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Blood Podcast
Review Series on the Structural Underpinnings of Hemostatic Plugs and Thrombotic Occulsions
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- [00:02]
Podcast Host
Welcome to this week's bonus episode of Blood Podcast, your source for innovative ideas and cutting edge information. In this episode, Associate Editor Dr. Thomas Ortel discusses the review series on the structural underpinnings of hemostatic plugs and thrombotic occlusions, with contributing authors Dr. Alyssa Wolberg, Jonas Emslie, and John Wiesel.
- [00:26]
Dr. Tom Martell
Welcome, everybody. My name is Tom Martell. I am chief of hematology at Duke, and I am responsible for putting this particular review series together. Today's podcast is going to be an introduction to a review series that we've put together that's looking at the structural underpinnings of hemostatic plugs and thrombotic occlusions. I've been involved with a couple of review series that have looked at individual clotting proteins, some that have looked at a couple of clotting proteins. This one looks at the complete formation of hemostatic plugs, the cells, the proteins, the other molecules that are involved in putting these things together, and how when things go awry, you may have a thrombotic occlusion resulting in potentially patient death, potentially chronic changes in those individuals. What we did was we put together a review series that included an introductory article that talks about the structure of thrombotic occlusions, particularly in different vascular beds such as venous circulation, such as arterial circulation. Then we brought in a couple of review articles that focused on individual constituents and that includes platelets factor 13, and then also the contact activation system. The latter actually also kind of links this in with inflammation and inflammatory processes which are involved with thrombotic occlusions as well. Then we closed out this review series with an article that's on novel approaches to try to control hemostasis, recognizing that bleeding, particularly when you're not in a hospital, can be extremely difficult to treat, and so developing novel approaches to try to control bleeding. Today we're going to hear about several of these articles. Dr. Alyssa Wolberg from University of North Carolina gave us a beautiful article on factor 13, and Dr. Emslie from University of Nottingham gave us an excellent overview of the molecular structures of the constituents of the contact phase pathway. And at this point, what I'd like to do is turn this over to Dr. Wahlberg to give us an overview of the article that she put together for this review series.
- [02:57]
Dr. Alyssa Wolberg
Thanks, Tom. The article that we put together is on one, especially hearing you talk on one level, very, very focused on a single protein. But we've come to learn over the last 50, 60 years but in particular in the last 10 years or so, that factor 13 plays a major role in determining essentially everything about the composition and components of a clot and therefore the way a clot functions in hemostatic situations, as well as when it goes awry, as you said, in thrombotic situations and others. And so our article talks about fundamental biology of factor 13. Things you just need to know in order to know Factor 13. And it's interesting biochemistry and locations in physiology. It's in plasma, it's in platelets, it's in cells, it's in all the components constituents that produce clots. But then talks a lot about what it does, including its traditional roles in which differ from other coagulation proteases. Instead of cleaving proteins, it glues them together. It's actually unofficially known as a meat GL glue, which makes it really, really unique. And its role then in how it holds together the clots and then the downstream implications of that, how it determines what's inside the clot retaining proteins, retaining red cells, allowing for these cells to be compressed and produce a solid clot. And what happens when that process isn't taking place. And we include some diseases that are associated with coagulation complications, including inflammatory diseases and liver diseases that are also affected by this protein and its role in producing a clot and producing stable fibrin.
- [04:41]
Dr. Tom Martell
Thank you. Now I'd like to turn to Dr. Emslie to give us an overview of the review article that he prepared for this review series.
- [04:53]
Dr. Jonas Emslie
My review article was regarding the contact system. So this system plays a role in coagulation and inflammation, but is not thought to be critical for blood hemostasis. It was a complicated review because there are four proteins involved, Factor 12, Factor 11, precalicrine and high molecular kininogen. And we felt we had to consider all four of these in this quite short review article to really get an overview of how the system is assembled and activated. And so we reviewed what was known about the structures of the enzymes, focusing on how they maintain their inactive or zymogen conformations for Factor 12 and Factor 11 and precalicorine. And we also spent a lot of time on the main cofactor as a critical linking factor, what was known about the structure of the cofactor and the protein protein interactions that it formed with the enzymes. So there is still a big mystery in the field in terms of what the precise membrane, where all the contact factors are assembled and the molecular mechanisms of how the enzymes become activated. And I think what I got most out of this review was to see where we were in understanding those molecular events and also identify what we didn't know so far. And I think the big takeaway was that we understand quite a lot about the structure of the zymogens of, say, Factor 12 and thinking about how they become disrupted and activated, but we really know very little about the higher rules done multiprotein complexes of contact system and very little about the molecular detail of substrate engagement.
- [06:57]
Dr. Tom Martell
Thank you very much for that overview. What I'd like to do now is turn to actually just asking a few questions about your articles and also asking you to maybe extrapolate a little bit farther out from your article and how it might interrelate with some of the others. And I'll start with you, Dr. Wahlberg. You do talk a little bit in your article at the end about potential therapeutic interventions. It strikes me as a clinician with my clinical hat on that patients with factor 13 deficiency usually don't have any bleeding until they get severely low, essentially non detectable factor 13. Are there strategies that you can think of that might might be antithrombotic or that could be antithrombotic that you could elaborate on a little bit that would potentially not cause the bleeding problems?
- [07:51]
Dr. Alyssa Wolberg
You're right that factor 13 levels can get quite low before they're considered clinically alarming. And that points to the idea that there may be an interesting window in which we can safely operate and manipulate maybe not just factor 13 levels, but its actual biochemical functions in even a more elegant fashion. So one of the things that we've noticed is that One of Factor 13's functions that we uncovered about 10 years ago was that Factor 13 cross links fibrin at a particular point in time when clot contraction is taking place. And its role in particular at that point is that the fibrin must be stabilized so that as contraction takes place, it can retain all of the cellular components of that clot, and in particular it can contain the red blood cells within that. So if one can uncouple that process either in a major fashion, like removing factor 13, or in a more elegant or delicate fashion like decreasing factor 13 or altering the timing of those interactions, one can produce clots that are smaller, that are smaller because they don't contain red blood cells. So they may be less likely to become occlusive and may actually give a better window for preventing thrombosis, either alone or in concert with conventional anticoagulants that have proven to be quite effective, but come with a risk of bleeding. And I think that part is well established. And so we've been very interested in trying to understand more about the implications of eliminating factor 13, decreasing its levels in a more safe fashion, or targeting the mechanisms that lead to the way it has this concerted relationship with fibrin cross linking, platelet mediated clot contraction and ultimately determination of that clot. And all of this at this point is still speculative. We are basic in translational laboratory, but haven't gone further. The field hasn't gone too much farther in exploring that from a pharmacologic perspective at this point. In particular, limited by the lack of really good drugs to target factor 13 that have the necessary pharmacologic properties to even do those experiments. But we're learning quite a bit from, as you said, observations of patients who can tolerate reduced levels of factor 13 reasonably safely. And learning from those patients then what may be developable in terms of potential therapeutics. We'll see what happens. We're excited about it. That's so it's an interesting question.
- [10:27]
Dr. Tom Martell
I'd like to turn to Dr. Emslie for a question which would just be again, putting my clinical hat on. There are definitely patients that I've seen over the years with venous thrombosis who have a very profound inflammatory response involving a leg. And there are other patients who really don't have any inflammatory response at all. Do you know of any data or is, is anybody looking at potentially polymorphisms or differences in these individual proteins that may mediate or ameliorate an inflammatory response in somebody with a venous thrombosis?
- [11:07]
Dr. Jonas Emslie
The one that I know of is the principal cofactor, high molecular weight kininogens. So this has a polymorphism associated with venous thrombosis that that's quite common in the population. So I think half of us here have one that has a more rapid form of contact activation and the other half is like a 50, 50 split in the population of this single amino acid mutation. And potentially this kind of variance could lead to, as you described different, some patients having, because the contact system drives bradykin inflammation, which is a potent inflammatory mediator as well as intrinsic pathway coagulation and also has these interactions with platelets and the immune cells, then these variants in the population that really have not been characterized and full loads could have this type of effect.
- [12:12]
Dr. Tom Martell
And to follow up, twisting it a little bit currently, as you know, there's a lot of excitement about inhibition of factor 11a and factor 11 as an antithrombotic strategy. Do you see that potentially stirring up any problems with the complement side of all of this or the inflammation side of all of this, or involvement with bradykinin. Could there be any downstream effects from inhibiting factor 11 that we need to be more aware of?
- [12:41]
Dr. Jonas Emslie
So far, what has been observed is a less bleeding side effect and a dampening down of immune processes that has been really encouraging, as well as obviously inhibition of the intrinsic pathway. I think one of the things we discovered when we were doing the review article is that level of redundancy in the system. So both factor 11 and precalicarine are homologous. They both cleave factor 9 part of the intrinsic pathway. And although precalicrine is the main driver for inflammation and bradykinin formation, factor 11 also seems to drive these inflammatory processes. So I know companies are trying to design dual inhibitors or of 11 and PK's because the factors, there's a level of they consume each other. So the concern is if you knock one out, the other may upregulate. So these kind of things are going to be interesting to observe as more and more data comes out of the use of the factor 11 inhibitors.
- [13:51]
Dr. Tom Martell
Interesting question that either of you or both of you could address would be in the introductory article from Dr. Wiesel. He provides actually a lot of structural information about thrombus formation in different circulatory beds and how he finds a lot of differences in the composition of clots depending on if it's in a vein or in a pulmonary artery, or in another artery, a stroke or an MI. Just curious, with either factor 13 or the components of the contact factor pathway, do we have any information that there are differences in these constituents that might contribute to why does a clot look different if it's formed in a vein versus if it's formed in an artery?
- [14:41]
Dr. Alyssa Wolberg
I think what you're touching on to me is one of the least appreciated elements of climate clots in general, at least to the public. And I love teaching this to students when they come. People learn of someone who had a clot or died of a clot without necessarily acknowledging that not all clots are the same. And Dr. Wisel does a really nice job of summarizing the differences between these. And in particular, this boils down to clots that form in arteries being particularly platelet rich. They're traditionally known as white clots. That's a little bit inaccurate. They're pink, they're lighter, they have fewer red cells, but they're very platelet driven. And the venous clots of course have predominant red cell component and they're red clots. And when you look at them, you can readily appreciate these. And what I have always told the students is that, you know that these are different because the anticoagulants or antithrombotic drugs that are used to prevent these two kinds of clots are very different. And so that's instructive from a fundamental science perspective and how we think about designing experiments that recapitulate what, what's actually taking place in the people when we do this in vitro or using other models within the lab. The interesting thing for me as a person who studied fibrinogen and now factor 13 for a number of years, is that clearly these components have roles in both of these. And we know that again because of the kinds of drugs that can be used to prevent and treat each of these kinds of clots. We've done studies looking at the role in particular factor 13 since that's affecting focus of this article in arterial thrombosis and venous thrombosis. And this falls on the heels of many groups that have done this before us to show that arterial clots seem to form even if there's no factor 13 there. But the ability of factor 13 to cross link and really stabilize those clots can vastly affect whether those arterial clots mi, stroke clear or can undergo thrombolysis. So, so that's particular role of factor 13 there and then in venous thrombosis because those clots form much more slowly and depend much more on the Presence of Factor 13 Cross linking Fibrin and really retaining the red cells in a process that is very deliberate and very temporally orchestrated. Then we think removing factor 13 in that case would actually affect the way that clot forms in the first place, even in addition to whether or not it could be clear cleared by conventional mechanisms. So there are some similarities and some differences and I find this actually really fascinating and instructive as we think about the potential roles that these proteins play in regular physiology, but also how we might target them therapeutically.
- [17:23]
Dr. Tom Martell
At this point in time, I'd like to introduce another one of our authors for this review series that we have managed to bring in. Loop in to talk a little bit about this review series. And this is Dr. John Wiesel, who is going to be talking to us about an overview of thrombus formation in different vessels, the structure of this and what information that gives us. And with that I'm going to turn it over to Dr. Wiesel to give an overview of his article.
- [17:55]
Dr. John Wiesel
This is a review article I wrote with my colleague Rustem Litvinov. And I'd say it has three different levels, three different layers. The first is that we highlighted some lessons that we learned from studying the structure and composition of various human thrombi. And the second is that we described some of the basic science research to study the underlying mechanisms that are responsible for the observations. And the third layer is that we mentioned various potential therapeutic opportunities that arise as a result of these observations. The major structural elements of thrombi are platelets, erythrocytes and fibrin, each playing a critical role in the determination of the biological and physical properties of thrombi, such as their permeability, stiffness, lytic and mechanical stability. And the relative amounts of the components are perhaps unexpected, especially in the case of arterial thrombi, in that we found there were a lot more fibrin and red blood cells than we expected. So I'd like to start by describing the components of thrombi, how they change in space and time, and I'm going to use the composition of thrombi from myocardial infarction patients as an example and how they change as a function of time. And by time, I mean the time from the start of chest pain of the patient until the extraction of the thrombus. And we saw dramatic changes as a function of time, with about a two fold increase in fibrin per hour of ischemic time at a 50% reduction in the quantity of platelets per hour of ischemic time. When we look at thrombus from myocardial infarction patient that is less than an hour old, we see almost entirely platelets. But if we look at older thrombi, we see then one end of the thrombus has almost entirely platelets, whereas the other end has almost entirely red blood cells and fibrin. And the fibrin is mostly on the surface. And the red blood cells have a unique shape that we have described. The red blood cells become polyhedral, which means they have a polyhedral shape, and we've named them polyhedrocytes. And this is the result of clot contraction. So in general, we found that there's very little free space in thrombi, both arterial and venous thrombi, and the structures are very tightly packed as a consequence of clot contraction or retraction. What happens is that platelets pull on fibrin and platelets make a kink in the fibrin fiber and pull it towards the platelets or the platelet aggregates and the fibrin agglomerates around the platelets, and then platelets pulling on fibrin compresses the red blood cells so that they change shape from being biconcave to being polyhedral. This is something we see in nearly all human thrombi. We've examined, both arterial and venous, that most of the red blood cells are polyhedral, what we call polyhedrocytes. And there are very few blood biconcave red blood cells in these thrombi. Also, I'd say we've learned that fibrin is the main structural scaffold of all these clots and is responsible for the mechanical properties of thrombi. The mechanical properties are important because blood clotting is basically a mechanical process to stop bleeding. And fibrin forms the basis of the mechanical properties of all clots and thrombi. And what we have learned also is that fibrin is a very resilient structure. And when there are mechanical forces in the vasculature, that those forces are impinging on thrombi or clots, the fibrin is pulled and actually the fibrin molecules unfold. And this unfolding is part of the function of fibrin as the mechanical basis of clots. Just to describe some of the new findings that we highlight in our review article, we found that in thrombotic patients, platelets become continuously partially activated and they become exhausted. So the ATP levels go down, the mitochondrial membrane potential decreases, and the platelets become refractory, so they're less able to respond. And this impaired contraction or retraction has at least three pathogenic consequences. First of all, there's increased thrombus obstructiveness because there's lack of clot contraction. So the thrombi are more obstructive. There is also reduced internal fibrinolysis because internal fibrinolysis is increased as a result of clot retraction. And finally, there's increased mechanical instability, and there's an increased predisposition to rupture or embolization. That clot contraction helps to reduce the possibility of rupture and embolization. So the reduced clot contraction in these thrombotic patients means there's more likelihood of embolization of thrombi. Also, I'd say another relatively new finding is that fibrin is what is called an equilibrium polymer. And what this means is that the fibrin molecules and oligomers can dissociate from fibrin and reassociate before they're cross linked by factor 13A. This also means that the addition of the synthetic peptide glyproarg, which is the knob that's responsible for fibrin polymerization. Fibrin polymerization occurs by knobs interacting with holes in adjacent molecules. And if that knob peptide is introduced to a clot or a thrombus, that can compete with the knobs that are on the fibrin molecules and help to dissolve a clot or thrombus. So this is another potential mechanism for treating thrombosis in that a synthetic fibrin knob memetic might potentially be used to dissociate fibrin and make the clot more easily dissolvable. I also mentioned that it's been difficult to study the functions of platelets because they're anucleate cells. And so it's very difficult to do any genetic manipulation except with mouse models and or other animal models. But recently it's been discovered that megakaryocytes derived from induced potent stem cells display many functions of platelets, including activation by thrombin, various signaling pathways, binding of fibrinogen, and especially contraction of clots. So this means that we can now possible to study platelet functions using CRISPR Cas9 technology to make mutations or knockouts of various cytoskeletal membrane or signaling proteins to study platelet functions by studying these I megakaryocytes.
- [25:49]
Dr. Tom Martell
I really found this very interesting that the thrombi structurally are different in different circulations and they change over time. I'm wondering, do any of these recapitulate normal hemostasis? If you have hemostasis, do we know much about the structure of the occlusion that is formed on that? Is it similar to any of these?
- [26:13]
Dr. John Wiesel
Yes, there are a lot of similarities. And here we're mostly relying on some various mouse models that have been used in recent years to study hemostasis, and they've been very useful. So we see a lot of similarities in terms of the role of platelets in forming an initial platelet plug and then the formation of fibrin that stabilizes the clot, and then usually especially in larger vessels, accumulation of red blood cells. But there are also some differences in the process. And one of the challenges, I think, for people who are using mouse models or microfluidic models is to be able to understand how we go from those mouse models to what we see in human thrombi. So that's one challenge that I would throw out for various scientists that are working in this area to try to make a better connection between their models and what we see in human thrombi.
- [27:18]
Dr. Tom Martell
Okay, well, thank you very much for that overview, and these articles are all very interesting. I'd like to close at this point in time by thanking the three authors who joined us on this podcast, Dr. Wisel, Dr. Wahlberg, and Dr. Emslie. And also acknowledge that we have a total of five articles in the review series that's going to appear here in the journal Blood. And I hope that you have found this podcast to be particularly exciting and interesting to lead you to want to read all of the papers. So I'd like to thank my authors, the authors who put together these papers. I'd like to thank Blood for allowing us to put together this review series. And I'd like to thank those people who are reading the articles and finding them interesting and learning new things about hemostatic plugs and thrombotic. Thank you very much, everybody.
- [28:15]
Podcast Host
Thank you for listening to this bonus episode of the Blood podcast. To read these articles, visit bloodjournal. Org this episode is copyrighted by the American Society of Hematology.