IWR-1 Inhibits Collagen-Induced Platelet Activation and Protects against Thrombogenesis

Abstract

Platelets play a crucial role in haemostasis and several pathophysiological processes. Collagen is a main initiator for platelet activation and aggregation. Given that Wnt signalling negatively regulates platelet function, and IWR-1 (a small molecule inhibitor for Wnt signalling) has the potential of inhibiting collagen synthesis, it is essential to investigate whether IWR-1 regulates collagen-induced platelet activation and protects against thrombogenesis. In the present study we found that IWR-1 pretreatment effectively suppressed collagen-induced platelet aggregation in a dose-dependent manner. In addition, IWR-1 also resulted in a decrease of P-selectin and phosphatidylserine surface exposure using fluorescence-activated cell sorting analysis. In vitro studies further revealed that IWR-1 had a negative effect on integrin a2β1 activation and platelet spreading. More importantly, the results from in vivo studies showed that IWR-1 exhibited a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Taken together, current results demonstrate that IWR-1 could effectively block collagen-induced platelet activity in vitro and in vivo, and suggest its candidacy as a new antiplatelet agent.

Keywords

IWR-1 - collagen - platelet activation - thrombogenesis

^∗ Note: Yanqing Wu and Ling Wang have made equally important and outstanding contributions to this paper and thus share senior authorship.

• References

• 1 Yun SH, Sim EH, Goh RY, Park JI, Han JY. Platelet activation: the mechanisms and potential biomarkers. BioMed Res Int 2016; 2016: 9060143
• 2 Thomas MR, Storey RF. The role of platelets in inflammation. Thromb Haemost 2015; 114 (03) 449-458
• 3 Jennings LK. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009; 102 (02) 248-257
• 4 Poole AW, Watson SP. Regulation of cytosolic calcium by collagen in single human platelets. Br J Pharmacol 1995; 115 (01) 101-106
• 5 Santoro SA, Walsh JJ, Staatz WD, Baranski KJ. Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation. Cell Regul 1991; 2 (11) 905-913
• 6 Kühl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000; 16 (07) 279-283
• 7 Miller JR, Hocking AM, Brown JD, Moon RT. Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways. Oncogene 1999; 18 (55) 7860-7872
• 8 Dejana E. The role of Wnt signaling in physiological and pathological angiogenesis. Circ Res 2010; 107 (08) 943-952
• 9 van Gijn ME, Daemen MJ, Smits JF, Blankesteijn WM. The Wnt-frizzled cascade in cardiovascular disease. Cardiovasc Res 2002; 55 (01) 16-24
• 10 Macaulay IC, Thon JN, Tijssen MR. , et al. Canonical Wnt signaling in megakaryocytes regulates proplatelet formation. Blood 2013; 121 (01) 188-196
• 11 Steele BM, Harper MT, Macaulay IC. , et al. Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A 2009; 106 (47) 19836-19841
• 12 Martins-Neves SR, Paiva-Oliveira DI, Fontes-Ribeiro C, Bovée JVMG, Cleton-Jansen AM, Gomes CMF. IWR-1, a tankyrase inhibitor, attenuates Wnt/β-catenin signaling in cancer stem-like cells and inhibits in vivo the growth of a subcutaneous human osteosarcoma xenograft. Cancer Lett 2018; 414: 1-15
• 13 Zhou MW, Yin WT, Jiang RH. , et al. Inhibition of collagen synthesis by IWR-1 in normal and keloid-derived skin fibroblasts. Life Sci 2017; 173: 86-93
• 14 Xu XR, Wang Y, Adili R. , et al. Apolipoprotein A-IV binds αIIbβ3 integrin and inhibits thrombosis. Nat Commun 2018; 9 (01) 3608
• 15 Cramer R, Dri P, Zabucchi G, Patriarca P. A simple and rapid method for isolation of eosinophilic granulocytes from human blood. J Leukoc Biol 1992; 52 (03) 331-336
• 16 Xu YR, Wang GY, Zhou YC, Yang WX. The characterization and potential roles of bone morphogenetic protein 7 during spermatogenesis in Chinese mitten crab Eriocheir sinensis . Gene 2018; 673: 119-129
• 17 McCarty OJ, Zhao Y, Andrew N. , et al. Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion. J Thromb Haemost 2004; 2 (10) 1823-1833
• 18 Hensch NR, Karim ZA, Pineda J, Mercado N, Alshbool FZ, Khasawneh FT. P2Y₁₂ antibody inhibits platelet activity and protects against thrombogenesis. Biochem Biophys Res Commun 2017; 493 (02) 1069-1074
• 19 Lin OA, Karim ZA, Vemana HP, Espinosa EVP, Khasawneh FT. The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function. PLoS One 2014; 9 (01) e87026
• 20 Zhang G, Xiang B, Dong A. , et al. Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation. Blood 2011; 118 (13) 3670-3679
• 21 Yu H, Chen Y, Wu H, Wang K, Liu L, Zhang X. Synthesis of huaicarbon A/B and their activating effects on platelet glycoprotein VI receptor to mediate collagen-induced platelet aggregation. Am J Transl Res 2017; 9 (02) 499-506
• 22 Farndale RW, Siljander PR, Onley DJ, Sundaresan P, Knight CG, Barnes MJ. Collagen-platelet interactions: recognition and signalling. Biochem Soc Symp 2003; (70) 81-94
• 23 Kuchay SM, Wieschhaus AJ, Marinkovic M, Herman IM, Chishti AH. Targeted gene inactivation reveals a functional role of calpain-1 in platelet spreading. J Thromb Haemost 2012; 10 (06) 1120-1132
• 24 Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci 2006; 119 (Pt 19): 3901-3903
• 25 Ghoshal K, Bhattacharyya M. Overview of platelet physiology: its hemostatic and nonhemostatic role in disease pathogenesis. ScientificWorldJournal 2014; 2014: 781857
• 26 Stegner D, Nieswandt B. Platelet receptor signaling in thrombus formation. J Mol Med (Berl) 2011; 89 (02) 109-121