Penn Researchers Use Laser Tweezers to Study Strength
of Ligand-Receptor Binding
(Philadelphia,
PA) - Using "laser tweezers," researchers
at the University of Pennsylvania School of Medicine
have measured the strength of the bond between a single
integrin molecule on the surface of a platelet and a
molecule of fibrinogen, a clotting protein found in
the bloodstream. An article detailing their findings
was published in the May 14th online early edition of
the Proceedings of the National Academy of Sciences
and will be featured in the May 28th print edition.
These findings refine the current paradigm of how blood
clots form. They show that changes in an integrin's
ability to bind to fibrinogen are regulated by the cell
as an all-or-none phenomenon with only one functional
state compatible with binding. The researchers also
offer a new application for laser tweezers in studying
the behavior of single molecules and the response of
cells to mechanical forces.
"Laser tweezers use the force of a focused laser
beam to trap and move particles. In this case, we used
the tweezers to play tug-of-war with a platelet-bound
integrin molecule on one side and a fibrinogen molecule
mounted on a tiny latex bead on the other," said
John W. Weisel, PhD, professor the Penn's Department
of Cell and Developmental Biology. "We were able
to measure the force that keeps blood clots together.
We can also determine the regulation of forces between
individual ligand-receptor pairs and the effects of
anti-clotting drugs, at the single molecule level."
Clotting is the body's first defense against damage
to blood vessels. When damaged, the cells that make
up blood vessels release chemicals that activate passing
platelets, causing them to adhere to the surface and
aggregate. When activated, platelets change shape and
expose integrin molecules - aIIbb3, to be specific.
The surface of a platelet contains approximately 80,000
copies of aIIbb3, and each copy binds to fibrinogen,
a fibrous protein that helps lash platelets together
to form a clot. While clotting may stop bleeding, the
formation of thrombi, or blood clots where they do not
belong, may also lead to a stroke or heart attack if
a clot blocks off blood vessels. So the subject of the
forces involved in how clots form and dissolve are important
to medical researchers. The regulation of activation
of these cellular integrins must be tightly controlled
to prevent thrombosis.
"Platelets are like multiple-watt lightbulbs: they
can be turned on to different degrees of activation,"
said Weisel. "Interestingly, our findings suggest
that no matter which setting you turn a platelet to,
integrin binds to fibrinogen with the same affinity."
Adenosindiphosphate (ADP) and thrombin are two platelet-activating
chemicals, each able to activate platelets to a different
proportion, depending on their concentration. According
to Weisel and his colleagues, even though more fibrinogen
binds to a platelet exposed to thrombin than to ADP,
it takes the same amount of force to break apart a single
pair of integrin and fibrinogen molecules. It takes
about 80-100 picoNewtons to separate the two molecules.
By comparison, a picoNewton is about the weight of a
single red blood cell and there are 500 million red
cells in a drop of blood. "You can change the degree
to which platelets are activated or the number of activated
integrin molecules, but not the strength of the integrin
bonds with fibrinogen," said Weisel.
Platelet-activating chemicals cause platelets to change
shape dramatically, turning the round discs of platelets
into multi-tentacled balls. This transformation allows
platelets to form complex aggregates via interactions
between activated integrins on these tentacles and fibrinogen
in the blood. Platelet activation increases the percentage
of activated integrins on the surface but the strength
of integrin-fibrinogen binding is the same for each
individual pair.
The trick to uncovering the strength of a single bond
between proteins was the use of the laser tweezers.
Laser tweezers cannot manipulate individual molecules,
as such, but Penn scientists developed a new model system
using tiny pedestals and beads that can be trapped and
moved. To measure the bond strength, the researchers
actually attached fibrinogen to microscopic plastic
beads and exposed them to integrin that was either attached
to pedestals or as they sat on the surface of living,
reactive platelet cells.
"Laser tweezers are a remarkable tool for cell
biology," said Weisel. "For the first time,
we can actually measure the force of a single ligand-receptor
bond, as it exists in real world, and study the cellular
regulation of activation of these specific receptors."
Penn researchers involved in these findings include
Rustem I. Litvinov, Henry Shuman, and Joel S. Bennett.
Funding for this research was provided through grants
from the National Institutes of Health.
# # #
Editor's Note: For a colorized scanning
electron microscope image of platelets in various stages
of activation, click here.
Please credit the University of Pennsylvania School
of Medicine for the image.
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