Determinación de los mecanismos moleculares de proteínas pro-coagulantes humanas mediante cristalografía de rayos X de macromoléculas biológicas, -Fase II-.

Cardiovascular diseases, including heart attacks and strokes, remain the main cause of morbidity
and mortality in developed and developing countries (Wilkins et al., 2017), which translates into a
strong economic impact on the health sector, and Costa Rica is not exempt from this situation. In
part, this is due to the fact that the structural and functional bases of the processes of blood clot or
thrombus formation are only incompletely and fragmentarily understood. Thrombin plays an
essential role in these processes, and the atomic-molecular basis of its interaction with other factors
involved in the coagulation process is poorly understood, in particular those with critically important
substrates such as cofactor VIII and the platelet protease-activated receptor (PAR1), which are
essential in the hemostatic processes involved in clot formation upon any signal of vascular
damage. On the other hand, X-ray crystallography allows the resolution of the 3D structure of protein
complexes, but a prerequisite is the crystallization of the sample to be analyzed. With the aim of
promoting the crystals formation of FVIII and PAR1, respectively, complexed with inhibited
thrombin, given the importance of these proteins that modify blood coagulation processes, and
continuing with the results obtained from previous projects within the roadmap of this line of
research, and with the collaboration of the research group of Dr. Pablo Fuentes-Prior (Institute for
Biomedical Research of the Hospital Sant Pau, Barcelona), the aim is to determine the atomicmolecular interactions between specific mutants of FVIII and PAR1 receptor complexed with
reversibly and irreversibly inhibited human thrombin. For this purpose (1) site-directed mutagenesis
of FVIII and PAR1 receptor will be generated, overexpressed and purified, (2) protein complexes of
site-directed mutants of FVIII and PAR1, respectively, with reversibly or irreversibly inhibited
thrombin will be prepared looking for the crystallization conditions of these complexes, (3) the
obtained crystals will be X-ray diffracted at the ALBA synchrotron in Barcelona (Spain) to obtain
crystallographic data, and (4) from the collected data the 3D structures of the prepared protein
complexes will be solved. This research is perfectly intertwined, and it is complementary to the
roadmap of this line of research, allowing to advance towards a better understanding of the
processes of interaction between thrombin and these protein factors; -and therefore- of the
mechanisms of thrombus formation.

Determinar las interacciones atómico-moleculares en los complejos FVIIITrombina-PPACK/Benzamidina y PAR1-Trombina-PPACK/Benzamidina mediante cristalografía
de macromoléculas.

Este proyecto de investigación se enmarca en el área de Aplicaciones Biomédicas, área definida como
prioritaria por la Escuela de Biología.