The Involvement of Copper Transport in Von Willebrand Factor Multimer Assembly

In the event of vascular injury, the underlying prothrombotic extracellular matrix becomes exposed to circulating blood. Endothelial cells at the site of injury secrete multimeric von Willebrand factor (VWF) that will bind to the exposed extracellular matrix and create a scaffold to which platelets can adhere, forming a platelet plug. These critical steps after vascular injury are essential for creating a stable clot. Intracellular VWF multimer assembly is required for its extracellular function, and defects in VWF assembly cause the bleeding diathesis, von Willebrand disease. In a cell-free in vitro system, we and others find that EDTA or EGTA inhibits multimer formation, and calcium ions have been proposed to be required for this process. However, we find that the binding affinity of calcium ions for VWF D domains is very weak (K_dof 300-400 μM) and that buffers containing less than 2 μM calcium ions support multimer assembly even though few calcium binding sites could be occupied. These results suggest a role for cations other than calcium in VWF multimer assembly. Here we show that copper ions support VWF multimer assembly in a cell-free system and that the intracellular copper transporter, ATP7A, is required for VWF multimer assembly in cultured cells.

Some calcium binding proteins undergo a conformational change upon binding calcium ions and exhibit a decrease in the apparent molecular weight (an electrophoretic mobility shift) as assessed by SDS-PAGE and Western blot. Although the D domains of VWF bind calcium ions, they do not exhibit this behavior. We tested a panel of other divalent cations including: iron, magnesium, manganese, zinc, and copper. We found that the VWF N-terminal D'D3 dimer bound copper ions as demonstrated by a shift in electrophoretic mobility. We therefore assessed the effect of copper ions on VWF multimer assembly in vitro. Briefly, addition of monensin (1.5 μM) to human umbilical vein endothelial cells (HUVEC) caused secretion of C-terminal dimers of proVWF into the media. Pretreatment of the conditioned media with the divalent cation chelator, EGTA, or the copper chelator, tetraethylenepentamine (TEPA), preceded immunopurification of C-terminal dimers of proVWF using a rabbit polyclonal anti-hVWF antibody (DAKO A0082) bound to protein A magnetic beads. Beads were incubated at pH 7.4 or pH 5.8, and in the presence or absence of divalent cations for 3 hours at 37°C before analyzing the VWF by gel electrophoresis. Addition of calcium ions (100 μM) to proVWF dimers pretreated with only EGTA allowed some VWF multimer assembly; however, copper ions (100 μM) induced the assembly of higher molecular weight multimers from proVWF dimers pretreated with either EGTA or TEPA. With either divalent cation, multimers assembled at low pH, but not at neutral pH. These findings support a role for copper ions in VWF synthesis.

In vivo, free copper ions are toxic; therefore, they are sequestered by a system of chaperones and transporters. We examined the role of the intracellular copper transporter, ATP7A, in VWF multimerization because it is expressed in the trans Golgi network of endothelial cells, the site of VWF multimer assembly. Mick Petris (University of Missouri-Columbia) provided both wildtype and ATP7A knockout mouse embryonic fibroblasts (MEFs). We stably expressed full length human VWF and analyzed the secreted multimers by agarose gel electrophoresis. Wildtype MEFs produced multimeric VWF; however, culturing the MEFs in the presence of the copper chelator, TEPA, markedly impaired VWF multimer assembly. Moreover, ATP7A-null MEFs secreted only VWF dimers. These results indicate that copper ions and ATP7A contribute to VWF multimer assembly in cells.

The dependence of VWF assembly on ATP7A in cultured cells suggests that humans with congenital ATP7A deficiency (Menkes disease) could have detectable abnormalities in VWF multimer structure, although dietary copper and parenteral copper replacement therapy could affect this phenotype. Our preliminary analyses have identified a Menkes disease patient with low plasma levels of VWF antigen, multimers and activity. Further study will be required to determine the prevalence and significance of altered VWF multimer assembly in Menkes disease.