FAM20c-Mediated Serine 1613 Phosphorylation in the A2 Domain of Von Willebrand Factor Regulates ADAMTS13 Activity and Thrombus Formation

Protein phosphorylation represents a common mechanism to regulate the structure and function of proteins. Although vast amount of extracellular proteins including secreted plasma proteins are phosphorylated, historically, phosphorylation has been intensively investigated for intracellular proteins. The plasma and subendothelial protein von Willebrand factor (VWF) undergoes post translational modifications such as glycosylation and sulphation to reach the mature protein product. However, phosphorylation of VWF has not been described. We have used mass spectrometry to analyze purified plasma VWF, and identified that serine 1613 within the A2 domain was phosphorylated. A natural occurring mutation on this residue (S1613P) causes von Willebrand disease Type 2A by increasing the susceptibility of VWF to be cleaved by ADAMTS13. Notably, S1613 overlapped with the S-X-E/pS motif, which is the consensus site for phosphorylation by an atypical kinase, FAM20c (family with sequence similarity 20, member C). Localized to the inner lumen of the golgi/endoplasmic reticulum, FAM20c is secreted and likely responsible for the phosphorylation of several secreted proteins bearing the S-X-E/ps motif. Therefore, we further investigated whether VWF can undergo phosphorylation by FAM20c and how such modification impacts the function of VWF, particularly on the activity of ADAMTS13. In vitro, recombinant FAM20c directly phosphorylated recombinant VWF-A1A2A3 domain protein and purified plasma VWF. Further analysis revealed that the isolated A2 domain but not A1 or A3 domain was phosphorylated by FAM20c. Phosphorylation was assessed employing ³²P labeling of proteins, protein shift in phospho tag gel and mass spectrometry. Treatment with  λ phosphatase diminished phosphorylation and a defective FAM20c kinase mutant failed to phosphorylate A2 and VWF proteins, confirming the phosphorylation event. In addition, FAM20c-mediated phosphorylation was markedly reduced in a non-phosphorylatable A2 S1613A mutant. Thus, all these outcomes indicate that the secreted kinase FAM20c can phosphorylate S1613 in the A2 domain of VWF. To explore the functional effect of S1613 phosphorylation, we compared the plasma-mediated cleavage of wild type (WT)A2, phosphomimetic S1613D mutant and the nonphosphorylatable A2 S1613A mutant. Unexpectedly, and in sharp contrast to the WT and S1613A variants, the S1613D mutant was effectively cleaved in the presence of the enzyme inhibitor, EDTA. In addition, cleavage of the S1613D mutant was robust and slightly faster than that of the WT and S1613A. These studies suggest that phosphorylation of  S1613 in VWF may facilitate the cleavage of VWF multimers. To further explore the physiological relevance of phosphorylated VWF in thrombosis, we generated phospho VWF S1613 and nonphosphorylated S1613 VWF antibodies and studied their effect on thrombus formation. In a microfluidic perfusion system, whole blood supplemented with 50 μg/ml of phosphoVWF antibody but not the nonphosphoVWF antibody, markedly potentiated thrombus formation on a collagen-coated surface. Collectively, these studies suggest that S1613 phosphorylation of VWF suppress thrombus formation, in part by facilitating cleavage of the VWF multimers. These studies identify for the first time that VWF can undergo phosphorylation and opens new avenues for regulation of VWF function by phosphorylation.