Molecular Mapping of the Chloride Binding Site in von Willebrand Factor (VWF): Energetics and Conformational Effects on the ADAMTS-13 Interaction with Wild Type and Type 2B R1306W VWF Forms.

Physiological concentrations of NaCl inhibit the hydrolysis of von Willebrand Factor (VWF) by ADAMTS-13. This effect is linked to the specific binding of chloride ions to VWF. Urea-induced unfolding was measured in the presence of NaCl, CH3COONa and NaClO4 at pH 8.0, 25°C, for multimeric VWF, the recombinant A1-A2-A3 VWF domains and the A1 domain. Chloride stabilizes the folded conformation of the A1-A2-A3 and A1 domains more efficiently than acetate but less strongly than perchlorate. Spectrofluorometric studies showed that chloride binds to both the A1 and A1-A2 domain, but not to the isolated A2 domain. Binding of Cl⁻ to both wild type (WT) and the natural mutant (2B VWD) p.R1306W A1-A2-A3 domains of VWF has a large heat capacity change equal to −1.0 and −0.4 Kcal mol-1 K-1 for WT, and p.R1306W A1-A2-A3 domains, respectively. This result implies that a burial of a vast apolar surface area is caused by conformational transitions linked to chloride binding. At any temperature, chloride affinity was higher for WT than for the mutant p.R1306W form. Chloride ions inhibit hydrolysis by ADAMTS-13 of the A1-A2-A3 and A1-A2 domains in the presence of either urea or high shear stress (40 dyn/cm2), while this effect was either absent or negligible in experiments using A2 and A2-A3 domains. Steady-state kinetic experiments showed that chloride ions inhibit allosterically the cleavage by ADAMTS-13 of the WT A1-A2-A3, whereas this effect was significantly reduced in p.R1306W form, as a consequence of the reduced chloride affinity (Figure 1). On the whole, these findings showed the existence of a conformational linkage between the A1 and A2 domains in the VWF molecule. The p.R1306W natural mutant, that has an higher affinity for GpIb, bears a mutation which stabilizes a conformation of the A1 domain in a GpIb-bound like state and reduces at the same time the A1 domain affinity for chloride. These findings suggest that in some type 2B VWD forms chloride ions bind with lower affinity and the rate of hydrolysis by ADAMTS-13 increases. This can contribute, along with the enhanced binding of high molecular weight VWF multimers to platelet GpIb molecules, to the depletion of these VWF forms and to the hemorrhagic diathesis usually observed in these patients.

Figure 1

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Linkage graph showing the k_cat/K_m values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k⁺ = 7.26 ± 0.2 × 10⁴ M⁻¹ sec⁻¹ k^CI = 1.23 ± 0.3 × 10⁴M⁻¹ sec ⁻¹, K_d = 35 ± 5 mM (WT A1-A2-A3); k⁺ = 1.36 ± 0.1 × 10⁵ M⁻¹ sec ⁻¹ k^CI = 3 ± 0.5 × 10⁴ M⁻¹ sec⁻¹ , k_d = 158 ± 20 mM (p R1306W), k⁺ 7.47 ± 0.3 × 10⁴ M⁻¹ sec⁻¹ k^CI = 1.38 ± 0.3 × 10⁴ M⁻¹ sec⁻¹, K_d = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains

Figure 1

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Linkage graph showing the k_cat/K_m values of hydrolysis of WT A1-A2-A3 (•), p. R1306 W(○) WT A1-A2 (□) isolated A2 domain (•), and A2-A3 domains (▿). The continuous lines were drawn to eq 4 with the best - fit parameter values k⁺ = 7.26 ± 0.2 × 10⁴ M⁻¹ sec⁻¹ k^CI = 1.23 ± 0.3 × 10⁴M⁻¹ sec ⁻¹, K_d = 35 ± 5 mM (WT A1-A2-A3); k⁺ = 1.36 ± 0.1 × 10⁵ M⁻¹ sec ⁻¹ k^CI = 3 ± 0.5 × 10⁴ M⁻¹ sec⁻¹ , k_d = 158 ± 20 mM (p R1306W), k⁺ 7.47 ± 0.3 × 10⁴ M⁻¹ sec⁻¹ k^CI = 1.38 ± 0.3 × 10⁴ M⁻¹ sec⁻¹, K_d = 36.2 ± 8 mM(A1-A2). No significative NaCl effect was observed for both isolated A2 and A2 -A3 domains

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