The mechanism of VWF-mediated platelet GPIbalpha binding

Abstract

The binding of Von Willebrand Factor to platelets is dependent on the conformation of the A1 domain which binds to platelet GPIbalpha. This interaction initiates the adherence of platelets to the subendothelial vasculature under the high shear that occurs in pathological thrombosis. We have developed a thermodynamic strategy that defines the A1:GPIbalpha interaction in terms of the free energies (DeltaG values) of A1 unfolding from the native to intermediate state and the binding of these conformational states to GPIbalpha. We have isolated the intermediate conformation of A1 under nondenaturing conditions by reduction and carboxyamidation of the disulfide bond. The circular dichroism spectrum of reduction and carboxyamidation A1 indicates that the intermediate has approximately 10% less alpha-helical structure that the native conformation. The loss of alpha-helical secondary structure increases the GPIbalpha binding affinity of the A1 domain approximately 20-fold relative to the native conformation. Knowledge of these DeltaG values illustrates that the A1:GPIbalpha complex exists in equilibrium between these two thermodynamically distinct conformations. Using this thermodynamic foundation, we have developed a quantitative allosteric model of the force-dependent catch-to-slip bonding that occurs between Von Willebrand Factor and platelets under elevated shear stress. Forced dissociation of GPIbalpha from A1 shifts the equilibrium from the low affinity native conformation to the high affinity intermediate conformation. Our results demonstrate that A1 binding to GPIbalpha is thermodynamically coupled to A1 unfolding and catch-to-slip bonding is a manifestation of this coupling. Our analysis unites thermodynamics of protein unfolding and conformation-specific binding with the force dependence of biological catch bonds and it encompasses the effects of two subtypes of mutations that cause Von Willebrand Disease.

Figure 1

(A) Size exclusion chromatography of 10 μM WT and RCAM A1. Elution volume of bovine serum albumin (molecular mass = 68 kDa) indicated by arrowhead. (Inset) Nonreducing (− β-ME) and reducing (+ β-ME) SDS-PAGE of WT and RCAM A1. (B) Far-UV CD spectra of 10 μM WT and RCAM A1 in the presence of 0.5 M urea. Spectra represent the average of 10 individual scans with a pathlength of 1 mm at 25°C. (C) Urea unfolding of WT and RCAM A1 monitored at 222 nm.

Figure 2

The binding of WT (circles), R1306 (squares), I1309V (diamonds), G1324S (triangles), and RCAM A1 domain (solid circles) to fixed platelets at 37°C. Data are representative of three experiments each done in triplicate. (Error bars) Standard deviation of the mean. (Lines) Best fit. (Shaded areas) Representative of the fitting error. The binding affinities reported in Table 2 were obtained by fitting the data to Eq. 2 weighted according to the error on each data point.

Figure 3

Thermodynamic cycle defining the equilibrium binding of A1 to GPIbα and the catch-to-slip bond model of forced dissociation from two bound conformational states.

Figure 4

(A) Comparison of our previously reported catch-to-slip bond lifetime data as a function of force with the model fit to Eq. 8. (Lines) Best fit. (Shaded areas) Representative of the fitting error. (B) Deconvolution of the overall bond lifetime for each variant of the A1 domain into the native (τ_N) and intermediate (τ_I) state specific bond lifetimes as a function of force. (Open symbols) Native state. (Solid symbols) Intermediate state. (Shaded curve) Bond lifetime and the binding probability of the intermediate state. R1306Q (squares), I1309V (diamonds), WT (circles), and G1324S (triangles).

Figure 5

(A) Force dependence of the dissociation rates from the native and intermediate states. (B) Force dependence of the probability of binding (B) to each state. (C and D) Native and intermediate state flux as a function of force. (C, inset) Magnification of the J_N curves at <1 s⁻¹. (A, B, and D, shaded curve) Force dependence of the rate of dissociation (A), the binding probability (B), and the flux through the intermediate state (D). (Open symbols) Native state; (solid symbols) intermediate state for the A1 domain variants, R1306Q (squares), I1309V (diamonds), WT (circles), and G1324S (triangles).

Figure 6

(A) Equilibrium (zero force) dissociation rates of GPIbα from the A1 domain native state (open symbols) and intermediate state (solid symbols) for each of the variants shown in Fig. 4 as a function of the thermodynamic stability of the A1:GPIbα complex (ΔG⁰_NG⇌IG). The k⁰_N transitions from k⁰_I at low stability to a maximal rate k⁰_N, ∞ at high stability, as described by Eq. 9. The value k⁰_I = 1.83 ± 0.03 s⁻¹, k⁰_N,∞ = 8.7 ± 0.1 s⁻¹, ∂ln(k)/∂ΔG⁰ = 2.7 ± 0.3 mol s⁻¹ kcal⁻¹, and ΔG⁰_1/2 = 0.51 ± 0.05 kcal mol⁻¹. (B) The resulting equilibrium native, intermediate, and total bond lifetimes. (C) The native and intermediate binding probability. R1306Q (squares), I1309V (diamonds), WT (circles), and G1324S (triangles).