Background: Thiol isomerases facilitate the rearrangement of protein disulphide bonds required for the correct folding of secreted proteins. They typically localize in the endoplasmic reticulum but some cells have thiol isomerases in granular structures or on the cell surface. Thiol isomerases are key regulators of platelet activation and blood coagulation and this requires that they are exposed on the surface of activated platelets. However, understanding the origin and localization of platelet thiol isomerases and the mechanisms underlying their release to the platelet surface is necessary to further elucidate their role in thrombosis and haemostasis. Aim: we explored the biogenesis and trafficking of the thiol isomerases PDI and ERp57 in megakaryocytes, identified the compartment where they reside in platelets and characterized the cellular events responsible for their movement to the platelet surface. Methods and Results: Using immunofluorescence microscopy we observed the distribution of thiol isomerases throughout mouse and human megakaryocyte development. We determined that the biogenesis of thiol isomerases is distinct from that of cargo proteins destined for the platelet granules, that are trafficked through the trans-Golgi defined by TGN46 (red) or recycling endosomes containing the transferrin receptor (TFR). Immunofluorescence microscopy and subcellular fractionation of platelets revealed that thiol isomerases are mainly present in the inner cell-surface membrane region defined by sarco/endoplasmic reticulum calcium ATPase 3 (SERCA3) and their distribution does not change in the absence of platelet α-granules (Nbeal2 -/- mouse platelets or platelets of patients affected by Gray Platelet Syndrome). Thiol isomerases were secreted to the surface of activated platelets, as measured by flow cytometry and confocal microscopy, and their movement was prevented by Latrunculin A that blocks the polymerization of actin (46±8 % and 77±12% inhbition, for PDI and ERp57, respectively), but not by the absence of membrane fusion mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4 (absent in platelets from Unc13dJinx mice). Conclusions: PDI and ERp57 are synthesized and packaged into a compartment that is distinct from known granules and localize to the inner surface of the paletelet outer membrane identified by SERCA 3. The movement of thiol isomerases on the surface of activated platelets is driven by the polymerization of actin, but does not depend on the vesicular-membrane fusion mediated by Munc13-4. Defining the origin, trafficking and mechanism of secretion of platelet thiol isomerases may aid the development of selective inhibitors that could represent new antithrombotic strategies.