A role for the thiol isomerase protein ERP5 in platelet function

Abstract

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding. Analysis of human platelet membrane fractions identified the presence of the thiol isomerase protein ERP5. Further study showed that ERP5 is resident mainly on platelet intracellular membranes, although it is rapidly recruited to the cell surface in response to a range of platelet agonists. Blocking cell-surface ERP5 using inhibitory antibodies leads to a decrease in platelet aggregation in response to agonists, and a decrease in fibrinogen binding and P-selectin exposure. It is possible that this is based on the disruption of integrin function, as we observed that ERP5 becomes physically associated with the integrin ␤ 3 subunit during platelet stimulation. These results provide new insights into the involvement of thiol isomerases and regulation of platelet activation. IntroductionIn classical terms, reduction/oxidation systems within a cell have been represented very simply. The cytoplasmic environment is hypoxic and reducing, whereas the extracellular environment is normoxic and oxidizing. The generation of a disulfide bond from 2 cysteine residues is an oxidation reaction. To correctly generate these bonds inside the cell, there are, therefore, a group of enzymes known as the thiol isomerases. These are capable of the formation, reduction, and rearrangement of the disulfide-bonding patterns of proteins, often as part of folding of nascent proteins. The thiol isomerase enzymes are anchored to the endoplasmic reticulum via KDEL-receptor proteins. [1][2][3] Recent studies have suggested additional functions for thiol isomerase enzymes: on the surface of cells, where they participate in receptor activation and remodeling, and substrate processing. [4][5][6] Protein disulfide isomerase (PDI) is the best-characterized thiol isomerase to demonstrate this dual functionality. A number of cell types, including bovine aortic endothelial cells, 7 rat hepatocytes, 8,9 and human B cells, 5,10 have been shown to secrete PDI, which associates with the cell surface. Cell-surface PDI has been implicated in the reduction of the disulfide-linked diptheria toxin heterodimer 11 and events triggering the entry of HIV into lymphoid cells. 6,12 On the basis of a series of investigations, initially by Detweiller and coworkers, a role for PDI in platelet physiology is now established. 4,[13][14][15][16] Early studies demonstrated PDI was present on the external membrane of activated and resting platelets, and proteins with thiol isomerase activity were secreted f...

Platelet Glycoprotein Ibβ/IX Mediates Glycoprotein Ibα Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear

“…␣/␣ or ␤/␤), is most likely. Investigations have shown that platelet function could be regulated by a redox switch, a thiol-disulfide interchange catalyzed by enzymes, such as the protein disulfide isomerase (35) and other thiol isomerases (36). Several major platelet surface receptors have been reported to utilize this mechanism to regulate the affinity to their ligands (37), including fibrinogen receptor ␣ IIb ␤ 3 , collagen receptor ␣ 2 ␤ 1 , and ADP receptor P 2 Y 12 .…”

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

“…Indeed, it is likely that both RGD peptides and Ero1␣ compete for fibrinogen binding through occupancy of the RGD binding sequence. Recently, additional thiol isomerase enzymes, called endoplasmic reticulum proteins 5, 29, 44, 57, and 72 (ERP5, ERp29, ERp44, ERp57, and ERp72) and transmembrane thiol isomerase 3 (TMX3), were identified in platelets and megakaryocytes (35,36). ERP5 was found to behave similarly to PDI because of the following: (a) its levels on the platelet surface are rapidly elevated upon activation with agonists; (b) antibodies that block the thiol isomerase activity of ERP5 abolish platelet activation, and (c) ERP5 becomes associated with ␣ IIb ␤ 3 upon platelet stimulation.…”

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

“…Free thiols become available on the platelet surface upon activation [10] and those present on integrin α IIb β 3 provide a direct target for redox modification [33]. Thiol-disulphide exchange within α IIb β 3 , catalysed by an endogenous isomerase activity [34], by PDI [11,35], or by ERP5 [36] is necessary for transition to its active conformation. Thus the thiol redox state of α IIb β 3 influences platelet adhesive function and any agent that modifies this redox state may interfere with platelet aggregation.…”