Blood Flow Beneath a Microscope

By Jason Socrates Bardi

"[Mars] showed Jove the immortal blood that was flowing from his wound, and spoke piteously, saying, 'Father Jove, are you not angered by such doings?'"

——Homer, The Iliad, Book V (Samuel Butler translation), 800 B.C.E

"I'm going to show you the best way to visualize platelets," says Professor Zaverio Ruggeri, giving an impromptu lesson in blood clotting last month in a darkened laboratory in the Molecular and Experimental Medicine (MEM) building at The Scripps Research Institute (TSRI).

Ruggeri holds the Roon Chair in Cardiovascular Research and heads MEM's Division of Experimental Hemostasis and Thrombosis. He also directs TSRI's Roon Center for Research in Arteriosclerosis and Thrombosis, endowed in 1981 with private funds from the Roon family—Leo and Anna Miesem Roon, their son Donald Roon and daughter-in-law Lois Roon. The Roon Center supports researchers in thrombosis and arteriosclerosis, and sponsors an annual lecture by a prominent scientist in the field.

Ruggeri continues his demonstration, showing how an all-day video can record the results. For the last several years, he has done similar experiments in this room, the Roon Research Laboratory in Artheriosclerosis and Thrombosis, three or four times a week.

"I have thousands of hours of video," he smiles.

The videos show the movement of blood cells over a flat surface under the microscope. In particular, they show the movement of platelets—those flat, molecule-filled cytoplasmic disks in the blood that are necessary for clotting.

On the surface of the microscope stage is a chamber over flows which blood exposed to proteins, cells, and other materials. The surface is engineered to mimic the surface of a blood vessel—with a layer of endothelial cells on top of collagen and other "matrix" components.

Ruggeri uses these systems to look at the interaction of platelets with various surfaces encountered in the circulation, and to study the interaction of individual platelets with the endothelial cells and wounds.

Above the noise of the cooling fans, Ruggeri points out how the computer scans slices of the sample flowing over the surface in successive one-micron slices, from the matrix material on the bottom, through the layer of endothelial cells, and finally above the endothelial cells where the platelets are flowing and accumulating.

To mimic a flesh wound, an artificial wound has been made on this surface by scraping away some of the endothelial cells, exposing the matrix components underneath. In real life, the exposed endothelial matrix affects the flow of the platelets, ultimately leading to clotting of blood. Here, Ruggeri is able to monitor and record these microscopic events.

"We'll see how the platelets interact with this 'lesion'," says Ruggeri as he starts the flow, "where they are accumulating in relation to the cut."

There, on the screen are cascading blips not much larger than a single pixel. Those blips, Ruggeri says, are single platelets. As they rush over the scarred surface of the artificial wound and encounter the subendothelial layer, they clump into bright spots several micrometers across (composed of thousands of platelets). The platelets are clotting.

"These are very dynamic events, and we are interested in [capturing] the real-time dynamic aspects of them," says Ruggeri.

The Two Faces of Clotting

Scientists are interested in the molecular mechanisms that govern clotting because many individuals suffer from diseases related to these mechanisms, such as bleeding disorders.

The most famous such bleeding disorder is hemophilia. Ruggeri, however, has spent most of his career studying a disorder that is more common but less-well-known: von Willebrand disease—a genetic defect caused by mutations in a large, sticky protein called von Willebrand factor, which interacts with platelets to initiate clotting. Von Willebrand disease is less severe than hemophilia. It is, however, the most common hereditary bleeding disorder, affecting at least one percent of the population.

Another reason scientists are interested in clotting is its potential for therapeutic intervention in vasculature disease. Clotting is an essential physiological process, but at the same time, the blood components that heroically stop bleeding, also nefariously cause diseases such as heart attacks and stroke, which are the most common causes of death in the United States today.

Platelet adhesion and clotting are central to acute and chronic arterial diseases, since platelets have a predominant role in initiating acute adhesion in coronary arteries. For many years, people like Ruggeri have been trying to design a way to prevent the von Willebrand factor protein from initiating platelet adhesion and clotting in the hopes of ameliorating arterial disease.

"As yet, there is no small molecule inhibitor," says Ruggeri. "But my prediction is that in the future, there will be a specific anti-platelet drug targeting this interaction."

Ruggeri is contributing to this effort by elucidating the molecular mechanisms of these physiological processes. Recently, in collaboration with TSRI Staff Scientist Reha Celikel and TSRI Associate Professor Kottayil Varughese, he published a paper in the journal Science describing the structure of a platelet receptor called glycoprotein Ib bound to the coagulation protein thrombin, which induces blood clotting by producing the sticky protein fibrin.

"We are trying to understand, at the structural level, how glycoprotein Ib, thrombin, and von Willebrand factor could assemble into complexes that influence one another," says Ruggeri. "There are some interesting points of convergence."


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TSRI Professor Zaverio Ruggeri heads the Department of Molecular and Experimental Medicine's Division of Experimental Hemostasis and Thrombosis, and directs the Roon Research Center. Photo by BioMedical Graphics.