Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M

<div><p>Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.</p></div

Clinical and laboratory parameters of the patients with VWF/p.V1316M mutation.

<p>Clinical and laboratory parameters of the patients with VWF/p.V1316M mutation.</p

Expression of apoptotic proteins in mice expressing VWF/pV1316M.

<p>For each experiment, 12 mice were used: 2 injected with pLIVE, 4 injected with WT and 6 injected with VWF/p.V1316M mice. Washed murine platelets (2.5 x10⁸ platelets/mL) were lysed and then an equal number of platelets was loaded. Apoptotic proteins were assessed by immunoblotting with anti-Bak, anti-Bax and anti-14-3-3ζ antibodies. Data are expressed as the ratio of apoptotic protein expression versus 14-3-3ζ expression. Then, the ratio for mVWF/p.V1316M or WT mVWF was compared with the corresponding ratio for control pLIVE plasmid (100%) and results are presented as means ± SEM from three independent experiments.</p

Expression of apoptotic proteins in patients with VWD-type 2B.

<p>Washed platelets (2.5 x10⁸ platelets/mL) from controls (C) or P1 and P2 were lysed and then equal numbers of platelets were loaded. Apoptotic proteins were assessed by immunoblotting with anti-Bak, anti-Bax, anti Bcl-xL and anti-14-3-3ζ antibodies. Data are expressed as the ratio of apoptotic protein expression versus 14-3-3ζ expression. Then, the ratio of an apoptotic protein for P1 or P2 was compared with the corresponding ratio for control (100%). Results are means ± Standard Error of the Mean (SEM) from three independent experiments. *p = 2.8.0x10^-2, **p = 2.3x10^-3, ***p = 2.9x10^-4.</p

PS exposure in patients with VWD-type 2B.

<p>PS exposure was determined <b>A)</b> in washed platelets and <b>B)</b> in blood pretreated or not with ABT-737 (10 μM) for 60 minutes at 37°C. Then washed platelets and blood were further incubated with annexin V-PE and an anti-CD41a-FITC (a platelet marker for blood) for 15 minutes and then analyzed by flow cytometry. Results are expressed as a percentage of positive Annexin V. Means ± SEM from three independent experiments are shown, **p = 1.4x10^-3 and ***p = 5.8x10^-8 (paired Student t test).</p

ΔΨm depolarization in mice expressing VWD-type 2B.

<p>ΔΨm depolarization was determined in washed platelets pretreated for 30 minutes with TMRE-PE (500 nM final concentration) at 37°C. Then platelets were incubated further for 60 minutes with or without ABT-737 (10 μM final concentration) and analyzed by flow cytometry. Results are expressed as a percentage of depolarized cells. Means ± SEM from three independent experiments are shown. *p = 2,5 x 10⁻² and **p = 5.0 x10^-3 (unpaired Student t test).</p

PS exposure in mice expressing VWD-type 2B.

<p>PS exposure was determined A) in washed platelets and B) in blood pretreated or not with ABT-737 (10 μM) for 60 minutes at 37°C. Then washed platelets and blood were further incubated with annexin V-PE and an anti-CD41-FITC (a platelet marker for blood) for 15 minutes and then analyzed by flow cytometry. Results are expressed as a percentage of positive annexin V. Means ± SEM from three independent experiments are shown.</p

<i>Δ</i>Ψm depolarization in patients with VWD-type 2B.

<p>ΔΨm depolarization was determined A) in washed platelets and B) in blood pretreated for 30 minutes with TMRE-PE (50 nM final concentration) at 37°C. Then platelets were further incubated for 60 minutes with or without ABT-737 (10 μM final concentration) and analyzed by flow cytometry. Results are expressed as a percentage of depolarized cells. Means ± SEM from three independent experiments are shown,**p = 1.4x10^-3 and ***p = 6.0x10^-5 (unpaired Student t test).</p

Caspase 3 activity in patients with VWD-type 2B.

<p>Washed platelets (2.5 x10⁸ platelets/mL) pretreated or not with ABT-737 (10 μM) were lysed and caspase-3 activity was assessed by immunoblotting with <b>A</b>) an anti-caspase-3 antibody <b>B)</b> with an anti-gelsolin antibody and C) with anti-calpain 1 antibody. Results are representative of two or three independent experiments.</p