Glycoprotein Ib clustering in platelets can be inhibited by α-linolenic acid as revealed by cryo-electron tomography

Figures

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  Figure 1.. Platelet adhesion to von Willebrand factor is inhibited by α-linolenic acid. Blood collected into EDTA and pre-incubated with vehicle or α-linolenic acid (ALA) for 1 h was perfused on von Willebrand factor (vWF) at high-shear rate and platelet adhesion monitored by fluorescence microscopy. Platelet-covered area measured at the end of the perfusion (A) or every minute during the perfusion (B) was significantly reduced by the ALA treatment (n=6, P=0.0039). The first time-point in B corresponds to 5 s after the start of the flow. (C) Single platelet rolling velocity measurements showed an increased speed in the ALA-treated samples, and a correspondingly increased distance until firm adhesion (D) (n=6, P=0.04 for C and P=0.0009 for D). (E) Representative projection images of a vehicle (VEH)- or ALA-treated sample showing single platelets rolling over vWF. (F) Membrane cholesterol depletion by pre-incubation of EDTA-blood with methyl-β cyclodextrin MbCD) completely abolished platelet adhesion to vWF (n=6, P<0.0001).

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  Figure 2.. High-shear induces von Willebrand factor binding in platelet-rich plasma but not in washed platelets. (A) Washed platelets were exposed to high-shear flow and analyzed for GpIb abundance by flow cytometry, which did not reveal any difference between vehicle- and α-linolenic acid (ALA)-treated platelets (n=3). MFI: mean fluorescence intensity. (B) Platelet-rich plasma was either left untreated or exposed to high-shear flow, and platelet-bound von Willebrand factor (vWF) analyzed by flow cytometry. In both vehicle- and ALA-treated samples, high-shear flow induced vWF binding, as shown by a 3-fold increase compared to that of samples not exposed to the shear force (n=3, P<0.05). (C) Exposing washed platelets (WP) to high-shear flow did not lead to vWF binding; addition of exogenous human vWF before shear exposure resulted in robust binding (n=3).

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  Figure 3.. High-shear induces a local rearrangement of GpIb as revealed by cryo-electron tomography. (A–C) Immunogold labeling and (E–G) neighborhood analysis for the control (A, E), shear-activated (B, F) and α-linolenic acid (ALA)-treated shear-activated (C, G) platelets. (A–C): Projection images (~70 nm in thickness) obtained from tomograms of platelets on which GpIbα has been labeled with immunogold. The labeling was detected with protein G conjugated to 6 nm gold. To make the gold identification easier, the gold has been labeled with red circles. The yellow arrows indicate crowded neighborhoods in the pictures. All the projections have the same scale and the scale bar in A is 100 nm. (E–G) Analysis of the immunogold labeling. The three-dimensional coordinates of the 6 nm gold particles from six tomograms for each condition were selected and the distances to the “neighboring receptors” within a radius of 50 nm were calculated. The number of neighbors each receptor has is depicted in the histogram. (D) Integrin αIIbβ3 immunogold labeling was performed on spread platelets and (H) the same neighboring analysis performed.

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  Figure 4.. Comparison of GpIb labeling by super-resolution microscopy and cryo-electron tomography reveals a similar receptor distribution. (A) The GpIb receptors in shear-activated platelets were labeled and imaged by ground state depletion (GSD) as described in the Methods. (B) In order to compare the localization of the receptors determined by cryo-electron tomography and GSD microscopy, a synthetic model of platelet perimeter was generated by merging 17 tomograms from the gold-labeled platelets. The gold particle coordinates from the synthetic platelets were filtered to the same resolution as the GSD (20 nm), represented here in red. White arrowheads indicate local clustering areas. Scale bar: 2.5 μm for both A and B.

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  Figure 5.. Analysis of GpIb distribution by total internal reflection microscopy. (A-D) Superresolution analysis of GpIb in platelets treated with vehicle (A), α-linolenic acid (ALA) (B) or methyl-b cyclodextrin (MbCD) (C) showed no detectable differences in the distribution of the receptor as measured by particle size (D). Scale bar: 2.5 μm.