Protein Disulfide Isomerase Negatively Regulates Platelet Function through Modulation of the Thromboxane A2 Receptor

Protein disulfide isomerase (PDI) is an intracellular oxidoreductase that cleaves and forms disulfide bonds in substrate proteins to facilitate their proper folding. However, platelets and endothelium release PDI upon vascular injury. This extracellular PDI serves a critical function in platelet activation and thrombus formation. A PDI antagonist is currently in phase II/III clinical trials for thrombosis prevention. Yet the mechanisms of how PDI enables platelets to respond to the local redox environment remain poorly understood. In particular, the substrates on which PDI acts to modify platelet function have yet to be elucidated.

To identify platelet receptors that are controlled by PDI, we evaluated the ability of PDI to modify the effect of different platelet agonists on a platelet aggregation. Recombinant PDI (rPDI) had no effect on platelet aggregation induced by a dose curve of a PAR1 activating peptide, a PAR4 activating peptide, thrombin, collagen, or epinephrine. Unexpectedly, rPDI markedly inhibited aggregation induced by the stable thromboxane A₂ analog U46619 (13.8±5.6% of control). In contrast, ERp57, a related thiol isomerase, had little effect and a mutant PDI that lacks the active site cysteines responsible for disulfide bond rearrangement failed to inhibit U46619-induced platelet aggregation, implicating disulfide bond rearrangement. rPDI also blocked U46619-induced dense granule secretion (15.0±5.5% of control), α-granule secretion (24.4±0.1% of control) and α₂β₃ activation (65.0±0.1% of control). Conversely, inhibition of endogenous PDI augmented U46619-mediated platelet activation. To confirm that PDI was acting upstream in the U46619 activation pathway, we evaluated proximal signaling events. rPDI significantly decreased U46619-induced p38 MAPK phosphorylation in both platelets and HEK cells overexpressing the thromboxane prostanoid (TP) receptor, indicating inhibition of Gα_12/13 pathways. In contrast, there was little inhibition of Ca²⁺ flux, indicating lack of inhibition of Gαq-mediated signaling. These results suggest the presence of a functional disulfide bond within the TP receptor that is sensitive to PDI and required for Gα_12/13 signaling.

Evaluation of the amino sequence of the TP receptor demonstrates two cysteines, Cys-105 in the extracellular loop I and Cys-183 in the extracellular loop II, that form a disulfide bond and may be modified by redox environment. To determine if the platelet TP receptor is sensitive to modification of redox environment, we evaluated U46619-induced signaling in the presence of GSH and GSSG. Incubation with >0.25 mM GSH reduced U46619-induced platelet aggregation to 11.0±0.6% of control, whereas GSSG did not alter platelet aggregation in response to U46619. These results indicate a functional disulfide bond in the TP receptor that responds to changes in redox environment.

Evaluation of additional agonists demonstrated that ADP-induced aggregation was sensitive to rPDI. rPDI did not affect the initial response to 3.5 μM ADP, but completely abrogated the secondary wave of aggregation. This defect in the secondary wave could be overcome by using 5 μM ADP, thus suggesting impaired endogenous ADP release. Indeed, rPDI blocked dense granule release even in response to 5 μM ADP. This result is consistent with the known reliance of dense granule release on TP stimulation and suggests that the negative regulation of PDI on ADP-induced platelet activation is secondary to the inhibition of PDI on the TP receptor.

These are the first studies to our knowledge that demonstrate the ability of a thiol isomerase to modulate the function of a G-protein coupled receptor via disulfide bond rearrangement. This mechanism could provide a means to modify signaling through the TP receptor in respond to changes in redox environment.