Platelet translocation under shear stress and stability assessment. (A) Representative images of platelets translocating on immobilized wild-type A1A2A3 (left) and C1669S/C1670S A1A2A3 (right) at shear rates of 800 and 1500 s−1. (B) Platelet translocation pause times and (C) translocation velocities and total number of analyzed platelets as a function of shear rate for the wild type (blue symbols) and C1669S/C1670S A1A2A3 (red symbols). Asterisks indicate significance (P ≤ .05). Only single platelets were tracked in this analysis. (D) Survival fraction decay functions of platelet pause times at 1500 s−1 and 9000 s−1. (E) Dissociation rate constants as a function of shear rate obtained from biexponential fitting of the pause time survival fraction decay curves. (F) SPR sensorgrams for both tridomains at concentrations of 4.0, 3.0, 2.0, and 0.6 μM. Comparison of the equilibrium binding curves and GPIbα-binding affinities (inset) for wild-type A1A2A3 and C1669S/C1670S A1A2A3. The resulting apparent KD values are: 36.1 ± 0.8 μM for wild-type A1A2A3, 32 ± 0.7 μM for C1669S/C1670S A1A2A3; P value = .004. Values for A1 either expressed as a single domain in E coli (3.9 ± 0.1 μM) or in HEK293 cells (32.4 ± 0.1 μM) were taken from the study by Tischer et al.13 The global maximum binding signal, RMax, is 670 ± 9 RU. Note that a higher KD-value indicates a lower affinity. (G) Interaction of GPIbα, A2WT (HEK293 or E coli), A2 C1669S/C1670S (HEK293), and of A2 ΔCCS (E coli) with surface-immobilized wild-type A1 domain. Each protein (1 μM) was perfused over the surface immobilized A1 domain. Although GPIbα binds to the A1 domain, A2 does not interact with A1 regardless of the expression host used, plasmid construct, or the absence/presence of the vicinal disulfide. (H) Thermal denaturation of wild-type and C1669S/C1670S tridomains monitored via DSC (scan rate = 2.0°C/min). Heat capacity thermograms were background corrected relative to the buffer and normalized to a polynomial baseline to define the excess heat capacity. Excess heat capacity was fit using nonlinear least squares analysis and deconvoluted into individual A1, A2, and A3 domain contributions using Wolfram Mathematica per a thermodynamic model in which each domain thermally denatures in a 2-state manner. Both panels contain deconvoluted thermograms of the single domains within the tridomain. Fit residuals are shown at the bottom. Vertical lines and arrows indicate the destabilization of A1 and A2 and a small stabilization of A3 in C1669S/C1670S. (I). Thermal transition temperatures of the deconvoluted domains. A1: 56.2 ± 0.4°C in A1A2A3 vs 51.9 ± 0.4°C in C1669S/C1670S A1A2A3; P value ≤ .001. A2: 53.0 ± 0.3°C in A1A2A3 vs 48.0 ± 0.3°C in C1669S/C1670S A1A2A3; P value ≤ .001. A3: 61.6 ± 0.3°C in A1A2A3 vs 62.5 ± 0.3°C in C1669S/C1670S A1A2A3; P value = .014. Teq. for the single A domains expressed in E coli.12,14,15 A1: 51.8 ± 1.8°C single domain vs 56.2 ± 0.4°C in A1A2A3; P value = .014. A2: 51.8 ± 0.4°C single domain vs 53.0 ± 0.3°C A1A2A3; P value = .011. A3: 67.2 ± 1.2°C single domain. (See the platelet translocation videos and supplemental Figures 4-7 for an overview of the flow assay and images of translocating platelets at other shear rates; supplemental Figures 8-10 for a comparison of pause time histograms, velocity histograms, and survival fractions at all shear rates; supplemental Table 1 for platelet translocation parameters and statistical analysis using a 2-tailed t test; supplemental Figure 11 for SPR response curves of wild-type and C1669S/C1670S A1A2A3 fit to a simple binding model; supplemental Figures 12 and 13 for control experiments for the interactions in trans between A2 and A1 and between A2 and GPIbα; supplemental Figure 14 for thermal scan rate dependencies of the excess heat capacity of wild type, C1669S/C1670S, C1669A/C1670A, and C1669G/C1670G that demonstrate that destabilization is independent of the amino acid side chain; supplemental Figure 15 for reproducibility/repeatability of the DSC data; and supplemental Table 2 for DSC parameters in the supplemental materials.)
