The Lateral Diffusion And Fibrinogen Induced Clustering Of Platelet Integrin Alpha(Iib)Beta(3) Reconstituted Into Physiologically Mimetic Guvs

Platelet integrin alpha(IIb)beta(3) is a key mediator of platelet activation and thrombosis. Upon activation alpha(IIb)beta(3) 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 alpha(IIb)beta(3) behaviour away from the complexity of the platelet machinery. Here, a novel method of alpha(IIb)beta(3) insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. alpha(IIb)beta(3) 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 Mn2+ or DTT does not influence the diffusion coefficient of alpha(IIb)beta(3). Similarly, exposure to alpha(IIb)beta(3)'s primary ligand fibrinogen (Fg) alone does not affect alpha(IIb)beta(3)'s diffusion coefficient. However, addition of Fg with either activator reduces the integrin diffusion coefficient from 2.52 +/- 0.29 to mu m(2) s(-1) to 1.56 +/- 0.26 (Mn2+) or 1.49 +/- 0.41 mu m(2) s(-1) (DTT) which is consistent with aggregation of activated alpha(IIb)beta(3) 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-Delbruck 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, alpha(IIb)beta(3) was also reconstituted into a "raft forming'' GUV with well defined liquid disordered (L-d) and liquid ordered (L-o) phases. Using confocal microscopy and lipid partitioning dyes, alpha(IIb)beta(3) showed an affinity for the DOPC rich L-d phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich L-o phase. Activation and Fg binding of the integrin did not alter the distribution of alpha(IIb)beta(3) between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound alpha(IIb)beta(3) into cholesterol rich domains is not responsible for the integrin clustering observed.