Blood Clot Mechanics at the Molecular Level

Cross-posted to Biocurious a blog about biology through the eyes of physicists.

The function and dysfunction of blood clots are often directly related to their mechanical properties: clots stop blood from flowing through wounds but can also break away (embolize) and block blood vessels causing stroke. Strength and plasticity are both important for ensuring the former is more common than the latter and so people have been studying the mechanics of clots for over 50 years. 

Despite this history, or perhaps because of it, new discoveries are being made all the time. Take the recent observations reported by Liu et al. last year in Science (abstract is free). They used a combined fluorescence and atomic force microscope (not unlike the one I've been working with recently!) to stretch single fibrin fibers--the ones that make up the protein mesh of blood clots shown in green in the image above--to see how far they could stretch. They found that some fibers could stretch up to 5 times their relaxed length before breaking! Check out the movies at Martin Guthold's site.

How can fibrin fibers accommodate so much stretch? For these extreme stretches especially, I think it's likely that protein unfolding will play a role. But without knowing anything about the mechanics of the proteins that make up fibrin fibers, this is just speculation. And that's where single molecule stretching comes in. For some background on single molecule mechanics you can see a previous post here. Basically, a protein is adsorbed on a surface and then pulled away using the sharp tip of the microscope's cantilever. The nonspecific attachment between the tip and protein of interest is sometimes strong enough to pull apart the relatively weak interactions that hold proteins together. To address clot mechanics at the molecular level, I started pulling on fibrinogen, the protein that forms fibrin fibers after activation by an enzyme called thrombin. See last November's molecule of the month for more details on fibrinogen and the clotting cascade.

Unlike most other proteins studied so far by single molecule AFM, fibrinogen has a more complicated and varied structure and this complicates the interpretation of single molecule experiments. To improve our chances, we took advantage of another enzyme that acts on fibrinogen called factor XIIIa that covalently attaches fibrinogen molecules together. When we mix fibrinogen and factor XIIIa together we get little chains of fibrinogen that are perfect for pulling on:

When these little chains (the scale bar in that image represents 50 nm) are pulled, we see a sawtooth force-extension curve that is consistent with the sequential two-state unfolding of protein domains in series. Before doing this experiment, it wasn't known _which_ domain of fibrinogen would unfold first but, based on the distance between the peaks of the sawtooth and the known structure of fibrinogen, we could rule out the globular end domains and conclude that it is likely the coiled-coil domains that are each unfolding independently. The next figure shows the length increase upon coiled-coil unfolding on the left and the crystal structure of fibrinogen on the right. Notice the rod shape with two end domains separated by a three stranded coiled-coil. It's quite a looker.

We've pulled on fibrinogen and found that its coiled-coils unfold at a force around 100 pN in a two-state like process (the two states being folded and unfolded). This fact, and some elaborations on it, are interesting as pure biophysics, but what does that mean for blood clot mechanics and therefore physiology? Good question, I'm glad you asked. Well, we know the stiffness of single fibrin fibers because of some other work in John Weisel's lab (~10 pN/nm^2) and we know the approximate cross sectional area of the molecules (~10 nm) so we can calculate that the force per molecule after a two fold stretch of a fiber is around 100 pN, i.e. enough to unfold coiled-coils! This unfolding could serve to absorb just over a two-fold stretch in addition to that available from network rearrangement and fiber bending and that might prevent clot breakage in some cases. But what about at more modest extensions? To be honest, I don't know, but living with (and reveling in) uncertainty is one of the joys of science. And I have hunches. Hunches are key.

For some more technical details and references, see our new paper in Biophysical Journal available free online here [pdf]