Biochemical Characterization of Recombinant Von Willebrand Factor (VWF) with a Type 2B Mutation (P1337L) Coexpressed with a Type 1 VWF Defect (C275R): A Complex Molecular Defect in a Patient Previously Diagnosed with Type 2A Von Willebrand Disease (VWD).
Authors: Baronciani Luciano , Federici Augusto B. , Beretta Matteo , Cozzi Giovanna , Canciani Maria T. , Mannucci Pier M. 
Published: April 2020

Abstract
In a patient with apparently type 2A VWD (mean values: FVIII:C = 32 U/dL, VWF:Ag = 7 U/dL, VWF:RCo = &lt; 6 U/dL, VWF:CB = &lt;1, RIPA &gt;2.0 mg/ml, loss of high and intermediate molecular weight multimers in plasma and low platelet VWF content) a transient thrombocytopenia occurred after an infusion test with desmopressin. The propositus brother showed a less severe laboratory data (mean values: FVIII:C = 36 U/dL, VWF:Ag = 17 U/dL, VWF:RCo = 6 U/dL, VWF:CB=1, RIPA = 1.2 mg/ml). In order to better characterize VWD type diagnosis, the complete VWF gene was screened by SSCP analysis and direct DNA sequencing. The two brothers resulted to be compound heterozygous for mutation P1337L and a novel candidate defect C275R. In their children, mutations were associated with either type 2B (P1337L) or type 1 (C275R) VWD in heterozigous state. Both mutations were introduced separately into an individual vector pSV-VWF by site direct mutagenesis. rVWF-P1337L and rVWF-C275R, were transiently expressed in Cos 7 cells, solely, together and with the rVWF-WT. All the rVWFs in cells supernatants were tested by VWF:Ag and multimer analysis. Binding of different rVWFs to the GpIb platelet receptor was evaluated by an ELISA method (Federici et al. Haematologica89:77, 2004), at increasing concentrations of ristocetin (0, 0.125, 0.25, 0.5, 0.8 and 1 mg/mL), and the rVWF bound to GpIb was revealed by anti-VWF antibody-HRP reading O.D. 492 nm. Only the expressed rVWF-C275R showed a strongly reduced VWF:Ag, in comparison with the rVWF-WT, in cells medium and a complete absence of large and intermediate multimers, only dimers being present. The other rVWFs (P1337L, P1337L/WT and C275R/WT) showed only a slightly reduced VWF:Ag, in comparison to rVWF-WT, with the exception of rVWF C275S/WT that was about 40% of the rVWF-WT; all of them, however showed a full set of multimers. GpIb binding assay (see Table) showed that rVWF obtained by co-expression of mutation P1337L with C275R behave very similarly to rVWF-P1337L, suggesting that rVWF-C275R subunits cannot be assembled into intermediate and large molecular weight multimers. Therefore, in the propositus and his brother, mutation P1337L, present in all the VWF subunits, strongly enhanced the VWF interaction with GpIb receptor. As a consequence, in vivo, these patients showed a reduced RIPA (&gt;2 mg/ml), as typically observed in VWD type 2A, because of the loss of VWF high- and intermediate multimers due to the spontaneous interactions of such “hemizygous” type 2B VWF with circulating platelets. Our data suggest once more the importance of mutation analysis within family members and in vitro expression studies especially when phenotypic data are not certain.
rVWFs Ristocetin 0 mg/mL Ristocetin 0.125 mg/mL Ristocetin 0.25 mg/mL Ristocetin 0.5 mg/mL Ristocetin 0.8 mg/mL Ristocetin 1 mg/mL WT 0.066 O.D. 0.071 O.D. 0.128 O.D. 0.154 O.D. 0.691 O.D. 0.924 O.D. P1337L 0.117 O.D. 0.310 O.D. 0.689 O.D. 0.984 O.D. 0.984 O.D. 1.039 O.D. P1337L/WT 0.063 O.D. 0.166 O.D. 0.409 O.D. 0.740 O.D. 0.894 O.D. 1.008 O.D. P1337L/C275R 0.084 O.D. 0.372 O.D. 0.635 O.D. 0.925 O.D. 1.105 O.D. 1.117 O.D.