Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets

Abstract

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbβ3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and Caldaggef1^-/- mice. Using αIIbβ3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B.

Graphical abstract

Figure 1.

VWD type 2B phenotypes in WT mice overexpressing VWF/p.V1316M. (A) Platelet counts in WT mice expressing only VWF/WT (n = 26, red circles; mean platelet count set to 100%) and mice expressing VWF/p.V1316M (n = 25, blue circles) 4 days after hydrodynamic gene transfer. The right axis shows absolute platelet count. (B) Platelet size; the left axis represents relative values, and the right axis represents FSC values. (C) Representative images of platelet aggregates in blood smears from WT-VWF/p.V1316M mice, stained with eosin and methylene blue. Images were acquired with a Nikon TE300 microscope and 100×/1.4 NA oil objective (Nikon Instruments, Melville, NY) equipped with a QImaging Retiga EXi CCD camera (QImaging, Surrey, Canada) using SlideBook software version 5.0, Intelligent Imaging Innovation. Total magnification ×1000.

Figure 2.

Platelets from IL4R-IbαTg mice are unaffected by the presence of VWF/p.V1316M. (A) Platelet counts in IL4R-IbαTg mice expressing only VWF/WT (100%, n = 4, red triangles) and mice overexpressing VWF/p.V1316M (n = 4, blue triangles) 4 days after hydrodynamic gene transfer. The left axis represents relative counts, and the right axis shows absolute platelet counts. (B) Platelet size in IL4R-IbαTg mice (100%, n = 4, red triangles) and IL4R-IbαTg-VWF/p.V1316M mice (n = 4, blue triangles). (C) Single platelets in blood smears from IL4R-IbαTg-VWF/p.V1316M mice. (D) Platelet counts and platelet size in Stim1^fl/flPF4-Cre⁺ (red circles), Caldaggef1^−/−P2Y12^−/− (blue squares), and talin1^fl/flPF4-Cre⁺ (green triangles) mice in the presence of VWF/WT (100%, open symbols) and VWF/p.V1316M (filled symbols). n = 3-7 mice per group. (E) Platelet aggregates found in blood smears of Stim1^fl/flPF4-Cre⁺, Caldaggef1^−/−P2Y12^−/−, and talin1^fl/flPF4-Cre⁺ mice expressing VWF/p.V1316M. Images in panels C and E were acquired as described in Figure 1.

Figure 3.

Sustained platelet activation is impaired in mice overexpressing VWF/p.V1316M. αIIbβ3 integrin activation measured by flow cytometry with JON/A-PE antibody (selectively binds to the activated form of the receptor). JON/A-PE was added with the agonist (PAR4p, 200 μM) (A) or 10 minutes after the agonist (B-C). The mean fluorescence intensity measured in stimulated platelets from WT (A-B) or Caldaggef1^−/− (C) mice expressing VWF/WT (red bars) was set to 100%. Red bars, platelets expressing VWF/WT only; blue bars, platelets expressing VWF/p.V1316M. Data are shown as mean ± SD; n = 3-13 mice per group. ***P ≤ .001. ns, not significant.

Figure 4.

VWF/p.V1316M affects platelet granule release. (A) αIIbβ3 integrin activation measured with JON/A-PE, added 10 minutes after stimulation with ADP (10 μM) and U46619 (5 μM). MFI in stimulated WT platelets (red bars) set as 100%. Red bars, VWD2B(p.V1316M) platelets. (B) Quantification of secreted [³H]serotonin from δ-granules of washed WT and VWD2B(p.V1316M) platelets (red and blue bars, respectively), stimulated or not with PAR4p (1 mM). (C) P-selectin surface expression on PAR4p-stimulated WT and VWD2B(p.V1316M) platelets (red and blue bars, respectively). Data are shown as mean ± SD of 3-17 mice per group. **P ≤ .01; ***P ≤ .001.

Figure 5.

Phosphorylation of PKC substrates is dysregulated in VWD2B(p.V1316M) platelets. (A) Immunoblotting for phosphorylated PKC substrates following sodium dodecyl sulfate–polyacrylamide gel electrophoresis of platelet lysates from WT-VWF/WT and WT-VWF/p.V1316M mice (resting and PAR4p activated [200 μM]). β-actin was detected in every sample as loading control. (B) Quantification of PKC substrate phosphorylation (whole lane), normalized to β-actin content, using Image Studio Lite software (LI-COR Biosciences). Data are shown as arbitrary units (a.u.); PKC substrate phosphorylation in resting WT-VWF/WT platelets was set at 1. Data are mean ± SD, n = 5-8 mice per group. (C) Immunoblotting for phosphorylated PKC substrates following sodium dodecyl sulfate–polyacrylamide gel electrophoresis of human platelet lysates (resting) from 1 patient affected by VWD (p.V1316M) type 2B and a healthy donor (control). 14.3.3ζ (anti-14.3.3 ζ 4.; Santa Cruz Biotechnology) was detected as loading control. Horseradish peroxidase–conjugated secondary antibodies were applied, and immunoreactive bands were revealed using Pierce ECL Western Blotting Substrate. (D) Quantification of PKC substrate phosphorylation, normalized to loading control, performed with ImageJ software. Data are shown as a.u.; PKC substrate phosphorylation in resting control platelets was set at 1. Data are mean ± SD of 3 patients and 3 healthy controls. Immunoblots for patients 2 and 3 are reported in supplemental Figure 7. *P ≤ .05; ***P ≤ .001.

Figure 6.

VWF/p.V1316M induces ADAM17-independent GPIbα shedding. Quantification of GPIbα (A) and GPIX (B) receptor levels by flow cytometry at the surface of circulating platelets collected from WT-VWF/WT (red bars, n = 26) or WT-VWF/p.V1316M (blue bars, n = 26) mice. (C) Platelet count in ADAM17^fl/flPF4-Cre⁺ mice expressing VWF/WT (100%, n = 4, red diamonds) or VWF/p.V1316M (n = 4, blue diamonds) 4 days after hydrodynamic gene transfer. The right axis shows the absolute platelet count. (D) Platelet size in ADAM17^fl/flPF4-Cre⁺ mice (100%, n = 4, red diamonds) and ADAM17^fl/flPF4-Cre⁺ -VWF/p.V1316M mice (n = 4, blue diamonds). The right axis shows FSC values. (E) Platelet aggregates found in blood smears from ADAM17^fl/flPF4-Cre⁺-VWF/p.V1316M mice. Images were acquired as described in Figure 1. (F) Quantification of GPIbα receptor levels by flow cytometry at the surface of circulating platelets collected from ADAM17^fl/flPF4-Cre⁺-VWF/WT mice (n = 4, lime green bar) and ADAM17^fl/flPF4-Cre⁺-VWF/p.V1316M mice (n = 4, light green bar). (G) Representative immunoblot for glycocalicin (GC; detected with anti-GPIbα antibody Xia.G7 from EMFRET Analytics, Würzburg, Germany) in plasma from WT and ADAM17^fl/flPF4-Cre⁺ mice expressing VWF/WT and VWF/p.V1316M (upper panel). Quantification of data in upper panel using Image Studio Lite software (lower panel); data are mean ± SD. n = 3-9 mice per group. ***P ≤ .001.

Figure 7.

VWF/p.V1316M decreases hemostasis in a laser-injury model. Quantitative analysis (SlideBook software) of platelet accumulation and bleeding time after laser injury to the saphenous vein of WT-VWF/WT (red) and WT-VWF/p.V1316M (blue) mice. Mice were injected with an Alexa Fluor 488–labeled antibody against GPIX to monitor platelet accumulation. Repeated vascular injury was induced by laser ablation. (A) Representative images at the indicated times after the first injury. Intravital microscopy was performed with a Zeiss Examiner Z1 microscope (Zeiss, Oberkochen, Germany) equipped with a Hamamatsu Orca Flash 4.0 camera (Hamamatsu Photonics, Hamamtsu City, Japan) and a 20× water immersion objective (numerical aperture 1, working distance 1.8 mm) (Zeiss, Jena, Germany). Scale bars, 50 μm. (B) Accumulation of platelets at the site of injury, quantified as sum fluorescence intensity (± SEM). (C) Bleeding time. (D) Bleeding time after the first laser injury to the saphenous vein of WT-VWF/WT mice (red bar), control mice with low peripheral platelet counts (low-PLT; light green bar), and WT-VWF/p.V1316M mice (blue bar). Low-PLT mice were generated by adoptive transfer of WT platelets into thrombocytopenic IL4R-IbαTg mice, yielding a PPC ∼0.7 × 10⁸/mL of blood. Data for WT-VWF/WT and WT-VWF/p.V1316M mice are the same as in (C). Total number of injuries at distinct locations: n = 30 (obtained in 3 WT-VWF/WT mice), n = 38 (obtained in 5 WT-VWF/p.V1316M mice), and n = 15 (obtained in 4 low-PLT mice). Data in panels C-D are mean ± SD. **P ≤ .01; ***P ≤ .001.