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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