Page 258 - Haematologica May 2020

N. Eaton et al.
The decreased GPIbα surface expression was due to internalization, and not to shedding, as total GPIbα expression was maintained during the course of the exper- iment, as shown by immunoblot analysis (Figure 3C). Taken together, the data show that DNM2 specifically regulates GPVI signaling, rather than GPIbα downregula- tion.
Impaired α-granule secretion and integrin αIIbβ3 activation in CRP-stimulated dynasore-treated and Dnm2Plt–/– platelets
Following activation by collagen or soluble agonists, platelets secrete their granule contents and activate their surface integrin αIIbβ3 in order to recruit circulating platelets and mediate platelet aggregation, respectively. The significance of DNM2 in these platelet hemostatic processes was assessed by flow cytometry (Figure 4). Control platelets, dynasore-treated platelets, and Dnm2Plt–/– platelets were activated with CRP (Figure 4A and C) or thrombin (Figure 4B and D), and analyzed for P-selectin (CD62P) expression, a marker for α-granule secretion (Figure 4A and B), and binding of fluorescently-labeled fib- rinogen, a marker for integrin αIIbβ3 activation (Figure 4C and D).19 Both CRP and thrombin induced a concentra- tion-dependent increase of α-granule secretion and inte- grin αIIbβ3 activation in control platelets, reaching about 80% of platelets expressing CD62P or binding fibrinogen with 25 mg/mL CRP or 0.25 U/mL thrombin, respectively. Dynasore treatment resulted in a significant decrease in platelet responses to CRP, as only 30-40% platelets expressed CD62P and bound fibrinogen with 50 mg/mL CRP. By contrast, platelet responses to thrombin were not significantly affected by dynasore treatment. CRP-depen- dent CD62P expression and fibrinogen binding were com- pletely abolished in Dnm2Plt–/– platelets and only about
AB
20% expressed CD62P and bound fibrinogen in response to 0.5 U/mL thrombin.
Altered spreading of dynasore-treated and Dnm2Plt–/– platelets
Following activation, platelets rapidly change shape from resting disc-like entities to morphologically distinct forms, first by rounding, then by extending finger-like
30 filopodia and spreading thin sheet-like lamellipodia. The
significance of DNM2 in platelet spreading was examined (Figure 5). Control platelets, dynasore-treated, and Dnm2Plt–/– platelets were activated with either 1 mg/mL CRP or 0.01 U/mL thrombin and allowed to adhere onto immobilized fibrinogen. In control platelets, stimulation with CRP or thrombin resulted in filopodia extension and lamellipodia spreading, as evidenced by phalloidin stain- ing, a marker for polymerized actin, with the greatest dif- ference being more angular or rounded appearance, respectively (Figure 5A). Treatment with dynasore miti- gated lamellipodia formation in CRP-stimulated platelets (Figure 5B), and to a lesser, non-statistically significant degree in thrombin-stimulated platelets (Figure 5C), although it did not prevent filopodia growth.
Dnm2Plt–/– platelets displayed great heterogeneity in shape change with either agonist (Figure 5A), wherein spread platelet surface area varied between below control levels or up to a 5-fold increase in size following stimula- tion, reflecting the increased size of these platelets.9 Dnm2Plt–/– platelets revealed extreme irregularity in their cytoskeletal and overall morphological arrangement, con- sistent with altered spreading capacity.
Absence of fibrinogen in Dnm2Plt–/– platelets
The fibrinogen content of platelet α-granules derives from the integrin αIIbβ3-dependent uptake of plasma-
Figure 3. Impaired GPIbα downregula- tion in CRP-stimulated dynasore-treat- ed and Dnm2Plt–/– platelets. Control platelets, platelets treated with 100 μM dynasore, and Dnm2Plt–/– platelets were activated for 3 minutes (min) at 37°C with 25 mg/mL CRP (A) or 0.1 U/ml thrombin (B), incubated with FITC- labeled anti-mouse CD42b antibody, and analyzed by flow cytometry. Results are expressed as percentage of CD42b
C
expression at
mean±standard error of mean (SEM) of three independent experiments, and are compared statistically to control (*P<0.05; **P<0.01; ***P<0.001). (C) Control, dynasore-treated, and Dnm2Plt–/– platelets were activated or not with 0.1 U/mL thrombin or 25 mg/mL CRP for 3 min at 37°C as indicated. Platelet lysates were subjected to SDS- PAGE and probed for GPIbα and β-actin as loading control, as indicated. Results are representative of three independent experiments.
rest, represent
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haematologica | 2020; 105(5)