Summary. In type 2N von Willebrand disease (VWD), vonWillebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D¢ domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site-directed mutagenesis and transient expression in COS-7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

Patient C.M. presented platelet function defects symptomatic of Glanzmann's thrombasthenia. However, analysis of surface-labeled platelets by SDS-polyacrylamide gel electrophoresis revealed the usual presence of the major glycoproteins, including GP IIb and GP i1la. Platelet fibrinogen was not detected. Analysis of Triton X-100 extracts of Ca2"-washed C.M. platelets by crossed immunoelectrophoresis (CIE) showed normal amounts of GP IIb-IlIa complexes. However, when samples were electrophoresed through an agarose gel containing "2I-fibrinogen, the usual binding of fibrinogen to GP fib-IIIa did not occur. Furthermore, the GP IIb-IIIa complexes showed an increased sensitivity to dissociation with EDTA, either after Triton X-100 solubilization or in the intact platelet membrane. For example, after incubation with EDTA at room temperature, the patient's platelets bound little of the monoclonal antibodies AP-2 or T10 (anti-GP IIb-IIIa complex) although normally binding Tab (anti-GP fib). Patient C.M. appears to represent a subgroup of thrombasthenia where platelets contain unstable GP Hib-HlIa complexes unable to support fibrinogen binding.

No abstract

Summary.We report the identification in five patients (three families) affected with type 2B von Willebrand disease (VWD) of three heterozygous nucleotide substitutions at the codon for arginine 543, 545 and 578 of the mature von Willebrand factor (VWF) subunit resulting in a glutamine, proline and leucine substitution, respectively. These mutations are located in the A1 loop where prevalent type 2B mutations (Arg543Trp, Arg545Cys and Arg578Gln) have been already identified at the same positions. By in vitro mutagenesis of full-length cDNA of VWF and transient expression in Cos-7 cells, we have shown that the six corresponding mutated recombinant VWFs (Gln543, Trp543, Cys545, Pro545, Leu578 and Gln578 rVWF) exhibited quantitatively normal expression and normal multimeric pattern but increased ristocetin-and botrocetininduced binding to platelets as compared with that for wildtype rVWF. The two mutations at position 545 induced the greatest reactivity for GPIb of corresponding rVWFs as compared to the two mutations at positions 543 and 578.

No abstract