Page 257 - Haematologica May 2020

DNM2 is required for GPVI signaling
CRP stimulation induced tyrosine phosphorylation of sev- eral proteins, including proteins at 125, 72, 68, 56, 52, and 38 kDa (Figure 2A). Dynasore-treated platelets had a mod- erate reduction of tyrosine phosphorylation of these pro- teins in response to CRP stimulation, and Dnm2Plt–/– platelets failed to increase protein tyrosine phosphoryla- tion, even at high doses of CRP.
Because of its proximal role in the GPVI signaling path- way,28 Lyn activation was probed using an antibody specifically directed against its phosphorylated activating tyrosine 396 (Tyr396) residue (Figure 2B). Stimulation of control platelets with CRP induced a 62±19% increase in Lyn Tyr396 phosphorylation that peaked at 2.5 mg/mL CRP (mean±SEM; n=3 in each group) (P=0.0408). Lyn Tyr396 phosphorylation was attenuated in dynasore- treated platelets and markedly reduced in Dnm2Plt–/– platelets.
GPVI expression in control, dynasore-treated, and Dnm2Plt–/– platelets was further evaluated by immunoblot analysis using the monoclonal antibody JAQ1 (Figure 2C). Following stimulation of control platelets with 25 mg/mL CRP, JAQ1 signal decreased by 15%, compared to resting levels. Dynasore treatment resulted in a 10% increase in JAQ1 signal, which was maintained following CRP stimulation. Taken together, the data show that
A
DNM2 contributes to GPVI homeostasis at rest and GPVI downregulation and Lyn activation following GPVI liga- tion. As reported previously, GPVI expression was markedly decreased in Dnm2Plt–/– platelets,9 and was unaf- fected by CRP.
Figure 2. GPVI signaling defects in dynasore-treated and Dnm2Plt–/– platelets. Control platelets, platelets treated with 100 mM dynasore, and Dnm2Plt–/– platelets were activated or not with CRP for 2 minutes at 37°C as indicated. (A) Platelet lysates corresponding to 2 mg of protein were subjected to SDS-PAGE and probed for phos- photyrosine (pTyr), phosphorylated Lyn Tyr396 (pLyn), Lyn, GPVI, and β-actin as a loading control. Results are representative of five independent experiments. Assessment of Lyn Tyr396 phosphorylation (B) and GPVI expression (C) in CRP-stimulated control, dynasore-treated, and Dnm2Plt–/– platelets. Results represent mean±standard error of mean (SEM) of 3-4 independent experiments, and are compared statistically to control (*P<0.05; **P<0.01; ***P<0.001).
Impaired GPIbα downregulation in CRP-stimulated dynasore-treated and Dnm2Plt–/– platelets
GPIbα is internalized during platelet activation,29 a phe- nomenon that is expected to negatively affect initial platelet adhesion to collagen-bound vWF and for which a role of dynamin has been reported, based on pharmaco- logical inhibition.22 The role of DNM2 in this process was investigated in response to the GPVI agonist CRP or the soluble G-protein-coupled receptor agonist thrombin (Figure 3). Expression of surface GPIbα decreased to about 30% of resting levels following stimulation of control platelets with CRP (Figure 3A) or thrombin (Figure 3B).
In response to stimulation with CRP, expression of sur- face GPIbα decreased to 60% of resting levels in dynasore- treated platelets, a 50% reduction compared to controls, and Dnm2Plt–/– platelets failed to down-regulate GPIbα (Figure 3A). By contrast, dynasore treatment or Dnm2 deletion did not affect GPIbα downregulation when platelets were stimulated with thrombin (Figure 3B).
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