Blood coagulation factor IX: structural insights impacting hemophilia B therapy.

Coagulation factor IX plays a central role in hemostasis through interaction with factor VIIIa to form the factor X-activating complex at the site of injury. The absence of factor IX activity results in the bleeding disorder hemophilia B. This absence of activity can arise either from a lack of circulating factor IX protein or from mutations that decrease the activity of factor IX. This review focuses on analyzing the structure of factor IX with respect to molecular mechanisms that are at the basis of factor IX function. Proteolytic activation of factor IX to activated factor IX(a) and subsequent structural rearrangements are insufficient to generate fully active factor IXa. Multiple specific interactions between factor IXa, the cofactor VIIIa, and physiological substrate factor X further alter the factor IXa structure to realize the full enzymatic activity of factor IXa. Factor IXa also interacts with inhibitors, extravascular proteins, and cellular receptors that clear factor IX(a) from circulation. Hemophilia B is treated by replacement of the missing factor IX by plasma-derived protein, a recombinant bioequivalent, or via gene therapy. An understanding of how the function of factor IX is tied to structure is leading to modified forms of factor IX that have increased residence time in circulation, higher functional activity, protection from inhibition, and even activity in the absence of factor VIIIa. These modified forms of factor IX have the potential to significantly improve therapy for patients with hemophilia B.

Multidimensional infrared diffusion-ordered spectroscopy in depletion mode distinguishes protein amyloids and monomers.

Conventional and two-dimensional infrared (2D-IR) spectroscopy are well suited to study amyloid aggregates, because the amide I mode is a sensitive probe of the aggregate structure. However, these methods are not so useful to study mixtures of aggregates and monomers, which generally have overlapping amide I spectra. Here, we show that IR-Diffusion-Ordered Spectroscopy can disentangle the contributions of protein monomers and aggregates (amyloids) in FTIR and 2D-IR spectra by separating the spectral contributions based on molecular size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient inside an IR sample cell and tracking its equilibration in an IR-frequency-resolved manner. The amyloid diffusion is too slow to be experimentally observable, so instead of tracking the arrival of molecular species diffusing into the initially empty region of the sample cell, we track the depletion of the more rapidly diffusing species as they leave the sample-filled region. This way, we can still obtain the spectrum of very slowly diffusing species, although we cannot determine their diffusion coefficient. We first demonstrate this depletion method on a mixture of two small organic molecules and then show how it can be used to separate the spectrum of a mixture of bovine-serum-albumin amyloids and monomers into its component spectra, both in the FTIR and 2D-IR case.

Infrared Diffusion-Ordered Spectroscopy Reveals Molecular Size and Structure.

Inspired by ideas from NMR, we have developed Infrared Diffusion-Ordered Spectroscopy (IR-DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR-frequency resolved manner. Analogous to NMR-DOSY, a two-dimensional IR-DOSY spectrum has IR frequency along one axis and diffusion coefficient (or equivalently, size) along the other, so the chemical structure and the size of a compound are characterized simultaneously. In an IR-DOSY spectrum of a mixture, molecules with different sizes are nicely separated into distinct sets of IR peaks. Extending this idea to higher dimensions, we also perform 3D-IR-DOSY, in which we combine the conformation sensitivity of femtosecond multi-dimensional IR spectroscopy with size sensitivity.