Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.
Keywords: Aggregation; CLEC-2; Disaggregation; GPVI; Platelets; Tyrosine kinase
The immunoglobulin receptor glycoprotein VI (GPVI) and C-type lectin-like receptors 2 (CLEC-2) activate platelets through Src tyrosine kinases and spleen tyrosine kinase (Syk) via an immunoreceptor tyrosine-based activation motif (ITAM) and hemITAM, respectively. An ITAM has two repeats of the amino acid sequence YxxL separated by 6–12 amino acids, whereas a hemITAM has a single YxxL motif. Phosphorylation of the conserved tyrosine residues in these motifs leads to the binding of Syk through its tandem SH2 domains (in the case of CLEC-2, bridging two receptors) and activation of a signaling cascade that culminates in the activation of phospholipase Cγ2 (PLCγ2) [[
Recently, Ahmed et al. have shown that inhibition of GPVI signaling by a blocking Fab or with inhibitors of Src and Syk kinases enhances disaggregation of platelets on a collagen surface at arterial shear in the presence of anticoagulation [[
GPIIb-IIIa antagonists, including eptifibatide, have also been shown to reverse aggregation under minimal shear following stimulation by ADP as measured by light transmission aggregometry (LTA) [[
There are several explanations for the failure of eptifibatide and Fab 9012 to fully reverse aggregation to collagen as measured by LTA, including poor penetration within the aggregate, the lack of flow, or masking by additional receptors. In the present study, we have asked whether small-molecule inhibitors of Src, Syk and Bruton's tyrosine kinase (Btk) that block signaling by GPVI and CLEC-2, and which have greater accessibility to the center of the aggregate, induce reversal of phosphorylation and aggregation.
Details on the source of materials and platelet preparation can be found in the Online Supplementary Method.
Washed platelets at 2x10
5x SDS reducing sample buffer (10% SDS, 0.5 M DTT, 50% glycerol, 0.125 M Tris, pH 6.8) was added to washed platelets (2x10
Washed platelets (2x10
Data are presented as mean ± standard deviation of the mean with statistical significance taken as p <.05 unless otherwise stated. Statistical analysis was performed using Welch's t-test and one-way ANOVA multiple comparison, as stated. All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software Inc. La Jolla, USA).
We first examined the role of Src, Syk and Btk kinases in the maintenance of platelet aggregation in LTA in response to stimulation by GPVI. Aggregation to a maximally effective concentration of the GPVI ligand, CRP (10 μg/ml), peaked within 120 sec and was maintained for up to 50 min (not shown). Kinase inhibitors, used at maximally effective concentrations, were given at 150 sec and aggregation was monitored for 20 min. The reversal in light transmission at this time was 1.5 ± 2.8% in the presence of vehicle and 3.0 ± 3.4 and 0.4 ± 0.5% in the presence of the Syk and Btk inhibitors, PRT-060318 and ibrutinib, respectively (Figure 1 Ai-ii). These values were not significantly different. In contrast, the Src inhibitor PP2 caused a small but significant reverse in light transmission which started after approximately 5 min and reached 11.4 ± 4.0% over 20 min (P <.05; Figure 1 Ai-ii), dasatinib also showed a tendency to reverse but this was not significant (7.4 ± 7.2 %). We further investigated the effect of Syk inhibition on aggregation at earlier times. The addition of the Syk inhibitor PRT-060318 at 15, 30, and 45 sec after agonist stimulation blocked further aggregation but did not induce reversal over 20 min (not shown), consistent with the previous report from Zou et al [[
Graph: Figure 1. Src kinase inhibitors partially reversed GPVI and CLEC-2 mediated platelet aggregation. Washed platelets at 2x108/ml were stimulated with (A) 10 µg/mL CRP or (B) 100 nM rhodocytin and incubated with PP2 (20 µM), dasatinib (10 µM), PRT-060318 (1 µM), ibrutinib (200 nM) or vehicle, after 150 sec of agonist stimulation. LTA monitored for 20 min. (i) Representative traces of seven identical aggregation experiments. (ii) Mean ± SD % disaggregation after 20 min of agonist stimulation. *(P <.05), **(P <.01) and ***(P <.001) calculated using Welch's t-test indicate statistically significant differences. ns, not significant. N = 7 separate donors.
The addition of inhibitors of Btk and Syk kinases at 150 sec post-agonist addition also had no significant effect on aggregation induced by rhodocytin over 20 min relative to the vehicle control (Figure 1 Bi-ii). However, as with GPVI signaling, the Src inhibitors PP2 and dasatinib caused a small but significant reversal of aggregation: the degree of reversal in the presence of vehicle over 20 min was 0.41 ± 0.5% compared with 10.8 ± 10.0 and 5.8 ± 5.7% in the presence of PP2 and dasatinib (P <.05), respectively (Figure 1 Bi-ii).
We extended the experiments to the G protein-coupled receptor agonist TRAP-6. As with CRP and rhodocytin, we observed that the Src inhibitor PP2 (9.9 ± 3.1%, P <.05) caused a small but significant reverse in light transmission relative to vehicle (1.0 ± 0.1%); in contrast, however, there no significant reversal of aggregation in the presence of dasatinib (6.0 ± 5.0%) and PRT-060318 (2.1 ± 0.9%) (Figure S1). The lack of significance with dasatinib, despite the clear trend, may reflect variation between donors and the relatively small nature of the reversal.
The results show that aggregation induced by CRP, rhodocytin and TRAP-6 is maintained for up to 20 min in the presence of inhibitors of Syk and Btk whereas there was a small but significant reversal of aggregation to CRP in the presence of Src kinase inhibitors.
Experiments were performed to measure the reversal of tyrosine phosphorylation upon kinase addition. Tyrosine phosphorylation of Syk at Y525/526, LAT at Y200 and Btk at Y551 was increased over 20-fold in the first 150 sec of stimulation by CRP (10 μg/ml) and maintained for up to 50 min (Figure 2 Ai-ii). Tyrosine phosphorylation of Btk at Y223 and PLCγ2 at Y1217 showed a slower and lower rate of increase of up to 5-fold over the first 150 sec, and this was also sustained over 50 min. The Src kinase inhibitors, PP2 and dasatinib, added at 150 sec, induced rapid dephosphorylation of Syk Y525/526, LAT Y200, Btk Y223, Btk Y551 and PLCγ2 Y1217, with phosphorylation declining to basal levels by 20 min (Figure 3 Ai-ii). One notable difference between the two kinase inhibitors is the loss of phosphorylation of a band at 50–60 kDa in the presence of dasatinib which corresponds to Src family kinases. This may be due to inhibition of Csk which phosphorylates Src kinases at their inhibitory tyrosine residue [[
PHOTO (COLOR): Figure 2. Tyrosine phosphorylation is sustained for 50 min in GPVI and CLEC-2 mediated protein phosphorylation. Washed platelets at 4x108/ml were stimulated with (A) 10 µg/mL CRP or (B) 100 nM rhodocytin in the presence of 9 μM eptifibatide. Platelets were lysed with 5x reducing sample buffer at stated time after addition of agonist. Whole cell lysates were probed for whole cell phosphorylation or kinase phosphorylation with the stated antibodies. (i) Representative blot and (ii) mean ± SD % of tyrosine phosphorylation from 3 experiments. *(P <.05), **(P <.01) and ***(P <.001) assessed by one-way ANOVA multiple comparison analysis indicate statistically significant differences. ns, not significant. N = 3 separate donors.
Graph: Figure 3. Kinase inhibitors reverse GPVI and CLEC-2 mediated protein phosphorylation. Washed platelets at 4x108/ml were stimulated with (A) 10 µg/mL CRP or (B) 100 nM rhodocytin CRP in the presence of 9 μM eptifibatide. Platelets were incubated with PP2 (20 µM), dasatinib (10 µM), PRT-060318 (1 µM), ibrutinib (200 nM) or vehicle after 150 sec of agonist stimulation. Platelets were then lysed with 5X reducing sample buffer 20 min after addition of agonist. Whole cell lysates were probed for whole cell phosphorylation or kinase phosphorylation with the stated antibodies. (i) Representative blot and (ii) mean ± SD % of tyrosine phosphorylation from 3 experiments. *(P <.05), **(P <.01) and ***(P <.001) calculated using Welch's t-test indicate statistically significant differences. ns, not significant. N = 3 separate donors.
A similar set of results were observed for the CLEC-2 ligand rhodocytin, with phosphorylation sustained for up to 50 min (Figure 2 Bi-ii), and rapid reversal of downstream substrates in the presence of inhibitors of Src, Syk and Btk kinases (Figure 3 Bi-ii). There were however several differences to those with GPVI, namely that PP2 did not inhibit LAT Y200 phosphorylation, and there was no significant difference on phosphorylation of Syk Y525/526 or PLCγ2 Y1217, when treated with PRT-060318 and ibrutinib, respectively. The explanation for these differences is not known but may be due to a small overall increase and the sensitivity of detection.
These results show that CRP and rhodocytin stimulate sustained tyrosine phosphorylation with rapid inhibition of their downstream substrates upon addition of kinase inhibitors. This indicates that the failure of the kinase inhibitors to induce a complete reversal of aggregation is not due to a lack of inhibition of tyrosine phosphorylation.
The observation that aggregation is maintained in the presence of inhibitors of Src, Syk and Btk shows that this is independent of GPVI or CLEC-2 signaling, which contrasts with the role of GPVI in supporting thrombus stabilization under flow. One potential explanation for this is the masking of the role of GPVI by the release of feedback messengers ADP and TxA
PHOTO (COLOR): Figure 4. Kinase inhibitors together with secondary mediators or GPIIb-IIIa antagonist partially reversed GPVI and CLEC-2 mediated platelet aggregation and reverse GPIIb-IIIa activation. Washed platelets at 2x108/ml were stimulated with (A) 10 µg/mL CRP or (B) 100 nM rhodocytin and incubated with PP2 (20 µM), dasatinib (10 µM), PRT-060318 (1 µM), ibrutinib (200 nM) or vehicle together with 10 µM indomethacin and 2.5 U/mL apyrase, after 150 sec of agonist stimulation and monitored by LTA for 20 min. (C) Washed platelets (2x108/ml) were stimulated with 10 µg/mL CRP and then treated with eptifibatide 9 µM alone or together with PP2 (20 µM) or PRT-060318 (1 µM), after 150 sec of agonist stimulation and monitored by LTA for 20 min. (i) Representative traces of six identical aggregation experiments. (ii) Mean ± SD % of disaggregation after 20 min of agonist stimulation. (N = 6) (D) Reversal of GPIIb-IIIa activation by tyrosine kinase inhibitors. Washed platelets (2x107/ml) were incubated with PAC-1-FITC, stimulated with CRP (10 µg/mL) and then treated with vehicle, PP2 (20 µM) or PRT-060318 (1 µM) 150s after agonist stimulation. Flow cytometry measurements were done at 20 min after agonist stimulation. (i) Flow cytometry histograms depict activation of GPIIb-IIIa in platelets that were unstimulated (blue), treated with CRP (10 µg/mL) and followed with vehicle (purple), PP2 or PRT-060318 (red). (ii) MFI fold change ± SD of four independent and identical experiments (N = 4). *(P <.05), **(P <.01) and ***(P <.001) calculated using Welch's t-test indicate statistically significant differences. ns, not significant.
The above experiments were repeated in the presence of GPIIb-IIIa antagonist eptifibatide to investigate the role of GPIIb-IIIa in GPVI-mediated aggregation. The maximal reversal in the presence of eptifibatide alone or in combination with Src and Syk inhibitors was 4.5 ± 2.9, 13.4 ± 11.6 and 13.7 ± 7.4%, respectively (Figure 4 Ci-ii), which were significant (P <.05) compared to vehicle (0.7 ± 0.6%).
Experiments were designed to investigate whether activation of GPIIb-IIIa in the absence of aggregation is reversible. Activation of GPIIb-IIIa was measured using PAC-1-FITC by flow cytometry in a dilute suspension of platelets to prevent aggregation. PP2 and PRT-060318 were added 150 sec after stimulation by CRP. Both inhibitors induced full reversal of GPIIb-IIIa activation in 20 min (1.1 ± 0.3 and 1.1 ± 0.5 fold change in MFI) compared to sustained activation in the presence of vehicle (12.8 ± 10.5 fold) (Figure 4 Di-ii). This demonstrates that activation of GPIIb-IIIa by GPVI is a reversible process in the absence of aggregation.
The present study shows that signaling through GPVI or CLEC-2 and the feedback agonists ADP and TxA
In contrast to the sustained aggregation, Src and Syk inhibitors rapidly induce reversal of activation of GPIIb-IIIa when measured in a dilute platelet suspension which does not support outside-in signaling by GPIIb-IIIa and platelet aggregation due to the absence of added fibrinogen and low platelet density. This shows that inside-out activation of integrin GPIIb-IIIa is a reversible process in contrast to the sustained aggregation monitored by LTA. This may reflect the absence of binding fibrinogen to activated GPIIb-IIIa, and subsequent outside-in signaling and that, once formed, the maintenance of platelet aggregation is also regulated by additional interactions between membrane proteins [[
In contrast to the present findings, an increased rate of disaggregation has been reported at arteriolar rates of shear in thrombi formed on a collagen surface upon addition of a GPVI-blocking Fab or inhibitors of Src and Syk kinase [[
The observation that inhibitors of GPVI and Src and Syk tyrosine kinase can promote de-aggregation under flow may have clinical significance including in the phase 2 trial of the GPVI-blocking Fab glenzocimab in combination with best treatment care in patients who have had a thrombotic stroke [[
These results provide an important extension of a recent finding that CRP-stimulated platelets can maintain aggregation for a prolonged time [[
In conclusion, the present study shows that strong aggregation appears to be an irreversible process when measured by LTA following activation by tyrosine kinase-linked receptors. Platelet aggregates are maintained despite inhibition of tyrosine kinase and G protein-coupled receptor signaling pathways, or blockade of integrin GPIIb-IIIa, and contrast to results observed on a collagen surface under the more physiological condition of arterial flow. The small reduction in aggregation measured by LTA in the presence of Src kinase inhibitors is likely to reflect loss of contraction as a result of inhibition of outside-in signaling from GPIIb-IIIa.
H.Y.F.C and L.A.M receive funding from the European Union's Horizon 2020 research and innovation program [Marie Sklodowska-Curie grant agreement (No. 766118)]. S.P.W. is supported by a British Heart Foundation chair [CH03/003]. L.A.M. is registered in a joined PhD program of the Universities of Birmingham and Santiago de Compostela, H.Y.F.C. is registered in a joined PhD program of the Universities of Maastricht and Birmingham. The authors would like to thank Martina Colicchia for advice in flow cytometry.
No potential conflict of interest was reported by the author(s).
Supplemental data for this article can be accessed online at https://doi.org/10.1080/09537104.2022.2069235.
By Hilaire Yam Fung Cheung; Luis A. Moran; Albert Sickmann; Johan W.M. Heemskerk; Ángel Garcia and Steve P. Watson
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