin αIIbβ3 outside-in signalling. We determined the interactions of ERp44 with substrates or client proteins using mass spectrometry and co-immunoprecipitation. It was demonstrated that ERp44 interacts with at least 10 proteins; in particular, with Ero1 α, IP3R1 and fibrinogen. In summary, this study provided new insights into the role of ERp44 in the inhibition of platelet function and provided evidence for potential interactions of ERp44 with different substrates in platelets.