In vitro phosphorylation of von Willebrand factor by FAM20c enhances its ability to support platelet adhesion

Abstract

Essentials Platelet adhesion to von Willebrand factor (VWF) is critical for hemostasis and thrombosis. Whether VWF can undergo phosphorylation is unknown. Family with sequence similarity 20 kinase phosphorylates VWF A2 domain at S1517 and S1613. Phosphorylation of VWF and VWF A1A2A3 domain at S1613 enhances platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) mediates platelet adhesion and contributes to hemostasis at sites of vascular injury as well as to arterial thrombosis. The A1A2A3 domains of VWF contain important sites that differentially participate in supporting platelet adhesion. FAM20c (family with sequence similarity 20, member C) has emerged as a serine/threonine kinase, which phosphorylates extracellular proteins containing the S-X-E/pS motifs that are also found within the VWF A domains. This is of interest because we and others have shown that structural modifications within these A domains influence the ability of VWF to support platelet adhesion. Objective We assessed if VWF A domains can be phosphorylated and the functional consequence of phosphorylated VWF. Results Here, we show that FAM20c phosphorylated purified plasma VWF, VWF A1A2A3 protein, isolated A2 domain, but not A1 and A3 domain proteins, in vitro. FAM20c phosphorylated the isolated A2 domain at S1517 and S1613 within the S-X-E recognition motif, with S1613 being the major phosphorylation site. Mass spectrometry analysis of purified plasma VWF from healthy donors revealed several phosphorylation sites, including the S1613 in the A2 domain. VWF A1A2A3 domain protein phosphorylated at S1613 promoted stable platelet adhesion and microthrombi at high shear stress. Lastly, under high shear stress VWF treated with FAM20c and ATP robustly supported platelet adhesion, compared to VWF treated with FAM20c in the absence of ATP. Conclusion These outcomes indicate that VWF can be phosphorylated by FAM20c in vitro, and this novel post-translational modification enhances the adhesiveness of VWF to platelets.

Keywords: blood platelets; cell adhesion; phosphorylation; protein kinase; von Willebrand factor.

Fig. 1.. In vitro phosphorylation of VWF A domains by FAM20c.

Incorporation of ³²P from γ−³²P ATP to VWF A1A2A3 protein (A) and the individual A domains (B) by FAM20c but not by the FAM20c kinase dead mutant (D478A). Samples from kinase assays were separated by SDS-PAGE and subjected to autoradiography (upper panel) or visualized by Coomasssie blue staining (lower panel). Degradation product of A1 protein in Figure 1B is shown as **. Phosphorylation of A1A2A3 was blocked by treatment with λ phosphatase. Autophosphorylation of FAM20c in this assay is labelled as FAM20c. Blots are representative of three independent experiments.

Fig. 2.. Identification of S1517 and S1613 in the VWF A2 protein as FAM20c phosphorylation sites.

Mass spectrometric analysis of the A2 protein subjected to FAM20c in vitro kinase assays. (A) LC/MS^e low energy (MS¹) precursor ion mass spectra showing A2 domain protein incubated with FAM20C kinase in the absence and presence of ATP. Lock mass corrected and charge-reduced, mass-centroid base peak spectra are shown where x-axis represents M+H⁺ (Da), and y-axis is relative ion intensities; numbers shown on top of spectrum peaks represent LC retention times. Peaks circled in the +ATP spectrum show the presence of phosphorylated peptides not found in the -ATP spectrum, indicating FAM20c-mediated specific phosphorylation. Phosphopeptides in circle i and circle ii correspond to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1517 and those in circle iii and iv to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1613. (B) LC/MS^E base peak high energy (MS²) product ion mass spectra showing all b- and y-ions observed in the MS2 spectrum for two of the phosphopeptide isoforms circled in ii and iv respectively. These MS2 spectra show unequivocally (as summarized in the peptide sequence display shown at the top of each spectra) the site of FAM20c- mediated phosphorylation on the A2 domain protein as being localized to Ser-1517 and Ser-1613. pS refers to site of phosphorylation, and oxM refers to an oxidized methionine residue. (C) Incorporation of ³²P from γ−³²P ATP to wild type (WT) VWF A2 and various A2 mutants by FAM20c and FAM20c kinase dead mutant. Samples were separated by SDS-PAGE and subjected to autoradiography (upper panel) or visualized by Coomassie blue staining (lower panel). Blots are representative of two independent experiments.

Fig. 2.. Identification of S1517 and S1613 in the VWF A2 protein as FAM20c phosphorylation sites.

Mass spectrometric analysis of the A2 protein subjected to FAM20c in vitro kinase assays. (A) LC/MS^e low energy (MS¹) precursor ion mass spectra showing A2 domain protein incubated with FAM20C kinase in the absence and presence of ATP. Lock mass corrected and charge-reduced, mass-centroid base peak spectra are shown where x-axis represents M+H⁺ (Da), and y-axis is relative ion intensities; numbers shown on top of spectrum peaks represent LC retention times. Peaks circled in the +ATP spectrum show the presence of phosphorylated peptides not found in the -ATP spectrum, indicating FAM20c-mediated specific phosphorylation. Phosphopeptides in circle i and circle ii correspond to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1517 and those in circle iii and iv to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1613. (B) LC/MS^E base peak high energy (MS²) product ion mass spectra showing all b- and y-ions observed in the MS2 spectrum for two of the phosphopeptide isoforms circled in ii and iv respectively. These MS2 spectra show unequivocally (as summarized in the peptide sequence display shown at the top of each spectra) the site of FAM20c- mediated phosphorylation on the A2 domain protein as being localized to Ser-1517 and Ser-1613. pS refers to site of phosphorylation, and oxM refers to an oxidized methionine residue. (C) Incorporation of ³²P from γ−³²P ATP to wild type (WT) VWF A2 and various A2 mutants by FAM20c and FAM20c kinase dead mutant. Samples were separated by SDS-PAGE and subjected to autoradiography (upper panel) or visualized by Coomassie blue staining (lower panel). Blots are representative of two independent experiments.

Fig. 2.. Identification of S1517 and S1613 in the VWF A2 protein as FAM20c phosphorylation sites.

Mass spectrometric analysis of the A2 protein subjected to FAM20c in vitro kinase assays. (A) LC/MS^e low energy (MS¹) precursor ion mass spectra showing A2 domain protein incubated with FAM20C kinase in the absence and presence of ATP. Lock mass corrected and charge-reduced, mass-centroid base peak spectra are shown where x-axis represents M+H⁺ (Da), and y-axis is relative ion intensities; numbers shown on top of spectrum peaks represent LC retention times. Peaks circled in the +ATP spectrum show the presence of phosphorylated peptides not found in the -ATP spectrum, indicating FAM20c-mediated specific phosphorylation. Phosphopeptides in circle i and circle ii correspond to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1517 and those in circle iii and iv to phosphopeptide isoforms which include FAM20c-mediated phosphorylation on Ser-1613. (B) LC/MS^E base peak high energy (MS²) product ion mass spectra showing all b- and y-ions observed in the MS2 spectrum for two of the phosphopeptide isoforms circled in ii and iv respectively. These MS2 spectra show unequivocally (as summarized in the peptide sequence display shown at the top of each spectra) the site of FAM20c- mediated phosphorylation on the A2 domain protein as being localized to Ser-1517 and Ser-1613. pS refers to site of phosphorylation, and oxM refers to an oxidized methionine residue. (C) Incorporation of ³²P from γ−³²P ATP to wild type (WT) VWF A2 and various A2 mutants by FAM20c and FAM20c kinase dead mutant. Samples were separated by SDS-PAGE and subjected to autoradiography (upper panel) or visualized by Coomassie blue staining (lower panel). Blots are representative of two independent experiments.

Fig. 3.. Identification of S1613 phosphorylation in the purified plasma VWF.

LC/MS spectra showing phosphorylation of the Ser-1613 site within the VWF A2 domain from VWF purified directly from human plasma. LC/MS^e low energy (MS¹) precursor ion scan of purified VWF after trypsin digestion shown in top spectra, with precursor ion of m/z 2412.1362 shown enlarged and circled within inset, corresponding to peptide sequence EQAPNLVYMVTGNPApS¹⁶¹³DEIKR confirmed by LC/MS^E high energy (MS²) sequence analysis shown in lower spectra. Four separate Mass Spectrometry experiments were performed with three different donors.

Fig. 4.. Increased platelet adhesion to A1A2A3 protein from FAM20c transfected cells

(A) Immunoblotting of A1A2A3 protein purified from HEK 293 cells that are transfected with A1A2A3 and dead FAM20c kinase mutant D478A (D478A) or A1A2A3 and FAM20c (FAM20c) with anti-VWF antibody. (B) Whole blood was perfused over A1A2A3 protein obtained from cells with D478A or FAM20c at a shear rate of 1500 S⁻¹ and adhesion visualized in a Bioflux system. (C) Quantification of platelet coverage is depicted in a logarithmic scale. Data are obtained from three independent blood donors.

Fig. 5.. A1A2A3 protein phosphorylated at S1613 enhances platelet-VWF interaction.

(A) Coomassie staining of the purified A1A2A3 wild type (WT) and A1A2A3 S1517A mutant. (B) Whole blood was perfused over immobilized A1A2A3 (WT), A1A2A3 (S1517A) mutant that was previously incubated with FAM20c in the presence of ATP (S1613 phosphorylated) and absence of ATP (S1613 not phosphorylated) at a shear rate of 1500 S⁻¹ and adhesion visualized in a Bioflux system. (C) Quantification of platelet coverage and stable microthrombi. Data are obtained from six independent blood donors.

Fig. 6.. In vitro phosphorylation of VWF supports stable platelet adhesion.

(A) Incorporation of ³²P from γ−³²P ATP to plasma purified VWF by FAM20c but not by D478A FAM20c kinase dead mutant (D478A). Samples were separated by SDS-PAGE and subjected to autoradiography (upper panel) or visualized by Coomasssie blue staining (lower panel). Autophosphorylation of FAM20c is indicated as FAM20c. Blots are representative of 3 independent experiments. (B) Whole blood from three healthy donors was perfused over a surface coated with purified VWF treated with FAM20c in the presence or absence of non-radioactive ATP at 1500s⁻¹ shear rate. (C) Quantification of platelet adhesion.