The lateral diffusion and fibrinogen induced clustering of platelet integrin α_IIbβ₃reconstituted into physiologically mimetic GUVs

Abstract

Platelet integrin αIIbβ3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbβ3 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 αIIbβ3 behaviour away from the complexity of the platelet machinery. Here, a novel method of αIIbβ3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid composition. αIIbβ3 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 αIIbβ3. Similarly, exposure to αIIbβ3's primary ligand fibrinogen (Fg) alone does not affect αIIbβ3'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 αIIbβ3 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, αIIbβ3 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, αIIbβ3 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 αIIbβ3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbβ3 into cholesterol rich domains is not responsible for the integrin clustering observed.

Glycan-decorated protocells: novel features for rebuilding cellular processes

“…In a recent publication, preferential partitioning of platelet integrin a IIb b 3 into L d domains of phase-separated GUVs has been observed in both inactivated and activated (presence of DTT and Mn 2þ ) states. Fibrinogen binding to activated integrins did not change the preference of the integrin a IIb b 3 heterodimer to L d domains [129]. Moreover, homotypic cell -cell adhesion was modelled by incorporation of epithelial cadherin (E-cadherin) from the cadherin family into protocells and supported lipid bilayers [130,131].…”

Delivery of membrane proteins into small and giant unilamellar vesicles by charge‐mediated fusion

“…Giant unilamellar vesicles are promising membrane scaffolds for biological investigations, but only few protocols for the incorporation of MPs into GUVs have been described [7][8][9][10]36]. Our results prompted us to test fusion of positively charged SUVs containing MPs, with empty GUVs containing negatively charged lipids.…”

“…Because of these advantages, GUVs are also promising membrane mimetic systems for the design and bottom‐up construction of synthetic cells from purified components . While the insertion of soluble components into the GUV lumen is established , finding general methods for the insertion of functional membrane proteins into the GUV lipid bilayer is an active field of research .…”

Methods of reconstitution to investigate membrane protein function

“…With these variations in place, many IMPs have been succesfully inserted and studied in GUVs. These include; Ca 2+ -ATPase and bacteriorhodopsin (143), a flippase from yeast endoplasmic reticulum (149), glycophorin and a K + channel (221,222), and many more (111,143,153,183,185,223). It has also been shown that IMPs and glycosphingolipids retain their natural orientation post electroformation in GUVs produced from native membranes (157).…”

“…The formation of GUVs with soluble proteins or nucleic acids in the lumen is well established (158,(180)(181)(182) (146) and this is currently an active research area (118,148,153,(183)(184)(185).…”