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).

In a patient with apparently type 2A VWD (mean values: FVIII:C = 32 U/dL, VWF:Ag = 7 U/dL, VWF:RCo = < 6 U/dL, VWF:CB = <1, RIPA >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 (