A soldier on the battlefield begins bleeding internally during combat. Under normal circumstances, he has an hour or two for a surgeon to repair his injuries before they become fatal. But instead, this soldier of tomorrow has a bioengineered nanodisc implanted in his body that triggers the blood clotting process immediately, saving his life.
For the past few years, four laboratories at the University of Illinois have been pursuing breakthroughs in the biochemistry of blood clotting that could make such technology possible. In addition to life-saving therapies, the research may pave the way for better diagnostics of blood clotting disorders, such as kits that measure coagulation factors, and more effective, fast-acting drug targets for patients with hemophilia and thrombosis.
At the center of these labs are four scientists at the top of their respective fields: One is a leading expert on the biochemistry of blood clotting. Another has developed a revolutionary synthetic test bed, enabling researchers to study nanoscale-sized molecules. A third is one of the world’s foremost authorities on solid-state nuclear magnetic resonance (NMR). And the fourth is a leader in an emerging field called computational biology, providing new theories about coagulation using supercomputing.
Already, they are shedding new light on a biochemical conundrum, that solved, would offer new hope to millions.
Coagulation: Unlocking the mysteries of clotting
Normally, the blood clotting cascade is a mind-blowing process that plays out on the nanoscale level.
When bleeding begins, the vascular system constricts, limiting blood flow to the injured area. Then, platelets congregate at the injury site and secrete proteins that create a loose plug. Next, a clot forms to stabilize the plug. After the tissue has been repaired, the clot dissolves and the tissue is good as new.
But the process can go wrong. Hemophiliacs, for example, lack one of the proteins essential for blood clotting. Conversely, when the process happens inside a blood vessel, unwanted clots can develop and induce heart attacks, strokes and other diseases caused by thrombosis.
“It’s a complicated balance of molecules that promote or inhibit clotting,” says Jim Morrissey, a professor of medical biochemistry and researcher in the College of Medicine. “When the balance is just right, everything stays fluid. But when it’s off, it can go wrong in a whole bunch of different directions.”
Morrissey has been studying the blood clotting cascade since the 1980s and is considered a foremost authority on coagulation.
But so much about coagulation remains a mystery, including one of the biggest unanswered questions: How do blood clotting proteins bind to membrane surfaces, thus initiating the cascade?
In 2006, this question drove him to reach out to his Illinois collaborators. By tackling the problem in a systematic way, he hoped to succeed where others had failed.
His efforts paid off.
Earlier this year, the group made several findings -- among the most significant is a better understanding of the ways that cell membranes control the coagulation process. Scientists have long known that the cell membranes play an essential role in the blood clotting cascade, but how they do this has remained something of a mystery. The Illinois collaboration has allowed the researchers to generate and test new ideas to explain how different kinds of membrane lipids on damaged cells can come together to trigger blood clotting at the site of a wound. Specifically, they have examined the role of a particular lipid, phosphatidylserine, and examined at unprecedented resolution how this molecule interacts with calcium ions and clotting proteins.
“We’ve known for decades that membrane surfaces play a critical role in blood clotting, but researching this process has been frustrating because there haven’t been good methods for examining these kinds of interactions at an atomic scale,” Morrissey said. “By better understanding how blood clotting proteins interact with membranes, we’re laying the groundwork for potential breakthrough applications.”
For patients with hemophilia and for those with thrombosis, the results could be a first step in creating pharmaceuticals that are more cost-effective and fast-acting than drugs currently on the market.
For patients with a high risk of unhealthy blood clots, the most commonly used medicine can make it difficult to produce clots when patients injure themselves, and therefore do need to form a normal clot.
And for patients with internal bleeding or severe hemophilia, a life-saving round of the most powerful drug on the market – an extremely high dose of Factor Vlla – could cost tens of thousands of dollars. It could also cause an imbalance of the proteins involved in coagulation and result in unwanted blood clots.
“Clearly, there is lots of room for improvement,” Morrissey says. “Our main goal is to understand how this process works, but the potential this research has for helping patients in the very near future is extremely exciting.”
Nanodiscs: A testbed for studying blood
From a research perspective, coagulation poses a myriad of problems.
Not the least of these is that the process is virtually impossible to study in a normal laboratory environment. Nearly every step of the blood clotting cascade happens on a cell membrane; providing a membrane surface with defined composition and size is difficult. In addition, if you release any membrane protein from its natural environment, it becomes far less active.
For nearly 20 years, Morrissey struggled against this biological barrier in his own research. Then about six years ago, he attended a seminar given by Stephen Sligar, director of UIUC’s School of Molecular and Cellular Biology and creator of nanodiscs, which are tiny artificial membranes. Membrane proteins, which “get scrambled up” outside their normal environment, according to Sligar, thrive in the genetically engineered nanodiscs.
“Here was a problem that had been challenging biologists for decades, and Steve’s lab has this tremendous breakthrough,” Morrissey says. “These nanodiscs have allowed us to get a much better understanding of the protein-membrane interactions.”
Nanodiscs consist of a discoidal bilayer ringed and stabilized by “Membrane Scaffold Protein” (MSP). They are prepared in self-assembly reactions by solubilizing phospholipids and MSP in a suitable detergent, and then slowly removing the detergent.
The beauty of nanodiscs is that researchers can use them to study nearly any membrane protein under precisely controlled experimental conditions, since the nanodiscs can include nearly any type of lipid. Morrissey and Sligar began working together to study Tissue Factor, one of the integral proteins involved in coagulation. Not only did it enable Tissue Factor to anchor to the membrane, but also allowed it to recruit Factor Vlla to the membrane surface. Using the nanodiscs to incorporate a precisely defined number of phospholipids with defined charge and composition of acyl chain and headgroup, the team was able to accurately define the assembly of the coagulation complexes, enabling an understanding of structure and activity.
“Because of the biophysics of coagulation, you need to provide a membrane surface where you precisely know the composition of the membrane,” said Sligar, a faculty member in the College of Medicine. “With nanodiscs, you can use very quantitative measurements to ensure proteins bind to surfaces.”
The researchers are currently using nanoscale bilayers to investigate the phospholipid dependence of other protease- cofactor pairs in blood clotting.
While Sligar’s technology could pave the way for breakthroughs in any number of areas – membrane proteins are the targets for about half of the pharmaceuticals on the market – he is particularly excited about his participation in the coagulation research.
“You have this sort of perfect collaboration of experts in their field,” he said. “It’s been almost magical watching it all come together.”
Solid-State NMR: A snapshot of membrane-protein interactions
While the nanodiscs gave the team a test bed for their research, the scientists still lacked the capability to measure the results.
“These interactions are taking place at a nanoscale level,” Morrissey said. “You can’t study this process with a microscope.”
As fortuitously as the meeting between Morrissey and Sligar, the researchers met Chad Rienstra, who several years earlier had helped advance a new state-of-the-art structural biology technique as a graduate student at MIT’s world-renowned Francis Bitter Magnet Laboratory.
Called solid state nuclear magnetic resonance (SSNMR), the technique enables scientists to study matter at an atomic level. It’s particularly useful for biological processes like coagulation because researchers can look at proteins in a lipid-bound state that is extraordinarily difficult to crystallize for X-ray diffraction studies, or to study by traditional NMR methods.
SSNMR is a technologically demanding process, requiring dozens of instrument components to work precisely. The payoff is that it provides researchers the ability to study cells in their natural environment at a much higher resolution.
“We can see how these proteins would behave in a cell in the human body,” said Rienstra, who joined the Illinois chemistry faculty in 2002. “It gives us very accurate data on how they interact in a real blood coagulation event.”
SSNMR works like this: Scientists take a sample – in this case the nanodiscs prepared by Sligar’s lab – and put it in a hard ceramic tube called a rotor. The rotor fits inside a probe that is installed in a large superconducting magnet. The rotor spins at approximately one million revolutions per minute, and the instrument generates pulsed magnetic fields to interrogate the interactions between nuclei. Because the components of the sample have been isotopically labeled, the researchers are able to capture accurate measurements of the interactions.
“We now have a detailed map of many of the events involving blood clotting,” Rienstra said, “It’s at a level of detail that is sufficient for starting to design drugs that could disrupt the process.”
Morrissey and Rienstra recently received a grant from the National Institutes of Health for their portion of the research, and the group is working to secure funding for all of the collaborators.
The collaborative nature of the project has enabled breakthroughs that the researchers would have never achieved on their own, according to Rienstra.
“We’ll get a result that we may not understand, but we’ll go back and talk to our collaborators,” he said. “Someone else inevitably has an idea and comes up with a way to test whether it’s correct. It’s a very exciting, dynamic process and it’s how we’ve been able to move forward with our research so quickly.”
Computational Biology: Coagulation in action
NMR has been a powerful tool in studying the blood clotting cascade, but it has a significant limitation: it can only look at the interaction between proteins and the phospholipid bilayer in microseconds.
To truly understand blotting clotting, researchers need the ability to study the process for an even shorter period of time and to provide a more detailed description of the complex interactions between lipids and proteins. That is where Emad Tajkhorshid comes in.
A professor of biochemistry, biophysics and pharmacology, Tajkhorshid specializes in computational biology, a field that uses computing power and molecular dynamics methodologies to conduct atomic simulations over shorter time scales -- nanoseconds.
“It brings molecules to life,” said Tajkhorshid, a researcher in the College of Medicine. “We can watch them move, watch how their interactions form and break, and better understand what kind of interactions are important. We connect this information to make predictions about interactions in the natural world.”
The technology is based on a very old physics problem, which uses Newton’s Second Law to describe molecular motion using classical mechanics. Researchers have been using the method for several decades, but Tajkhorshid’s group, which includes researchers at other universities, uses supercomputers to run algorithms in parallel. By using thousands of processors to distribute the computational tasks – including calculating the interaction between pairs of atoms -- the supercomputers can open up new revelations at an atomic level.
Tajkhorshid’s research focuses on the modeling and simulation of anything that interacts with biological membranes; in particular, his work targets an important family of proteins that transport materials across the membrane and any matter that binds to the membrane, including coagulation materials.
While the methodology can’t be used to fully describe biological processes, it is very effective in making atomic-detailed predictions about the process that point scientists in the right direction.
For the Illinois team, Tajkhorshid’s research has already revealed new insights into coagulation. One simulation showed researchers that proteins penetrated the membrane much more deeply than previously thought. It also revealed a new understanding of the role of the phosphate groups within the membrane in the protein’s binding to the cell surface. In addition, it demonstrated that calcium ions play a dual role in coagulation; some are important in gluing the protein to the membrane, while others are important for keeping the structure of the protein intact.
“I’m really excited about how the physics-based methods that have been around for a while combine with computer science to solve real problems in biology,” Tajkhorshid said.
He’s even more thrilled about the possibilities that the collaboration presents: “Nobody has ever approached this problem as we are. I really see great potential for our consortium to change the way we treat blood clotting disorders.”
*Note: An update on Morrissey and team's research can be found in an article under College of Medicine News.