Sep 1 2015
Last summer, SLU scientists made a breakthrough discovery about the way in which blood clots. Through X-ray crystallography, they solved the molecular structure of prothrombin, an important blood-clotting protein, revealing an unexpected, flexible role for a "linker" region that may be the key to developing better life-saving drugs.
This is Nicola Pozzi, Ph.D., researcher at Saint Louis University. Credit: Saint Louis University
Now, they're racing on to solve the next puzzle.
With a $214,500 grant from the American Heart Association, SLU researcher Nicola Pozzi, Ph.D., hopes to build upon these findings.
"What I absolutely love about my job is the domino effect that a new discovery brings," Pozzi said. "When we solved the three-dimensional crystal structure of prothrombin we soon realized that our discovery would change the way we see prothrombin.
"Since our results were totally unexpected, they were very challenging to interpret but at the same time very fascinating. It turned out that solving the structure was the beginning of a new unexplored chapter for prothrombin, which is full of unanswered questions.
"One at a time, we hope we will address all of them. And, thanks to this award from the American Heart Association, we will be able to continue our work, test our hypothesis and eventually figure out how the transition from prothrombin to thrombin occurs at the molecular level."
The toll of cardiovascular disease
Experts predict that cardiovascular disease and its blood clot-related complications, like strokes and heart attacks, are likely to remain the leading cause of death and disability, and also represent a major burden to productivity in the U.S. and worldwide, well into the year 2020.
For example, ischemic stroke, which occurs when a blood clot (thrombus) blocks or plugs an artery leading to the brain, killed 6.7 million people in 2012. The consequences of strokes are catastrophic and impact the quality of life of millions of patients and their families. Direct and indirect costs associated with strokes in the U.S are estimated to be $43 billion a year.
Two sides of same coin
To address these challenges, SLU biochemists have focused their study on prothrombin, a protein that circulates in blood plasma. The protein is essential for life; no living patient has ever been reported with undetectable levels of prothrombin in the blood.
When the body is healthy, prothrombin circulates as an inactive molecule. Upon vascular injury, like a cut, after a surgical incision or a rupture of an atherosclerotic plaque (a common complication of cardiovascular disease), prothrombin is converted to thrombin. Thrombin, in turn, generates solid blood clots, physically blocking the loss of blood.
"It saves your life in that way, and it does a lot of other things, too," Pozzi said. "Prothrombin is the most important factor when it comes to clotting. We also believe its role likely goes beyond coagulation and may also play important roles in development and immunity."
And, as often happens in biology, thrombin plays a dual role even in the formation of blood clots. It can function both as a procoagulant and as an anticoagulant.
"Studying thrombin offers a look at two sides of the same coin. We study how blood clots, and how the clots are removed from circulation," Pozzi said. "The body's needs vary by situation. If you have a cut on your hand, the body activates the blood clotting process, turning prothrombin into the active thrombin, in order to stop the body from losing blood. On the other hand, the body would like to avoid forming blood clots in the veins and arteries that will interfere with circulation. The body also naturally has processes to avoid clots from forming and to remove them.
"And so, thrombin can be good and bad at the same time," Pozzi said. "Eliminating thrombin from circulation might be effective at stopping blood clots, but, since it also activates the anticoagulant pathway, it is not ideal to completely block its function."
A need for better drugs
Last century, two major classes of anticoagulants -- heparin and warfarin -- began to be used to treat thrombotic disorders. Not much changed for a long time and, today, these drugs are still commonly used.
Recently, some additional medications have been added to the market. These new drugs are very powerful, but, by blocking the bad and good functions of thrombin they can cause a number of serious side effects. For example, they can cause too much bleeding and currently there are no antidotes. Also there is uncertainty about dosing in some patients, like those with renal dysfunction.
"We believe that there are better strategies to keep blood-clotting in check," Pozzi said. "In particular, if we understand how prothrombin is converted to thrombin, we could identify the critical steps that control the rate of the reactions and could try to interfere with those steps. This way we would be able to potentially slow down, on demand, the generation of thrombin without completely killing the enzyme. Prevention could be key. We are looking for a more balanced approach.
"This is our goal for this project. We will apply state-of-the-art biophysical and biochemical techniques, such as X-ray crystallography and single molecule spectroscopy, to solve this long overdue mystery."
Next up: Prothrombin, the movie
To really understand how and when prothrombin is activated, the investigators need to observe how its structure changes.
"When we were able to trap prothrombin and solve its structure, it was like getting one good snap shot that gave us a lot of information. Now what we really need are lots of stills that we can piece together, like a movie. This will show us the exact way in which prothrombin changes into thrombin.
"The key, we believe, will be in the flexible linker. Using both X-ray crystallography and single molecule spectroscopy, we will begin to see how the molecule moves."
The project's success, Pozzi suggests, depends on its scientists' flexibility, as well.
"You work on something so hard. You have built in your brain a solution and then you see something different and you are so fascinated. You have to regroup, readjust your thoughts, be humble and think 'what I thought wasn't correct.'
"There are a lot of unanswered questions. It's very exciting.
"I believe that we are close to reaching another milestone in the field: the true dynamic look of prothrombin will soon be revealed," Pozzi said. "Stay tuned. We have such a great environment here at SLU that I am positive we will succeed."