Page 90 - Haematologica May 2020

M. Chimen et al.
Animal experiments
All experiments were performed in accordance with the Home Office Guidelines. In each experiment C57BL/6 IL4R/GPIbα−Tg or ApoE-/- or wild-type (WT) animals with the same background were allocated at random to experimental groups. Mice from the same litter were randomly distributed amongst experimental groups.
Results
Platelet activation in whole blood leads to formation of PEV and their adhesion to monocytes
duction and adhesion to monocytes (Online Supplementary Figure S4A-D).
PEV binding to monocytes is rapid
Accumulation of PEV on monocytes after stimulation of whole blood was progressive over a prolonged period of time (i.e. 30 min) (Figure 1B-C and Online Supplementary Figure S4). An important question is whether this pattern of accumulation is dependent upon the dynamics of PEV- monocyte interaction and adhesion, or whether the gen- esis of PEV from activated platelets is the rate-limiting step. Here we used the addition of isolated and pre- labelled PEV (1x109/mL) generated by stimulating platelets (3x108) with CRP-XL (1 μg/mL), to unstimulated whole blood to investigate this. After CRP-XL activation of isolated platelets, GPIbα stained PEV were readily dis- cernible by flow cytometry in platelet supernatants (Figure 3A and Online Supplementary Figure S5A-E). A sim- ilar pattern was observed for CD41 on PEV (Online Supplementary Figure S5C and S6). Interestingly, ~25% of the large vesicles detected by flow cytometry contained mitochondrial fragments, as previously described34 (Online Supplementary Figure S7A). Analysis using nanoparticle tracking showed that 3x108 platelets could yield 1.2±0.3x109 PEV compared to an average 1.3x108±2.8x107 vesicles in untreated conditions (Figure 3B and Online Supplementary Figure S5D-E) with a mean diameter of 274±188 nm. To date it has not been possible to simultaneously count vesicles, size them and analyse protein cargo using a single platform. The Exo View-R100 is a new platform which allows such analysis providing previously unattainable information in a single protocol.44 Using this assay we observed that PEV from CRP-XL stimulated platelets captured by a CD9 antibody had a mean size of 54 nm while those captured by a CD41a antibody had a mean size of 82.3 nm (Online Supplementary Figure S7B). Upon analysis using the Exo View system, we observed the majority of PEV were cap- tured by CD41a and CD9 (which are abundant on platelets), but not CD63 or CD81 (which are expressed on exosomes) (Online Supplementary Figure S7C). This was also confirmed by secondary labelling of captured PEV using fluorescent antibodies against CD9, CD63 and CD81 (Online Supplementary Figure S7D). Labelled PEV were added to whole blood at a concentration of 1x109/mL and their interactions with leukocytes assayed by flow cytometry. Many monocytes acquired GPIbα within 5 min, but neutrophils or lymphocytes did not (Figure 3C-D). The proportion of monocytes acquiring GPIbα slowly increased thereafter, while intensity of GPIbα staining increased steadily (Figure 3C-D). Interestingly, we found that most of the GPIbα signal detected by flow cytometry was intracellular (~80%) on both monocyte subsets (Online Supplementary Figure S8A- B) and in agreement with the confocal imaging data described in Figure 2C. We also analysed the adhesion of PEV labelled with the lipophilic dye PKH67 to exclude antibody-mediated interaction of PEV with monocytes. PKH67 labelled all of the PEV in the activated-platelet supernatant (Figure 3E). The dynamics of PEV binding to monocytes, neutrophils or lymphocytes (Figure 3F) was similar to that for the antibody-labelled PEV (Figure 3C).
The mechanistic basis for the preferential accumulation of GPIbα on monocytes was investigated using adhesion- blocking reagents. Inclusion of a function-neutralising
We investigated the effect of platelet activation on platelet-leukocyte interactions in whole blood. When thrombin receptor activating peptide (TRAP), an agonist of the platelet protease activated receptor-1 (PAR-1), was added to sheared whole blood, a time dependent increase in the percentage of monocytes bearing the platelet recep- tor GPIbα (CD42b) as well as CD41 (GPIIb) and in the intensity of GPIbα and CD41 staining, was observed (Figure 1A-C; Online Supplementary Figure S1A-C and S2). In unstimulated blood, few monocytes (~5%) possessed measurable levels of GPIbα, showing that shear did not activate platelets. During analysis monocytes were subdi- vided into two subsets using standard markers as previ- ously described.43 Classical monocytes (CD14+CD16–) rep- resent 90% of cells in the circulation and non- classical/intermediate monocytes (CD14+/dimCD16+) 10% (Online Supplementary Figure S1A). In our studies, we have compared classical to non-classical/intermediate mono- cytes grouped together. This is because the low numbers of isolated intermediate and non-classical monocytes do not allow appropriate functional testing of these subsets individually in our assays. The interaction between platelets and monocytes was similar when classical and non-classical/intermediate monocytes were assayed, showing similar patterns of GPIbα and CD41 accumula- tion over time (Figure 1B-C; Online Supplementary Figure S1A-C and S2). Interestingly, only a modest accumulation of GPIbα was evident on neutrophils stimulated with TRAP and even less when whole blood was stimulated with CRP-XL (Figure 1D and Online Supplementary Figure S1D-E). We observed no accumulation of GPIbα on lym- phocytes (Figure 1D and Online Supplementary Figure S1D).
The median fluorescent intensity (MFI) of GPIbα on monocytes after 30 minutes (min) of TRAP stimulation was well below the intensity on individual platelets (Figure 2A). Moreover, the time course of the acquisition of GPIbα by monocytes demonstrated a progressive accu- mulation that ruled out the binding of whole platelets (Figure 2B). This pattern of accumulation is consistent with the adhesion of PEV, which was confirmed using confocal microscopy (Figure 2C and Online Supplementary Figure S3). For comparison, we show a monocyte bearing whole platelets generated under different experimental conditions i.e. in the absence of shear (Figure 2D).
Here, we have reported the formation of PEV in response to thromboxane A2, ADP and cross linked colla- gen related peptide (CRP-XL). Activation of platelets in whole blood using CRP-XL, ADP, the thromboxane mimetic U46619, or the C-type lectin-like receptor (CLEC-2) agonist, rhodocytin, resulted in the same pattern of accumulation of GPIbα on monocytes, showing that different routes of platelet activation resulted in PEV pro-
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