Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans
Ana Marin D. Carneiro, … , Dennis L. Murphy, Randy D. Blakely
Ana Marin D. Carneiro, … , Dennis L. Murphy, Randy D. Blakely
Published March 3, 2008
Citation Information: J Clin Invest. 2008;118(4):1544-1552. https://doi.org/10.1172/JCI33374.
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Research Article Hematology

Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans

Abstract

The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism.

Authors

Ana Marin D. Carneiro, Edwin H. Cook, Dennis L. Murphy, Randy D. Blakely

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Figure 3

Physical interactions between SERT and integrin αIIbβ3.

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Physical interactions between SERT and integrin αIIbβ3. Western blots sh...
Western blots showing the association of SERT with members of the focal adhesion complex. Human platelet lysates were prepared under low- (A, 1% Triton X-100, n = 2 shown) and high-stringency (B, RIPA buffer) conditions, and SERT immunocomplexes were isolated (normal rabbit serum [lane 1, NRS] or 2 different anti-SERT polyclonal sera [nos. 48 and 50; see ref. 49 for details] were used). The focal adhesion proteins integrin αIIbβ3, vinculin, talin, and actin were identified by immunoblotting with specific antibodies. Only integrin αIIbβ3 interacts with SERT under high-stringency conditions. (C) GST pulldowns performed in human platelets under high-stringency conditions (RIPA buffer) demonstrate that the C terminus of SERT (CSERT) mediates the SERT/αIIbβ3 interaction. (D) GST pulldowns performed using purified αIIbβ3 (Enzyme Research) demonstrate that the β3 subunit directly interacts with the C terminus of SERT. (E) The fibrinogen-mediated enhancement of SERT uptake activity is not mediated by increased interactions with the focal adhesion complex. No significant differences were found between collagen and fibrinogen samples. For A–E, representative blots from at least 3 independent experiments are shown.