Page 203 - Haematologica - Vol. 105 n. 6 - June 2020

GpIb clustering in shear-activated platelets
membranes and reduction in the formation of typical pro- tein complexes/lipid rafts, among other effects.16-18 Based on our previous observations that ALA reduces platelet reactivity,10,19 and on studies showing the presence of GpIb in lipid rafts,20-22 we hypothesized that ALA might interfere with the distribution of GpIb on the plasma membrane in high-shear flow and, therefore, alter binding to vWF.
Statistical analysis
Data are plotted as the mean ± standard error of mean of at least three independent experiments. They were analyzed by a paired, two-tailed Student t-test with GraphPad Prism version 7 (GraphPad Software, La Jolla, CA, USA). P values <0.05 were con- sidered statistically significant.
Results
Pre-incubation of blood with the n-3 FA ALA at dietary relevant concentrations23 reduced the GpIb/vWF interac- tion under pathological high-shear flow (10,000 s-1, corre- sponding to the flow rate in an 80% stenosed artery), as measured by the platelet-covered area (106,963±15,892 μm2 with vehicle vs. 75,519±16,254 μm2 with ALA) (Figure 1A, B and Online Supplementary Videos S1 and S2). Analysis of single fluorescently labeled platelets showed that their speed was doubled when pre-incubated with ALA (Figure 1C), while the distance traveled before stopping was increased (8.89±4.0 μm with vehicle vs. 13.36±7.2 μm with ALA) (Figure 1D, E).
It has been reported that GpIb resides in cholesterol-rich membrane domains termed lipid rafts,20,21,24,25 and that it appears to cluster in conditions of high-shear flow.22 In agreement with these findings, cholesterol depletion with methyl-β-cyclodextrin (MβCD), able to remove 50-90% of membrane cholesterol,26 greatly reduced platelet adhe- sion to vWF, demonstrating the pivotal role of membrane cholesterol in GpIb-vWF adhesion under high-shear flow (188±16 μm2) (Figure 1F).
To exclude that the reduced adhesion of ALA-treated platelets was due to lower levels of membrane GpIb, vehi- cle- and ALA-treated washed platelets were exposed to high-shear flow in a viscometer (10,000 s-1 for 1 min) and analyzed by flow cytometry. Levels of membrane GpIb were not different between vehicle- and ALA-treated platelets (Figure 2A); rather, pre-treatment with the n-3 FA preserved GpIb levels, as shown by higher fluorescence values, suggesting it had an inhibitory effect on GpIb cleavage, as previously shown by our group.19
Next, we analyzed whether the effect on GpIb-vWF binding was specific for GpIb, vWF, or both. Whole blood was pre-treated with ALA or vehicle for 1 h, followed by isolation of platelet-rich plasma and exposure to high- shear flow (10,000 s-1 for 1 min). Flow cytometric analysis of platelet-bound vWF showed that pathological high- shear flow was able to induce GpIb-vWF binding, and that this was not influenced by the presence of ALA (Figure 2B). When the same experiment was performed with washed platelets, we could not detect any platelet-bound vWF, demonstrating the plasmatic origin of the bound vWF (Figure 2C); however, addition of exogenous human vWF to washed platelets was able to restore vWF-platelet binding (Figure 2C). These results show that ALA has no effect on vWF itself, and suggest that its inhibitory effect is exerted through platelet GpIb and is specific for binding to anchored vWF (as typically exposed in vivo by endothe- lial cells after injury).
High resolution imaging of GpIb receptors at the plasma membrane of intact platelets was conducted using cryo- ET27 (Figure 3A-C). Adherent platelets were incubated with anti-GpIb antibodies decorated with 6-nm gold-pro- tein A and imaged with cryo-ET. The coordinates of the gold nanoclusters were identified (Figure 3A-C). While the
Methods
Blood samples
Blood from healthy volunteers was obtained from the Blood Center of the Swiss Red Cross at the Cantonal Hospital Baden with informed consent according to the Declaration of Helsinki. The study was approved by the Institutional Review Board. EDTA or citrated blood was kept at room temperature until assays were performed (within 2 h of drawing). Blood was incu- bated with vehicle (0.1% ethanol) or ALA 30 μM for 1 h at room temperature before being used for subsequent experiments. The n-3 FA dose was chosen based on a previous study showing this to be a dietary reachable concentration.23 Platelet adhesion to vWF was performed on a Bioflux 200 system (Fluxion Bioscience, San Francisco, CA, USA) according to the manufacturer’s proto- col (see the Online Supplementary Data file for details).
Immunofluorescence staining for ground state deple- tion microscopy
Washed platelets isolated from vehicle or ALA-treated sam- ples were fixed with 4% paraformaldehyde for 15 min, then spun on a 1.5 coverslip in a Cytospin (Thermo Fisher Scientific, Waltham, MA, USA) and stained for ground state depletion (GSD) microscopy. For details, see the Online Supplementary Data file.
Flow cytometric analysis of von Willebrand factor binding
Platelet-rich plasma or washed platelets from vehicle- or ALA- treated blood samples were fixed with 4% paraformaldehyde for 15 min, then incubated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing a rabbit anti-human vWF antibody (1:500, Dako A0082) and an anti- GpIbα-APC (BD Bioscience, San Jose, CA, USA) for 1 h. In some experiments, exogenous human vWF (100 ug/mL, Hematologic Technologies) was added to washed platelets. Samples were washed three times in 1% BSA in PBS and then stained with anti-rabbit 488 (1:250, Jackson Immunoresearch, West Grove, PA, USA) for 30 min. After three washes in 1% BSA in PBS, sam- ples were resuspended in 300 μL PBS and analyzed on a LSR Fortessa (BD Bioscience).
Cryo-electron tomography
Resting and sheared platelets in Tyrode buffer were seeded on gold grids coated with a silicon mesh (R 1/4, 200 mesh, Quantifoil, Jena, Germany). Platelets were allowed to adhere for 10 min and then fixed with 4% formaldehyde for 5 min at room temperature, before being processed for cryo-ET. GpIb and integrin αIIbβ3 receptors were detected by immunogold labeling (details are given in the Online Supplementary Data). Data were acquired using an FEI Titan Krios. Tomograms were acquired with a magnification of 42,000× corresponding to a pixel size of 0.34 nm. The receptor density was analyzed using MATLAB scripts and the receptor distributions were plotted and statistically analyzed by OriginLab software (Northampton, MA, USA).
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