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Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 June ; 36(6): 1056–1057. doi:10.1161/ATVBAHA.116.307625.

How Does Protein Disulfide Isomerase Get into a Thrombus? Meenakshi Banerjee and Sidney W. Whiteheart Department of Molecular and Cellular Biochemistry, University of Kentucky

Keywords

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editorials; platelets; megakaryocytes; secretion; thiol isomerases

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Numerous studies have demonstrated a role for thiol isomerases (TI), such as protein disulfide isomerase (PDI), in thrombosis (reviewed in1). Using antibodies, inhibitors, inactive recombinant proteins, and transgenic animals, several groups have convincingly shown that platelet and endothelial cell enzymes contribute to normal platelet activation and deposition at vascular lesions. Based on these data, anti-TI therapeutics are being developed for use in humans.2 Platelets and endothelial cells contain at least 7 thioredoxin-like TIs; some of which appear to have overlapping functions.3 PDI, ERp57 and ERp5 have been studied in the most detail. For PDI, it appears to be released into the vascular microenvironment and retained via interactions with β3 integrins.4 Though important for thrombosis, it is unclear what critical substrates are modified by these enzymes or how they get into the extracellular space. To address the second question Crescente et al.5 examined how two TIs, PDI and ERp57, are packaged into platelets and how they are released upon platelet activation.

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Normally, TIs are present in the lumen of the endoplasmic reticulum (ER) or of other compartments of the secretory pathway. They facilitate the reshuffling of disulfide bonds in an oxidizing environment to mediate protein folding and maturation. As such, these enzymes are crucial to protein quality control and to the general trafficking of many membrane and secreted proteins. Given this important intracellular role, what are the TIs doing on the outside of an activated platelet in a growing thrombus? Data suggest that these enzymes could mediate β3 integrin activation to promote adhesion.6 Surface TIs may decrypt tissue factor thereby activating the coagulation pathway.7,8 GP1b, α2β1, thrombospondin-1 (TSP-1), vitronectin, thrombin, anti-thrombin, Factor XI, and fibrin have also been considered as potential substrates (reviewed in1). Additionally, TIs can denitrosylate NOmodified cysteines9, potentially reactivating inactive proteins. Despite these efforts, it is unclear whether a specific set of critical substrates or a generalized alteration in local redox environment accounts for the role of TIs in thrombosis.

Correspondence: Sidney W. Whiteheart, Ph.D., Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone, Lexington, KY, USA 40536, ; Email: [email protected] Disclosures: None.

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Our old images of platelets as simple, cell fragments containing granules, mitochondria and some membranes are gaining more definition (Figure 1). Recent studies demonstrate that platelets contain many of the organelles, excepting nuclei, which are generally associated with intact cells. These organelles contribute to platelet function in ways that we are only beginning to appreciate. The dense tubular system (DTS) is thought to be a calcium store and is analogous to ER.10 Golgi glycosyltransferases and sugar nucleotides have been shown to be present and active.11 Endosomal compartments12, multivesicular bodies (MVBs)13, and autophagosomes14 have all been detected in platelets. To determine where the TIs reside, Crescente et al.5 did an elegant series of immunofluorescence studies of both megakaryoctyes and platelets. They showed that PDI and ERp57 did not localize with classical granule markers nor with Golgi or endosomal markers. Consistently, no change in TI distribution was seen in NBEAL2−/− or Gray platelet syndrome platelets, lacking αgranules. The TIs did co-localize with SERCA3, calreticulin, and partially with calnexin, suggesting that the isomerases were present in an ER-like compartment (e.g., DTS). In megakaryoctyes, PDI and ERp57 were found in punctate structures in the cytoplasm and then concentrated at the tips of growing proplatelets. Taken as a whole, these data argue that PDI and ERp57 are in an ER-like structure that is transferred to proplatelets during differentiation.

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How are these thiol isomerases released from platelets and what does that tell us about the complexity of the platelet secretory system? To address these questions, Crescente et al.5 examined PDI and ERp57 exposure on stimulated platelets. As with P-selectin, TI exposure on activated platelets was inhibited by treatment with latrunculin A suggesting a role for actin polymerization in the secretion process. Interestingly, PDI exposure was not inhibited by the loss of the tethering factor, Munc13-4. Munc13-4 is essential for dense granule release and important for α-granule release.15 It is thought to work with the small GTPase, Rab27, to bring granule and plasma membranes together and thus promote Soluble NSFAttachment Protein Receptor (SNARE) protein-mediated membrane fusion.16 Does this mean SNAREs are not involved in PDI exteriorization? Perhaps, but there are a number of SNAREs present in platelets, not all of which may require Munc13-4 for function. Additionally, other Munc13 isoforms have been detected in platelets (Schraw and Whiteheart, 2004 unpublished). Does this suggest that PDI release is inherently different than classical granule cargo release? Probably, and it will be interesting to determine the mechanisms of this distinct exocytosis process.

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This final point begs questions about what platelets can release and from where it comes (Figure 1). The platelet releasate contains hundreds of proteins and small molecules. Are these components coming from classical secretory granules, i.e., dense and α-granules, or can activated platelets release the contents of their other organelles? From Crescente et al.5 it is clear that platelets release ER contents, i.e., PDI and ERp57. Other reports suggest that MVB, Golgi, and lysosomal contents are also released.17–19 Are different SNAREs used for each of these membrane fusion events? Are the various secretion steps similarly regulated and is there cross-talk? More importantly, how does this mixture of granule and organelle contents (containing signaling molecules, enzymes, and substrates) affect the microenvironment of a growing thrombus? Clearly the complex cell biology of these circulating fragments is enriching our views of what platelets can do and how they do it. Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 June 01.

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Acknowledgments Sources of Funding: This work was supported by National Institutes of Health (NIH) HL56652 and by American Heart Association 16GRNT27620001 (to SWW).

References

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Figure 1. The Complexity of Platelet Organelles

In resting platelets, the various cargo (depicted in the legend) are present in secretory granules, ER-derived compartments, and other organelles. Upon activation, granule/ organelle–plasma membrane fusion drives surface exposure and release of luminal cargo into the extracellular space.

Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2017 June 01.

How Does Protein Disulfide Isomerase Get Into a Thrombus?

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