Biocurious

/ a biophysics blog

New Perspective on Blood Clot Mechanics

Posted 25 April 2008 by Andre under

My collaborator John Weisel has just published a nice Perspective article in Science describing some of the recent progress that has been made in elucidating the mechanical properties of blood clots.

It’s a fascinating subject in part because of its relevance to health. As John puts it:

[A]lthough clotting is vital to the preservation of life, blood clots that impede the flow of blood in vivo—called thrombi—are responsible for most heart attacks and strokes and complicate other pathological conditions, including many types of cancer and peripheral vascular disease.

clot and fiber

But clots are also interesting purely from the materials perspective. In this context, they are a wonderful programmable material. A single protein monomer—fibrin—encodes the entire hierarchical structure of clots. A solution of fibrin will spontaneously self-assemble into a beautiful branched meshwork that fills a test tube but is still more than 99% water. To accomplish this feat, fibrin is more complex than some of the proteins that make up other networks like those found in the cellular cytoskeleton like actin and tubulin. This complexity makes studying fibrin both a challenge and a thrill. The challenge is to devise experiments that are sufficiently well controlled to interpret, as we tried to do in our single molecule pulling paper from last year (paper [free on Pubmed Central]; post at Biocurious). The thrill comes from the enormous variety of factors that modulate clot structure and function. Fibrin forms a smart material: it responds to external chemical cues found in blood that can cause it to grow or dissolve or to become stiffer and more impervious to mechanical insult. In fact, there are dozens of factors responsible for regulating the formation and degradation of blood clots in humans and these can be leveraged to perform useful experiments or to develop variations on biological clots with new properties.

All of these things are possible, and are indeed already happening, but we still don’t know in detail how the structure and properties of fibrin give rise to its macroscopic properties and that means that we still can’t be as clever as we would like in treating mechanical diseases involving fibrin and in designing new materials inspired by its properties.

But this too is starting to change, so stay tuned…

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How do you make and share scientific figures?

Posted 10 June 2007 by Andre under &

One of the most important parts of scientific communication is making figures that convey your results clearly and compellingly. These days, that means using software to arrange plots and images from other programs, add labels, and often draw simple cartoons or schematics to illustrate the central ideas. There are several features that I consider important for this kind of graphics software. It must support a variety of file formats and be able to export publication quality images. It should have convenient alignment tools so that your images can be easily centered with respect to each other or arranged into arrays. Importantly, the drawing tools should be flexible, give a clean professional looking end product, and not be to difficult to learn. These are not very stringent requirements as far as graphics applications go.

Since most science is collaborative (it’s rare to see single author papers in most journals), it’s also useful for each person involved in the project to have access to the same software for making corrections or suggestions for improvement. So the ideal scientific graphics software will also be compatible with several platforms (at least Windows and Mac) and cheap (preferably free).

Unfortunately, because of its ubiquity, Powerpoint is often the default choice for making figures despite its failure to meet most of the requirements outlined above. This is not acceptable to me, even though I’ve been using it recently because it’s what some of my collaborators use (before submitting anything to a journal I will most likely redo these figures in another program after the final versions have been agreed upon).

I’ve tried Omnigraffle and found that it was pretty good, but as far as I know, it’s only available for Macs. I haven’t used Illustrator but I’ve heard good things. Is it worth the money? Will I be able to convince collaborators to use it despite the cost?

What else is out there? What do you use for making figures?

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Veeco Bioscope II Application Note

Posted 1 June 2007 by Andre under

You may remember a post from last year describing some measurements we made on neurons using our combined atomic force/fluorescence microscope. Well, Veeco, the company that makes the AFM we used has put up their new Bioscope II product site and it uses some images I took:

You can also download the application note we wrote based on those measurements. It has essentially the same information as the blog post I linked to above, alas, it’s a pdf so it doesn’t have the movie.

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Blood Clotting Explained

Posted 15 April 2007 by Andre under &

Larry Moran, a biochemist at the University of Toronto, has a series of posts on his blog Sandwalk explaining the cascades of enzymes that regulate blood clotting. It’s an amazing process that must be carefully regulated. If the balance tips towards too much (or inappropriate) clotting, heart attacks and strokes result. Insufficient clotting leads to bleeding disorders. Moran guides us through from the basics, platelets, Extrinsic Activity and Platelet Activation, to intrinsic activity. It’s a free course in biochemistry and it provides the perfect background for some of our recent work on the mechanics of fibrinogen, the protein that actually forms the network of clots at the end of these enzyme cascades.

This kind of mini-tutorial is one of the great things that science blogging encourages and I hope we get more of it. The low number of comments on many science-heavy posts, even on very popular blogs like Pharyngula, shouldn’t be taken as a sign that there’s no interest in these topics. They don’t generate the same immediate reaction as posts on politics or religion, but they’re part of something larger that’s slowly developing. Since the Internet has become so easily searchable, finding scientific information written by experts for a lay audience is becoming much easier. Wikipedia is great, but the loosely nit archive of science that’s developing in science blogs has a different character. These posts often have interesting tangents about related topics and more narrative structure making them easier to read and, perhaps most importantly, they’re interactive so if you have a question about the subject of the post, blogs offer a great opportunity to directly ask experts for clarification without having to wait for the topic to come up in an “ask the experts” column in a science magazine.

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Blood Clot Mechanics at the Molecular Level

Posted 17 January 2007 by Andre under

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]

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Hybrid TIRF/AFM Imaging

Posted 26 October 2006 by Andre under

One of the things I’ve been working on in the last few months is simultaneous fluorescence and atomic force microscopy (AFM) of a variety of biological samples. I’ve talked about total internal reflection fluorescence microscopy, or TIRF, before and I’ve also shown you some AFM images of leaves and insect wings so I think the time is ripe to show you some work on combining the two. I should point out that all of the samples I’m going to show you were expertly prepared by Vidya Nadar in Peter Baas’s lab at Drexel University College of Medicine. Thanks to Vidya and Peter!

The first image shows the growth cone of an axon. Part (a) is a TIRF image of the stained microtubules that run through the body of the axon and splay into the growth cone. We know that everything in red is a microtubule because the labeling was done with tubulin specific antibodies. That’s the power of fluorescence combined with immunostaining. This technology has become indispensable in cell biology. To complement this information, AFM gives high resolution images with quantitative height information. Part (b) is a TappingMode AFM image of the same region and it is much easier to make out the cell extremities where there are no (or few) microtubules. It also contains information about the microtubule arrangement in the axon that is not available from the TIRF image alone. For example, two microtubule bundles are visible running through the axon in the TIRF image, but in the region of the AFM image highlighted with a white box, their relative position becomes clear: the lower bundle is actually broken and frayed, passing over its neighbor and terminating above the main part of the axon. Without the AFM image one might falsely conclude that the bundles simply merged and continued unobstructed through the rest of the axon. The line profiles in the inset illustrates this more clearly (these just show the intensities measured along the yellow lines in each image).

Here’s another image showing a glial cell from the same sample with the fluorescence overlaid on the AFM height image.

Using AFM for imaging cells is useful and, although you will often get nicer images from electron microscopy, AFM doesn’t require complicated sample preparation and cells can be imaged while they’re alive in ambient conditions. That eliminates some sample preparation artifacts and also makes it possible to make movies of processes occurring of the scale of several minutes.

But that’s not the most exciting thing to do with AFM. It is also possible to interact directly with samples using the AFM tip not for imaging, but for manipulation. Samples can be indented to measure their stiffness and the stiffness of their substrates, something that is increasingly recognized to be important for the regulation of several processes in different cell types (see Dennis’s review here) and most recently to direct stem cell differentiation (see Adam’s Cell paper [pdf]).

That work was done using a standard AFM, but now, using the combined instrument we can simultaneously visualize these manipulations. Here’s an example from a glial cell with labeled microtubules. Look how stretchy the fixed samples are! Sometimes I imagine fixed cells as almost vitrified, but that’s obviously not the case (nor would you expect it to be if you think about it: a crosslinked gel may be stiff, but it’s not solid). This same method was recently used to show that single fibrin fibers (the ones that form the scaffold of blood clots) are remarkably extensible (see also the movies on Martin Guthold’s site). But that’s not all. Since you can also measure the cantilever deflection during the manipulation, mechanical properties are also available from this method. This is a nice idea and you can expect to see a growing number of papers describing this kind of work in the next couple of years (maybe even some from me!).

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