Genetic disorders of blood coagulation, including hemophilia and von Willebrand's disease, affect approximately 1% of the U.S. The Tissue Factor (TF) pathway (or the extrinsic pathway) is believed to play a primary role in initiating blood coagulation during normal hemostasis as well as during many pathologic situations, including arterosclerosis and septicemia. However, there is indirect evidence that this scenario is not accurate. Human TF consists of 263 residues, the first 219 of which comprise the extracellular region. Extracellular TF (219) consists of two immunoglobulin-like domains connected by a single polypeptide linker (PRO 102 to ASN 107).

One way to help understand the motion in proteins is to use computer simulations, also called “virtual experiments”. Among the several techniques available within the computer simulations, there are the so-called Molecular Dynamics. Molecular Dynamics (MD) are techniques that permit the study of the motions of flexible molecules as a function of time. In this work, we have used MD simulations to predict the solution structure of free TF (fTF) and TF bound to factor VIIa (bTF). A complete refined solution equilibrated model for TF based on available crystal structures (1BOY, 2HFT, 1DAN) using MD simulations in aqueous medium for over 15 ns is presented.

The X-ray crystal structures (1BOY, 2HFT, 1DAN) were used initially as starting points in modeling the unbound and bound structures. The complete models were subjected to aqueous-phase MD simulations, where unconstrained dynamics were performed using AMBER9/PMEMD9 programs. The solute, ions/counter-ions and crystal water molecules, were immersed in rectilinear periodic boxes of at least 20Å each side. Standards steps of energy minimization and dynamics were carried out before beginning the “production run”. Finally, the post-equilibration NPT dynamics at 300 K, or production run, were completed for at least 15 ns depending of the study case.

The model sizes cover from 70,000 atoms up to 200,000 atoms, and a simulation length, for each system, up to 20 ns. All simulations were performed with (unless otherwise indicated): the ff99 and ff02, at 1 atm, 300 K, and 1fs time-step on the NPT ensemble. All proteins are solvated with TIP3P water molecules in rectangular periodic boxes, with 12, 15 or 20 Å between the protein and the box boundaries. Long range electrostatic interactions are calculated by the PME method. After a detailed study of the effect of the box's size in our simulations, we have found that these relatively large boxes are required to provide meaningful physical insight. We are using rectilinear boxes (due to the symmetry of the protein) of, e.g. 160x96x96 Å3 for a 134,422 atoms (TF-FVIIa complex). The size of the boxes is similar to the recently reported21 in the literature to model the lac repressor-DNA complex III DNA receptor with a total of 226,314 atoms.

Finally, these equilibrated solution structures for tissue factor should provide insight into the details of how tissue factor enhances the action of FVIIa. The possibility to predict these dynamic conformations could represent a crucial impact for the areas of

protein and drug design.