Author + information
- Bo Jiang,
- Jianhua Huang,
- Alokkumar Pathak and
- George Stouffer
Background: α-thrombin plays an important role in vascular healing responses, including cell proliferation, adhesion, spreading, contraction, and migration. We previously showed that αvβ3-integrin mediates thrombin-induced smooth muscle cell (SMC) proliferation and focal adhesion (FA) formation. Rap1, a small GTP-binding protein, has been shown to interact closely with αIIβ3 integrin after thrombin stimulation in platelets. We hypothesized that Rap1 mediates thrombin-induced proliferation and FA formation in SMC.
Methods: Rat and mouse aortic SMC were used for these studies. Activation of Rap1 was determined by GST pull-down assay. The causal role of Rap1 activation was determined by overexpression of GTPase-activating protein II and siRNA inhibition of Rap1A and/or Rap1B. Phosphorylation of focal adhesion kinase (FAK) and MAPKs including ERK1/2, JNK1, and p38, were assessed by western blotting. FA formation was determined by staining against vinculin.
Results: α-thrombin or thrombin receptor activating peptide (TRAP-6) activated Rap1 in SMC and this effect was abolished by the PAR-1 antagonist varopaxar, demonstrating that α-thrombin-activated Rap1 is mediated through PAR-1 receptors. Treatment with α-thrombin increased the activation of ERK1/2 and JNK1, FAK phosphorylation, FA formation, and proliferation in SMC. Overexpression of GTPase-activating protein II (Rap1GAP-II) completely obliterated endogenous and α-thrombin-induced Rap1 activation, and blunted more than 50% of α-thrombin-induced ERK1/2 and JNK1 activation. α-thrombin-induced activation of ERK1/2 and JNK1 was also inhibited by knocking down both Rap1A and Rap1B by a siRNA approach. α-thrombin-induced proliferation was reduced by overexpression of Rap1GAP-II or Rap1A + Rap1B siRNA. We also demonstrated that α-thrombin-induced FAK phosphorylation and FA formation were almost completely inhibited by overexpression of Rap1GAP-II.
Conclusions: Taking together, these data show that α-thrombin activates Rap1 via PAR-1 receptors and that α-thrombin-mediated MAPK activation, FAK phosphorylation, FA formation and cell proliferation in SMC was at least partially mediated through Rap1 activation.
Poster Hall, Hall C
Saturday, March 18, 2017, 9:45 a.m.-10:30 a.m.
Session Title: Cutting Edge Vascular Medicine
Abstract Category: 38. Vascular Medicine: Basic
Presentation Number: 1205-354
- 2017 American College of Cardiology Foundation