Immobilized collagen prevents shedding and induces sustained GPVI clustering and signaling in platelets

Figures & data

Figure 1. GPVI signaling remains constant in platelets spread on fibrous collagen for at least 3 h. (A) Confocal reflection images of washed human platelets spread on Horm collagen for the indicated times. Quantification of spread platelet surface area (B) and platelet number per field of view (C) for the specified time points from 5 FOVs from three independent experiments. Arrows indicate collagen fibers. Scale bar: 5 μm. (D) Western blot analysis of platelets spread for the indicated time points or non-adhered platelets (NA) probed for total phosphotyrosine (4G10), phosphorylated Syk (Tyr525/6; pSyk) and LAT (Tyr200; pLAT). The antibodies against α-tubulin, pan-Syk and pan-LAT were used as loading controls for total phosphotyrosines, phospho-Syk and phospho-LAT, respectively. Quantification of live-cell Ca²⁺ imaging of platelets spread for the indicated time points showing percentage of platelets exhibiting Ca²⁺ spiking (E), number of spikes per platelet in the 2 min imaging period (F), spike amplitude (G), spike duration (H). Data taken from three representative FOVs for each time point (~150 platelets in each FOV) from three independent experiments. All data are expressed as mean ± SEM. Significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test (* p < .05)

Figure 2. Phosphorylated Syk and LAT colocalize with GPVI along collagen fibers. (A) Confocal microscopy imaging of human washed platelets spread on collagen (10 µg ml⁻¹) for 1 h, labeled for (Ai) Pan-Syk, (Aii) pSyk Tyr^525/6, (Aiii) Pan-LAT, (Aiv) pLAT Tyr²⁰⁰. The enrichment of pSyk Tyr^525/6 and pLAT Tyr²⁰⁰ along collagen fibers is indicated by red arrows (Aii, iv). (B) Dual-color confocal imaging of platelets, pre-incubated for Pan-GPVI using 1G5-Fab (Bi), spread on collagen for 1 h and post-labeled for pSyk Tyr^525/6 (Bii, first panel) or pLAT Tyr²⁰⁰ (Bii, second panel). The position of collagen fibers is shown in the confocal reflection image (Biii). The qualitative colocalisation mask in (Biv) shows the colocalisation of GPVI and the respective phosphoprotein with enrichment at the collagen fibers (arrows). (C) Quantification of GPVI and phosphoprotein colocalisation using Pearson’s correlation coefficient for platelets spreading for 1 h and 3 h. Scatter plot represents mean ± SEM of n = 30 platelets taken from three independent experiments. Significance was measured using unpaired two-tailed t-test, * p < .05. Scale bar: 5 μm

Figure 3. dSTORM imaging and two-level DBSCAN cluster analysis of GPVI in platelets spread on collagen for 1 h and 3 h

(A) Washed human platelets spread on Horm collagen for 1 h and 3 h, labeled for GPVI using 1G5-Fab and imaged by TIRFM (Ai) and dSTORM (Aii). The localized data points from the dSTORM images were grouped into clusters using a two-level cluster analysis based on DBSCAN (Aii, iii). (Aiii) Level I clustering identified large clusters corresponding to collagen fibers. (Aiv) Level II clustering subdivided these large clusters into nanoclusters where different colors denote different clusters. (B) Quantitative analysis of cluster area for both Level I and Level II clustering at the different time points indicated. Bars represent mean ± SEM from three independent experiments. Significance was measured using unpaired two-tailed t-test p < .05: ns indicates non-significant difference. Scale bar: 5 μm (TIRF and dSTORM images) and 1 μm (cluster plots).

Figure 4. GPVI is not shed in platelets spread on fibrous collagen

(A) Western blot analysis of GPVI shedding using an antibody to the intracellular tail of GPVI (GPVI-tail) in washed human platelets that were either non-adhered (NA) or spread on collagen for the indicated time points. Adhered platelets treated with 2 mM NEM were the positive control for shedding, tubulin was the loading control. (B) Quantification of the percentage of shed GPVI. Bars represent the mean ± SEM from three separate experiments. (C) Dual-color epifluorescence imaging of spread platelets labeled for GPVI extracellular domain (1G5-Fab, Ci) and intracellular tail (GPVI-tail, Cii). The distribution of fibrous collagen is shown in the DIC image (Ciii). (Civ) Cololocalisation mask of the two domains of GPVI shows overlapping pixels and a high level of colocalisation along collagen fibers. (D) Dual-color epifluorescence imaging of spread platelets labeled for GPVI (Di) and ADAM10 (Dii) with collagen shown in the DIC image (Diii). (Div) Colocalisation mask of GPVI and ADAM10 shows no enrichment of colocalisation at collagen fibers. (E) Two-color dSTORM of GPVI (Ei) and ADAM10 (Eii). The magnified box region in the merge (Eiii) shows little colocalisation of GPVI and ADAM10 at a collagen fiber. Scale bar: 5 µm (whole FOVs and single platelet images) and 1 μm in (Eiv).

Figure 5. Effect of metalloproteinase inhibitors and NEM on GPVI clustering

TIRF (Ai) and dSTORM (Aii) images of GPVI in washed platelets spread on collagen for 1 h treated with DMSO (vehicle control), the metalloproteinase inhibitors GM6001 or GI254023 or NEM, which activates metalloproteinases. (Aiii) Level I clustering, where large clusters associated with the collagen fibers are isolated and (Aiv) Level II clustering where the large clusters are subdivided into nanoclusters. Different colors represent different clusters. (B) Quantification of the cluster size (area) at both Level I and Level II clustering. (C) Quantification of the number of fluorescent detections localized in dSTORM imaging of GPVI in control vs NEM-treated platelets. Bar graphs are mean ± SEM from three independent experiments. Cluster area was analyzed using one-way ANOVA with Tukey’s multiple comparisons test, ns: no significant difference. Number of detections was analyzed using unpaired two-tailed t-test, where * is p < .05. Scale bar: 5 μm (dSTORM images) and 1 μm (Level I and II cluster plots).

Figure 6. Effect of Src-family and Syk inhibitors on platelet spreading, GPVI signaling and clustering in response to fibrous collagen

(A) Confocal images of washed human platelets spread on collagen, labeled with phalloidin-Alexa488 to visualize F-actin. (Ai) Platelets pre-incubated with DMSO (vehicle control) or (Aii) pre-incubated with either PP2 (Src inhibitor) or PRT (Syk inhibitor) and then spread for 1 h. (Aiii) Platelets spread for 45 min before addition of either PP2 or PRT for 15 min (post-treatment). Red arrows highlight cells where the actin cytoskeleton is disrupted. Scale bar: 5 μm. (B) Quantification of platelet surface area in cells treated with inhibitors post spreading. Graphs are mean ± SEM from five whole FOVs from three independent experiments. Unpaired two-tailed t-test, ns: not significantly different. (C) Western blot analysis of the phosphorylation state of downstream GPVI signaling proteins following post-spreading treatment with the inhibitors. (D) Quantification of live-cell Ca²⁺ imaging of platelets labeled with Oregon green-488 BAPTA-1-AM Ca²⁺ dye, spread on collagen and then treated with vehicle control or kinase inhibitors PP2 or PRT. Bar graphs show the mean ± SEM of the percentage of spiking platelets per FOV, the number of spikes per platelets, the spike amplitude and the spike duration. In total, ~150 platelets within a whole FOV were analyzed for each individual experiment (n = 3). The significance was measured using one-way ANOVA with Tukey’s multiple comparisons test (*** p < .001, ns: not significantly different). (E) TIRF and dSTORM imaging and 2-level DBSCAN cluster analysis showing the effect of PP2 and PRT post-treatment on GPVI clustering and nanoclustering. Different colors represent different clusters. (F) Quantification of cluster area at both Level I and Level II clustering. Graphs are mean ± SEM from n = 3 experiments. Significance was measured using one-way ANOVA with Tukey’s multiple comparisons test (** p < .01; ns: not significantly different). Scale bar: 5 μm (TIRF and dSTORM images) and 1 μm (cluster plots).

Figure 7. Syk but not Src-family inhibitor impairs integrin α2β1 clustering on collagen in spread platelets

(A) TIRF (Ai) and dSTORM imaging (Aii) and 2-level DBSCAN cluster analysis (Aiii, iv) of integrin α2β1 in washed human platelets spread on collagen and post-treated with DMSO (vehicle control), the Src kinase inhibitor PP2 (20 μM) or the Syk inhibitor PRT (10 μM). Different colors indicated different clusters in the cluster plots. (B) Quantitative analysis of cluster area. Graphs are mean ± SEM from n = 3 experiments. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate the statistical significance (* p < .05). Scale bar: 5 μm (dSTORM images) and 1 μm (cluster plots).