The transmembrane protein disulfide isomerase TMX1 negatively regulates platelet responses

Abstract

Secreted platelet protein disulfide isomerases, PDI, ERp57, ERp5, and ERp72, have important roles as positive regulators of platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first described transmembrane member of the protein disulfide isomerase family of enzymes. Using a specific antibody, the recombinant extracellular domain of TMX1 (rTMX1) protein, a knockout mouse model, and a thiol-labeling approach, we examined the role of TMX1 in platelet function and thrombosis. Expression of TMX1 on the platelet surface increased with thrombin stimulation. The anti-TMX1 antibody increased platelet aggregation induced by convulxin and thrombin, as well as potentiated platelet ATP release. In contrast, rTMX1 inhibited platelet aggregation and ATP release. TMX1-deficient platelets had increased aggregation, ATP release, αIIbβ3 activation, and P-selectin expression, which were reversed by addition of rTMX1. TMX1-knockout mice had increased incorporation of platelets into a growing thrombus in an FeCl₃-induced mesenteric arterial injury model, as well as shortened tail-bleeding times. rTMX1 oxidized thiols in the αIIbβ3 integrin and TMX1-deficient platelets had increased thiols in the β3 subunit of αIIbβ3, consistent with oxidase activity of rTMX1 against αIIbβ3. Thus, TMX1 is the first identified extracellular inhibitor of platelet function and the first disulfide isomerase that negatively regulates platelet function.

Graphical abstract

Figure 1.

Extracellular platelet TMX1 inhibits aggregation of human platelets. (A) TMX1 expression on the platelet surface increases with platelet activation. TMX1 expression on nonactivated (NA) and thrombin-activated platelets. Representative line graph (left panel) and combined results (right panel). Data are mean ± standard error of the mean (SEM), n = 5 for each group. Human platelets were incubated with 1 U/mL thrombin for 5 minutes, followed by flow cytometry measurement using the anti-TMX1 antibody B01P. The anti-TMX1 antibody B01P increases platelet aggregation induced by SFLLRN (B), convulxin (C), and thrombin (D). In these studies, human platelets were incubated with IV.3 (10 μg/mL) for 5 minutes and then with B01P (20 μg/mL) for 5 minutes, after which they were activated. Representative tracings for aggregation and ATP release (left panels) and the combined results (right panels) are shown; data are mean ± SEM, n = 3 (B) and n = 4 (C-D). rTMX1 containing the CPAC-motif (rTMX1) inhibits convulxin-induced platelet aggregation (E) and thrombin-induced platelet aggregation and ATP release (F). Human platelets were incubated with rTMX1 for 5 minutes at the indicated concentrations before stimulation. Representative aggregation and ATP release (left panels) and combined results (right panels); data are mean ± SEM, n = 4 for each group. (G) Inactivated rTMX1 [TMX1(oo)] increases convulxin-induced platelet aggregation. Representative aggregation (left panel) and combined results (right panel); data are mean ± SEM, n = 6 for each group. *P < .05, **P < .01, ***P < .001, Student t test. IgG, normal mouse immunoglobulin G; MFI, mean fluorescence intensity.

Figure 2.

Deficiency in TMX1 potentiates platelet function and thrombosis and shortens bleeding times in mice. Convulxin-induced (A) and thrombin-induced (C) aggregation and convulxin-induced ATP release (B) for platelets from wild-type (TMX1^+/+) and TMX1-deficient (TMX1^−/−) mice. (C) rTMX1 (2 μM) was added to TMX1-null platelets 5 minutes prior to platelet activation. Representative tracings (left panels) and combined results (right panel); data are mean ± standard error of the mean (SEM), n = 4 (A-B), n = 5 (C). *P < .05, **P < .01, Student t test (A-B), analysis of variance (C). TMX1-deficient platelets have enhanced convulxin-induced activation of αIIbβ3 (detected by the JON/A activation-dependent antibody) (D) and P-selectin expression (E). Adding rTMX1 (2 μM) 5 minutes prior to platelet activation reverses the increased aggregation and P-selectin expression. Representative line graphs (left panels) and combined results (right panels); data are mean ± SEM, n = 4. Platelets from wild-type littermate control or TMX1-deficient mice were stimulated with convulxin for 5 minutes, followed by flow cytometry analysis. ***P < .001, analysis of variance. (F) Incorporation of platelets into a growing thrombus in TMX1^+/+ and TMX1^−/− mice was detected by Alexa Fluor–488 anti-CD41 after an FeCl₃ (3.5%)–induced mesenteric arterial injury. Images were taken at 3, 7, 12, and 20 minutes. Dashed lines mark the vessel wall. Scale bars, 200 μm. Mean vessel diameters: TMX1^+/+ mice, 94.34 ± 3.20 μm; TMX1^−/− mice, 93.71 ± 5.71 μm (P = not significant). (G) Composite of fluorescent intensity per area (FI/μm²) in TMX1^+/+ mice (n = 13 from 4 mice) and TMX1^−/− mice (n = 13 from 3 mice); data are mean ± SEM. **P < .01, ***P < .001, Student t test. (H) Tail bleeding time in TMX1^+/+ and TMX1^−/− mice; data are mean ± SEM. ***P < .001, Student t test. NS, not significant.