von Willebrand Disease (VWD), described in 1926, is currently the most frequent inherited hemorrhagic defect, although only a small percentage of affected patients have relevant clinical problems. VWD is caused by a reduction or by an altered activity of von Willebrand Factor (VWF), a multimeric glycoprotein with an high molecular weight essential for primary haemostasis. It is important both for platelet adhesion to the sub-endothelial matrix after a wound (through the binding to collagen and platelet receptor complex GPIb-IX-V) and for platelet aggregation (through the interaction with platelet receptor GPIIb/GPIIIa). Moreover, VWF stabilizes coagulation factor VIII, increasing its halflife.
VWF protein is encoded by VWF gene (178 Kb on chromosome 12) and it is synthesized by megakaryocytic and endothelial cells. Each monomer is 2813 amino acid long and it is organized into functional domains which allow the specific interaction with some sub-endothelial matrix components, with platelet receptors and with factor VIII. After a complex cellular biosynthesis, VWF is secreted as multimeric proteins with a molecular weight up to 20,000 KDa.

VWD is divided into 3 main groups. Quantitative defects of VWF in patients’ plasma can characterize VWD type 1 (partial defect; dominant genetic transmission) or VWD type 3 (no detectable traces of VWF in patients’ plasma; recessive transmission).
As concern VWD type 2, which include all the qualitative defects of VWF, we can distinguish 4 different sub-types.
VWD type 2A is characterized by the absence of high molecular weight multimers due to an increased plasmatic proteolysis or to a higher intracellular degradation.
In VWD type 2B, patients’ VWF has an increased affinity to platelet receptor GPIb-IX-V.
VWF of patients with VWD type 2M has a normal multimeric pattern but has a reduced ability in binding platelets.
VWD type 2N is characterized by defects which affect the binding to factor VIII.
Particularly important for this study is to note that mutations causing VWD type 2B are all distributed in a specific region of VWF A1 domain and generally they allow the entrance of water molecules (this structural changes lead to a stronger interaction between VWF and GPIb) or they change the binding to heparin (inducing an higher interaction with GPIb).

The aim of the study was to compare 2 different mutations on the same amino acid (R1308), responsible of VWD type 2B (with different severity). Since it was not possible to study the plasmatic proteins of the patients carrying these defects (because they have different destinies), it was decided to express in vitro the corresponding recombinant VWF in order to study the phenotype caused.
Once the vectors encoding these 2 mutations were prepared, we expressed the recombinant proteins (as well as the wt, as control) in COS7 cells. We analyzed the amount of protein produced (VWF:Ag measurement) and the multimeric pattern and we did not found significant differences.
Then, binding to GPIb for each of the recombinant VWF was investigated (VWF:RCo Elisa). With this method we recorded a hyper-platelet aggregation for VWF-R1308C, even if patients carrying this defect have a RIPA (ristocetin induced platelet aggregation) close to the normal range. On the other hand, VWF-R1308L has an higher affinity for GPIb than wt, but lower than VWF-R1308C, although patients with this mutation have a hyper-aggregant RIPA value. Our explanation for this in vivo and in vitro data is that in patients with R1308C, high molecular weight multimers (with a very high affinity for GPIb) can spontaneously bind to circulating platelets and as a consequence of that they are removed (retained into spleen). In patients with R1308L, the increased affinity for GPIb is not sufficient to cause the spontaneous binding to the platelets and thus the high molecular weigh multimers retention. We got the same indications also co-expressing the single mutations with the wild type VWF (mimicking the heterozygous state).
Another interesting result obtained is related to the binding ability of these recombinant molecules to collagen type I (VWF:CB I). Actually, this test revealed that both the mutants are able to bind collagen with a lower efficiency than wild type. This finding may also suggest the importance of the A1 domain in binding collagen (at least concerning the in vitro tests), although VWF A1 domain was not considered relevant for this activity so far.