Identification of new type 2B von Willebrand disease mutations: Arg543Gln, Arg545Pro and Arg578Leu

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.

Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia

“…The R1308P mutation (R545P in terms of an older nomenclature) has been located previously in a family with type 2B VWD, the principal reported propositus had a platelet count of 100 ϫ 10 9 /L, 15 with plasma lacking high-molecular-weight multimers. In this same study, recombinant mutated VWF with P1308 and C1308 substitutions bound spontaneously to platelets and had greater reactivity with GPIb compared with substitutions at positions 1306 or 1341.…”

“…Whereas calreticulin levels remained stable, levels of ␤3 increased with megakaryocyte maturation. A specific increase in SERCA3a protein expression was accompanied by a net decrease in PMCA4b expression during proplatelet formation (days [11][12][13][14][15][16][17]. The kinetics of this are shown in Figure 6B and compared with ␤3 expression.…”

“…Among the most affected codons is arginine (R)1308, which is commonly substituted by a cysteine (C), and in isolated cases by histidine (H), proline (P), or leucine (L) residues. 2,[11][12][13][14][15][16] Although the basic phenotype of type 2B VWD can be accounted for by the ability of the mutated protein to spontaneously bind to platelets, some reports highlight platelet abnormalities that are difficult to explain by this interaction alone. For example, the degree of reported thrombocytopenia is highly variable, with sometimes giant and morphologically abnormal platelets being observed.…”

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

“…The other group includes six structures, of which, one, labeled as "WT-NC," was a WT A1 reconstructed through taking the body of A1 from the crystal structure of the complex WT-A1/GPIb␣ (Protein Data Bank code 1SQ0) (20) and adding the N-terminal arm with the same protocol used in R543Q and I546V, and others, including a GOF mutant E501A (32) and four type 2B mutants (R578Q, R578W, R578P, and R578L) (33)(34)(35), were modeled per homology by replacing the corresponding side chain in WT A1 and subsequently performing 1000 steps of minimization in vacuo while all atoms except the mutated residue were fixed. The WT-NC A1 was used for a negative test to verify whether the reconstructed R543Q and I546V mutants were rational or not, and each GOF mutation (E501A, R578Q, R578W, R578P, and R578L) was selected to further examine whether it would cause a change of the dynamic properties of A1.…”

“…To verify this hypothesis, we further examined the dynamic properties of other five mutants (E501A, R578Q, R578W, R578P, and R578L) via MD simulations (see "Experimental Procedures"). The mutant E501A has gain of function phenotype (32), and R578Q, R578W, R578P, and R578L are known as the type 2B mutations (33)(34)(35).…”