Page 260 - Haematologica May 2020

N. Eaton et al.
with insoluble lipid rafts in cells expressing caveolin 1,38 the association of cavin 2 with detergent-resistant platelet lipid rafts was investigated using a sucrose gradient in human platelet lysates (Figure 6D), which also lack cave- olin 1 (data now shown). Cavin 2 did not associate with the insoluble sucrose gradient fractions rich in GM1 ganglio- side and flotillin 1. Taken together, the data show that platelets contained the endocytic machinery required for CME, but not for CavME.
Early, late, and recycling endosomal compartments are distinguished by their association with specific members of the Rab family of small GTPases. Platelets contained Rab5 (early), Rab7 (late), and Rab11 (recycling), as described previously,33,39 and their expression levels were not affected by the lack of DNM2 (Figure 6A).
Discussion
The cellular mechanisms and proteins regulating platelet and MK RME are poorly understood.39 Here, using both pharmacological and genetic approaches, we described
A
the role of the endocytic GTPase DNM2 in intracellular signaling via the collagen receptor GPVI and platelet hemostatic function.
In control platelets, ligation of the collagen receptor GPVI by its soluble agonist CRP induced an increase in protein tyrosine phosphorylation, including that of the proximal protein tyrosine kinase Lyn on its activating residue Tyr396, and a decrease in GPVI expression. Following dynasore treatment, phosphorylation of Lyn Tyr396 was attenuated and GPVI expression was main- tained. Recent studies have shown that common dynamin inhibitors, such as dynasore and Dyngo-4a, not only inhibit dynamin GTPase activity, but also disrupt the organization of cholesterol-rich membrane rafts in a dynamin-independent manner.40,41 Dynasore-treated platelets elicited defects in GPIbα downregulation, α-granule secretion, integrin αIIbβ3 activation, and spreading onto fibrinogen when stimulated via GPVI, but not by thrombin. By contrast, the cholesterol-lowering reagent, methyl-β-cyclodextrin, inhibits GPVI signaling, as well as platelet responses to the G-protein-coupled recep- tor agonists, thrombin and ADP.42,43 Hence, the data argue
BC
Figure 5. Altered spreading of CRP-stimulated dyna- sore-treated and Dnm2Plt–/– platelets. (A) Control platelets, platelets treated with 100 mM dynasore, and Dnm2Plt–/– platelets were activated with 1 mg/mL CRP or 0.01 U/mL thrombin and spread onto fibrino- gen-coated coverslips for 30 minutes (min) as indicat- ed. Fixed platelets were stained for polymerized actin (phalloidin; green) and β-tubulin (red) and analyzed by confocal microscopy. Images are representative of at least three independent experiments. Scale bars, 3 mm. (B) Assessment of platelet spreading in response to 1 mg/mL CRP. Total platelets scored were 374 control, 275 dynasore-treated, and 187 Dnm2Plt–/–. (C) Assessment of platelet spreading in response to 0.01 U/mL thrombin. Total platelets scored were 298 control, 226 dynasore-treated, and 192 Dnm2Plt–/– (*P<0.05; **P<0.01; ***P<0.001).
1420
haematologica | 2020; 105(5)