(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 – (alpha)IIb(beta)3, to be specific. The surface of a platelet contains approximately 80,000 copies of (alpha)IIb(beta)3, 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.
The above story is based on materials provided by University Of Pennsylvania Medical Center. Note: Materials may be edited for content and length.
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