Abstract/Summary

Introduction: Platelets are small a-nucleate blood cells that stop bleeding at vascular injury. Platelets form a haemostatic plug by sticking to exposed extracellular matrix proteins and aggregating at the site of injured vessel. Integrins are transmembrane receptors linked to actin cytoskeleton. Integrin αIIbβ3 activation and clustering is a vital part of platelet activation and is mediated in part through the association of kindlin-3 with integrin β3. Recent studies also revealed the interaction between kindlin-3 and β3 is necessary to regulate and support integrin activation in platelet at the site of injury. ILK is a known effector molecule which recruits kindlin-3 to bind to the cytoplasmic tail of β3. However, the temporal interaction between kindlin-3 - β3 and the mechanisms of αIIbβ3 clustering are not fully understood. The objective of this study was to develop a Stochastic Optical Reconstruction Microscopy (STORM) method for measuring β3-β3 clustering in human platelets and use this to investigate the time course of integrin β3 clustering, its association with kindlin-3 and the role of ILK in this process. Results: The localization of integrin β3 and kindlin-3 were analysed using the ImageJ plugin ThunderSTORM and a novel cluster analysis method was developed for defining clustering in R. The density of β3 molecules surrounding each β3 molecule (a measure of clustering) showed a biphasic pattern increasing from a low in resting platelets to peak at 60 seconds, falling at 90 seconds, then increasing again by 180 seconds before again declining by 300 seconds. β3-β3 FRET signals were observed in a biphasic fashion and confirmed our initial data presented on β3β3 clustering. The density of β3 molecules surrounding each kindlin-3 molecule showed the opposite trend of β3 clustering for the first 30 seconds (decreasing from a high in resting platelets) and then followed the same pattern as β3 clustering. The kindlin-3 - β3 co-cluster and β3-β3 cluster were positively III associated after 30 seconds stimulation but not in the first 15 seconds. (N-Methyl-3-(1-(4-(piperazin-1-yl) phenyl)-5-(4′-(trifluoromethyl) [1, 1′-biphenyl]-4-yl)-1H-pyrazol-3-yl) propanamide (22), Cpd-22, a selective ILK inhibitor, was characterised and found to inhibit different platelet functions such as platelet aggregation, fibrinogen binding and alpha granule secretion. Cpd-22 impaired thrombin mediated kindlin-3 - β3 co-clustering over time (in resting and after stimulation). Cpd-22 treated platelets displayed reduction in the percentage of β3 clustering in response to thrombin or CRP-XL. Therefore, real time of rate fibrinogen binding was measured and found to be inhibited and not totally dependent β3-β3 clustering. Cpd-22 impaired thrombin or CRP-XL stimulated integrin β3, Src, and Syk downstream signalling at late time points, corresponding with the second peak of β3 clustering. Conclusion: A STORM microscopy method was developed and used to quantify the temporal changes in the co-clustering of kindlin-3 with integrin β3, and the biphasic clustering of integrin β3 in human platelets following thrombin or CRP-XL stimulation. This was used to show that ILK has a role in platelet activation. Inhibition of ILK reduces kindlin-3 - β3 association. Thus, reduction of kindlin-3 - β3 leads to inhibition of β3-β3 clustering, and inhibition of β3 downstream signalling in late time points, corresponding with the second peak of β3 clustering.