Rienstra's lab then analyzed the samples using SSNMR, a technique that allows researchers to precisely measure the distances and angles between individual atoms in large molecules or groups of interacting molecules. His group found that one of every six or seven PS molecules was binding directly to the clotting factor, providing strong experimental support for the model derived from the simulations.
"That turned out to be a key insight that we contributed to this study," Rienstra said.
The team reasoned that if the PE head-groups were simply bending out of the way, then any phospholipid with a sufficiently small head-group should work as well as PE in the presence of PS. This also explained why only one PS molecule was actually binding to a GLA domain. The other phospholipids nearby were also interacting with the clotting factor, but more weakly.
The finding explained another mystery that had long daunted researchers. A different type of membrane lipid, phosphatidylcholine (PC), which has a very large head-group and is most abundant on the outer surface of cells, was known to block any association between the membrane and the clotting factor, even in the presence of PS.
Follow-up experiments showed that any phospholipid but PC enhanced the binding of PS to the GLA domain. This led to the "ABC" hypothesis: when PS is present, the GLA domain will interact with "Anything But Choline."
"This is the first real insight at an atomic level of how most of the blood-clotting proteins interact with membranes, an interaction that's known to be essential to blood clotting," Morrissey said. The findings offer new targets for the development of drugs to regulate blood clotting, he said.
|Contact: Diana Yates|
University of Illinois at Urbana-Champaign