Physical Proximity and Functional Association of Glycoprotein 1bα and Protein-disulfide Isomerase on the Platelet Plasma Membrane

Abstract

Platelet function is influenced by the platelet thioldisulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1b␣ (GP1b␣). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1b␣ presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by ϳ50% and PDI and GP1b␣ were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1b␣. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1b␣ on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1b␣ that resulted in exposure of a free thiol(s).The platelet thiol-disulfide balance is important for platelet function. Perturbation of platelet thiol status effects platelet aggregation and release. The low M r thiol compounds, reduced glutathione (GSH), cysteine, and 6-mercaptopurine, inhibit platelet aggregation induced by several agonists, while the disulfide-bond reducing agents dithiothreitol and ␤-mercaptoethanol promote aggregation (1). In addition, reaction of platelet sulfhydryl groups with the thiol specific compounds, diamide and N-ethylmaleimide, inhibits in vitro aggregation and clot retraction (2-5). These results imply that certain platelet thiol groups are critical for platelet aggregation. Furthermore, the observation that specific depletion of platelet GSH by 1-chloro-2,4-dinitrobenzene only marginally effects platelet aggregability implies that the critical thiol groups are associated with protein (6). In support of this notion, Yamada et al. (7) have shown that the anti-platelet aggregation actions of 2,2Ј-dithiobis(N-2-hydroxypropylbenzamide) are mediated through interaction of the compound with platelet protein thiol groups.Protein-disulfide isomerase (PDI) 1 is a noncovalent homodimer with a subunit molecular mass of 57 kDa that catalyzes thiol-disulfide interchanges that can result in formation, reduction, or rearrangement of protein disulfide bonds. It is generally considered that PDI is important for proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum (8 -10). PDI also functions as the ␤ subunits of prolyl-4-hyroxylase (11, 12) and the ␤ subunit of triglyceride transfer protein complex (13,14). Bovine aortic endothelial cells (15), rat hepatocytes (16), rat pancreatic cells (17), and human B cells (18,19) secrete PDI which associates with the cell su...

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

“…We studied changes brought about by GSNO in thiol redox state of MEG-01 cell surface proteins. Consistent with earlier reports [10,11] a number of such proteins were detectable by western blotting using MPB, an impermeable thiol specific probe. Cell treatment with GSNO caused a loss of thiol reactivity on most, but not all, proteins suggesting a selective process of GSNO-mediated thiol redox modification.…”

“…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.…”

“…For protein RSNOs (such as Snitrosoalbumin) to mediate platelet inhibition, prior exchange of NO onto a low molecular weight thiol appears necessary [8,9]. Free thiols are available on the platelet surface itself [10,11] and S-nitrosation of these thiols may be a further possible pathway of anti-platelet action. However the relatively unfavourable kinetics of transnitrosation reactions [12] suggest that some enzyme mediated system is required for the process to occur sufficiently rapidly to mediate signalling events.…”

Rutin- potent natural thrombolytic agent

“…Platelets are a rich source of extracellular PDI, expressing this protein on their surface and also secreting PDI in response to thrombin stimulation (Burgess et al, 2000;Cho et al, 2008). Endothelial cells also express PDI upon agonist stimulation or …”

“…Although having a C-terminal endoplasmic reticulum retention sequence, PDI has been identified at many diverse subcellular locations outside the endoplasmic reticulum. It has biological functions on the cell surfaces of lymphocytes, hepatocytes, platelets, and endothelial cells (Manickam et al, 2008;Hotchkiss et al, 1998;Essex, Li, 1999;Burgess et al, 2000;Bennett et al, 2000).…”