The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbβ3 reconstituted into physiologically mimetic GUVs. Issue 4 (27th February 2015)

• Record Type: Journal Article
• Authors: Gaul, Vinnie; Lopez, Sergio G.; Lentz, Barry R.; Moran, Niamh; Forster, Robert J.; Keyes, Tia E.
• Is Part Of: Integrative biology. Volume 7:Issue 4(2015:Apr.)
• Page Start: 402
• Page End: 411
• Publication Date: 2015-02-27
• Subjects: Biology -- Periodicals; Technology -- Periodicals; Biological systems -- Periodicals; 570.5
• DOI: 10.1039/c5ib00003c
• Languages: English
• ISSNs: 1757-9694

Abstract

Abstract : A novel method for reconstitution of Integrin αIIb β3 into GUVs with unrestricted lipid composition is described and the lateral diffusion and phase partitioning of the integrin on activation and ligand binding in biomimetic GUVs compositions is studied in GUVs with biomimetic formulations. Abstract : Platelet integrin αIIb β3 is a key mediator of platelet activation and thrombosis. Upon activation αIIb β3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIb β3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIb β3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIb β3 was inserted into two GUV lipid compositions that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn 2+ or DTT does not influence the diffusion coefficient of αIIb β3 . Similarly, exposure to αIIb β3 's primary ligand fibrinogen (Fg) alone does not affect αIIb β3 's diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 ± 0.29 to μm 2 s −1 to 1.56 ± 0.26 (Mn 2+ ) or 1.49 ± 0.41 μm 2 s −1 (DTT) which is consistent with aggregation of activated αIIb β3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin–Fg complex diffuses through the confocal volume in clusters. Using the Saffman–Delbrück model as a first approximation, the diffusion coefficient of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIb β3 was also reconstituted into a "raft forming" GUV with well defined liquid disordered (Ld ) and liquid ordered (Lo ) phases. Using confocal microscopy and lipid partitioning dyes, αIIb β3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIb β3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIb β3 into cholesterol rich domains is not responsible for the integrin clustering observed.