A University of Missouri-Rolla
researcher’s investigation into how proteins adhere to surfaces may lead the elimination of surface-induced thrombus formation, a phenomenon known to cause blood clots in artificial arteries, valves and veins.
For the past eight years, Dr. Daniel Forciniti, associate professor of chemical and biological engineering at UMR, and his students have been studying how and why proteins, particularly those present in human plasma, adsorb in solid surfaces.
“Many artificial materials that people use for constructing and building arteries may induce the formation of thrombus or blood clots,” Forciniti says. “It’s believed that the first steps in thrombus formation consist of the formation of these deposits of proteins on the surface.”
The UMR researchers began the project with a Whitaker Foundation grant and looked at a series of human plasma proteins. “Then we realized that the problem was really complex, and if we wanted to understand it better, we needed to somehow simplify it,” Forciniti says. “We have spent the last two years trying to synthesize peptides in the lab. They are smaller molecules (and therefore simpler) with the same chemical composition as the protein, but they can be tailored to enhance or diminish the surface interactions. Only now, after two years of work, we are able to have enough quantities of these peptides to begin looking at them at surfaces, which is the goal of the project.” The current research is supported by a three-year, $270,000 grant from the National Science Foundation.
At the beginning of their work, the researchers used a combination of three separate techniques to create a “picture” of fibrinogen adhered to solid surfaces in water. This particular protein, which is produced by the liver, is important because during the normal blood clotting process, fibrinogen is broken down by an enzyme called thrombin into short fragments of fibrin. Fibrin then clumps together with red blood cells and platelets to form blood clots.
By using atomic-force microscopy at Harvey-Mudd College in Claremont, Calif., Forciniti discovered that the molecule forms deposits on surfaces that have a very complex morphology and plenty of holes, which are always filled by a solvent – in this case water.
At Oak Ridge National Laboratories, Forciniti uses neutron reflectivity to look at the molecule’s reflection pattern of the protein adsorbed on glass and polystyrene. The technique shows that the protein’s deposits are soft and consist of several layers.
“To me, one of the coolest things is our investigation of protein adsorption using neutron reflectivity in combination with atomic-force microscopy,” Forciniti says. “People have argued for a long time that fibrinogen forms monolayer, meaning there’s just one molecule. We have overwhelming evidence using these two techniques that’s not true. Actually, fibrinogen forms multi-layers that are able to reach in water, to the extreme of having a water layer separating the first layer from the surface.”
The peptides that the researchers are currently using are made up of blocks of different amino acids. By carefully selecting the blocks, different portions of the peptide have different properties, Forciniti says. For example, some pieces have a net charge and are soluble in water while others are not.
“Since we know the chemical composition and where these pieces are, we can determine what kind of forces are actually playing a dominant role in the adsorption process,” Forciniti explains. “Depending on which one is dominant, the protein or peptide may lie flat, may stand up, or may incline.”
The research team has focused on water as the solvent because “all of the processes that happen in our body happen in an aqueous environment,” Forciniti says. Yet they also have started examining mixed solvents to address speculations of the relative weight on the adsorption process of the ability of proteins to form hydrogen bonds with themselves, the solvent, or the surface on the adsorption process. Using mixtures of either water and methanol and water and glycerol, the results have not been surprising. For example, the addition of methanol to water makes water a worse solvent for proteins, so they have a higher tendency to deposit on the surface, Forciniti adds.
All of the research team’s experimental work has been accompanied by theoretical work at an atomic level of detail. Forciniti believes this “marriage” between experimentation and theory can contribute to advances in this particular problem.
“A theoretical model that cannot be experimentally tested or experimental data that cannot be rationally explained is of little value,” Forciniti adds.