Misfolding Induced By the Mutations V1314D and F1369I Affects the Function and the Structure of the Von Willebrand A1-Domain

Von Willebrand disease (VWD) is the most common inherited human bleeding disorder. It is caused by deficiencies or defects in the plasma protein von Willebrand factor (vWF). The current classification of VWD consists of six distinct types. Type 1 and 3 result in a quantitative vWF deficiency in patients while the four type 2 variants (type 2A, 2B, 2M & 2N) are caused by qualitative defects in vWF. The von Willebrand factor is a multimeric multidomain glycoprotein that is secreted into blood from vascular endothelial cells. The protein initiates platelet adhesion at sites of cardiovascular injury. In vWF three essential domains could be identified which are responsible for the interaction with platelets and subendothelial tissue. While the A1 domain is responsible for the interaction with platelets, the A3-domain interacts with collagen from the subendothelial tissue. The A2-domain contains a cleavage site for the zinc protease AdamTS13 which regulates the size and the function of the vWF multimers by cleaving the A2-domain. We have recently studied the effect of several type 2B (= gain of function, stronger interaction with platelets) and type 2M (= loss of function, weak or no interaction with platelets) mutations in the vWF A1-domain and found clear evidence that these mutations cause a misfolding of this domain resulting in either gain- or loss of function (Tischer et al. (2014) Biophys. J. Accepted article). Hence type 2M and 2B VWD are clearly protein folding disorder diseases such like Alzheimer or various Amyloidoses. However since our group has obtained the A1-domain recombinantly from E.coli , it was unclear whether indeed misfolding of A1 is occurring or whether it is the result of the expression of the proteins in bacteria. To investigate this issue we have expressed triple domains in mammalian HEK293 cells consisting of A1, A2 and A3 and harboring the type 2B mutation V1314D and the 2B mutation F1369I in the A1 domain. The impact of the mutations on the biological function was determined in shear flow-dependent assays by observing the translocation of platelets on surface-immobilized A1- or triple domain. Using high speed video microscopy we were able to obtain statistical valid parameters for platelet translocation such as mean pause times and velocities. While F1369I did not support platelet translocation at all, V1314D was found to almost immobilize platelets in the flow chamber. The triple domain constructs harboring the mutations were found to have very similar functional features as the single domain mutants. F1369I triple domain did not support platelet translocation whereas V1314D triple domain immobilized platelets in the flow chamber at all applied shear rates. Structural characterization of the single A1 domains and of the triple domains resulted in evidence for massive misfolding in the A1 domain induced by the mutations. Therefore, all attempts to understand VWD and a potential drug development are required to account for non-native conformations of the A1 domain. Disclosures No relevant conflicts of interest to declare.