Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

Wakabayashi, Hironao; Monaghan, Morgan; Fay, Philip J.

doi:10.1074/jbc.m114.550566

Cited by 8 publications

(18 citation statements)

References 32 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nonetheless, the disulfide bond FVIIIa variant had altered FIXa affinity and FX Michaelis-Menten parameters, suggesting that the constraints of the disulfide bond do limit or alter some of the changes occurring. In contrast, an H281C/S524C variant linking A2 to A1 had only marginal effects on FIXa affinity and FX Michaelis-Menten parameters [22], perhaps indicating even less of a conformational change in this region upon activation.…”

Section: Discussionmentioning

confidence: 93%

“…An estimation of FVIIIa‐FIXa binding affinity ( K d ) and V max for FXa generation were calculated from initial rate data by fitting the data using nonlinear least squares regression analysis to a single‐site ligand binding model using the equation

\begin{matrix} F X a g e n e r a t i o n = \frac{V_{m a x}}{A_{T}} \\ ()(, \frac{A_{T} + B_{T} + K_{d} - \sqrt{{(A_{T} + B_{T} + K_{d})}^{2} - 4 A_{T} B_{T}}}{2}) \end{matrix}

where A T is the total concentration of FVIIIa and B T is the total concentration of FIXa .…”

Section: Methodsmentioning

confidence: 99%

“…FVIII preparations were resolved on an 8% polyacrylamide gel. Gels were stained with Gelcode Blue (Thermo Electron Corporation, Waltham, MA, USA) or transferred to a polyvinylidene fluoride membrane (PVDF) for probing with anti‐A2 (R8B12) and anti‐A3 (2D2) monoclonal antibodies as previously described . Band densities from dilutions of each purified protein were quantified and compared with standards on the same gel or Western blot to determine protein concentration.…”

Section: Methodsmentioning

confidence: 99%

“…The rate of conversion of FX to FXa was monitored in a purified system at 23 °C as previously described . Factor VIII (1 nM) was activated to FVIIIa with α‐thrombin (20 nM) for 1 min (or 15 s for rapidly decaying variants) in buffer containing 20 μM PSPCPE in FXa generation buffer (20 mM HEPES∙HCl [pH 7.2], 100 mM NaCl, 5 mM CaCl 2 , 0.01% Tween20 and 100 μg mL −1 BSA).…”

Section: Methodsmentioning

confidence: 99%

“…Previously, the FVIII structures and the homology model derived from ceruloplasmin have been guides for targeted mutagenesis to identify residues important for FVIIIa stability and A2 retention, as measured by biochemical analysis . A panel of polar residues at the A2 interface were mutated to examine the contribution that the residues made to FVIII/FVIIIa stability , providing evidence that several residues at both the A2‐A1 and A2‐A3 interfaces were important for FVIII and FVIIIa stability.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Stabilizing interactions between D666‐S1787 and T657‐Y1792 at the A2‐A3 interface support factor VIIIa stability in the blood clotting pathway

Monaghan

Wakabayashi

Griffiths

et al. 2016

Journal of Thrombosis and Haemostasis

View full text Add to dashboard Cite

EssentialsFactor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2-A3 interface. We present evidence for stabilizing interactions between D666-S1787 and T657-Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2-A3 linkage has a FVIIIa decay rate that is 1% of wild-type.Summary. Background: Factor (F)VIIIa activity and stability depends on the non-covalent association of the A2 subunit with the A1/A3C1C2 dimer, but the interactions that contribute to A2 association are not well defined. Previous work had shown that D666A and Y1792F mutations at the A2-A3 interface resulted in increased FVIIIa decay, suggesting that the residues were involved in bonding interactions important for FVIIIa stability. Objectives: Several potential hydrogen bonding partners of D666 and Y1792 across the A2-A3 interface were selected from the low-resolution FVIII crystal structure, and we used mutagenesis and biochemical analysis to examine the bonding interactions occurring at D666 and Y1792. Methods: Using a series of stability and functional analyses, we analyzed FVIII variants in which D666 and Y1792 were each swapped with the residues of potential bonding partners. Results and conclusions: We present evidence for hydrogen bonds between D666 and S1787 and between Y1792 and T657 that are important for FVIIIa stability. D666S/S1787D and T657Y/Y1792T variants each displayed wild-type (WT)-like FVIIIa stability and performed like WT FVIII in a series of functional analyses, whereas D666S, S1787D, and Y1792T single variants showed increased FVIIIa decay and a T657Y variant had little FVIIIa activity. These results suggest that WT hydrogen bonds are disrupted with the single mutations but maintained in the swap variants. Furthermore, mutation of D666 and S1788 to cysteine resulted in disulfide bond formation across the A2-A3 interface, confirming the close proximity of D666 and S1787, and this covalent attachment of the A2 subunit significantly increased FVIIIa stability.

show abstract

Section: Discussionmentioning

confidence: 93%

\begin{matrix} F X a g e n e r a t i o n = \frac{V_{m a x}}{A_{T}} \\ ()(, \frac{A_{T} + B_{T} + K_{d} - \sqrt{{(A_{T} + B_{T} + K_{d})}^{2} - 4 A_{T} B_{T}}}{2}) \end{matrix}

where A T is the total concentration of FVIIIa and B T is the total concentration of FIXa .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Stabilizing interactions between D666‐S1787 and T657‐Y1792 at the A2‐A3 interface support factor VIIIa stability in the blood clotting pathway

Monaghan

Wakabayashi

Griffiths

et al. 2016

Journal of Thrombosis and Haemostasis

View full text Add to dashboard Cite

show abstract

Effect of wavelength and filter set choices on fluorogenic thrombin generation assay: Considerations for interlaboratory differences

Jackson

Parunov

Monteil

et al. 2022

Research and Practice in Thrombosis and Haemostasis

View full text Add to dashboard Cite

Introduction: Insufficient agreement between results of various in-house and commercial thrombin generation (TG) assays complicates TG adoption and use. This can be caused by different assay variables, for example, volumes and concentrations of reagents. Although most modern assays employ the same fluorogenic thrombin substrate Z-Gly-Gly-Arg 7-amino-4-methylcoumarin (ZGGR-AMC), the excitation (Ex) and emission (Em) wavelengths used for fluorescence signal measurements can be different. Objectives:We sought to investigate the impact of wavelengths on the TG curves in commercial normal, hemophilia A, and antithrombin-deficient plasma samples.Methods: Fluorescence of ZGGR-AMC and AMC and TG curves were measured using different Em/Ex pairs to mimic settings used by common in-house and commercial TG methodologies. Results:The peak fluorescence of free AMC was measured at an Ex/Em of 360 nm/440 nm. The uncleaved substrate ZGGR-AMC produced a low fluorescence signal, which yielded a maximum value at an Ex/Em of 300 nm/390 nm. Interference of ZGGR-AMC fluorescence with that of AMC is abrogated at Em above 430 nm. Calibrated thrombin peak heights were higher at Ex/Em of 340 nm/440 nm and 360 nm/460 nm, but the overall difference in peaks between either wavelength pairs was less than 20%. Further, we found that the inner filter effect depended on the choice of Ex/Em wavelength pairs, demonstrating variable distortion in the shape of the TG curves, which was consistent with reduced linearity of calibrator curves. Conclusion:These data demonstrate that the choice and quality of fluorescence filters may have limited but measurable effects on the outcomes of TG analysis.

show abstract

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa

Majumder

2022

J Membrane Biol

View full text Add to dashboard Cite

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

Cited by 8 publications

References 32 publications

Stabilizing interactions between D666‐S1787 and T657‐Y1792 at the A2‐A3 interface support factor VIIIa stability in the blood clotting pathway

Stabilizing interactions between D666‐S1787 and T657‐Y1792 at the A2‐A3 interface support factor VIIIa stability in the blood clotting pathway

Effect of wavelength and filter set choices on fluorogenic thrombin generation assay: Considerations for interlaboratory differences

Phosphatidylserine Regulation of Coagulation Proteins Factor IXa and Factor VIIIa

Contact Info

Product

Resources

About