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- [00:02]
A
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. Beth Battinelli discusses the review series on platelet heterogeneity with authors Dr. Craig Morell, Dr. Larry Freilinger and Dr. Leo Nicolai.
- [00:25]
B
Thank you everyone for joining this blood podcast that is focusing on platelet heterogeneity and the idea that that there are more than one type of platelets. This would suggest that there are platelets that are different based on their site of origin, their content, receptor surface expression, and also in terms of functionality. Joining me today, I have the authors of three articles in this review series, Dr. Craig Morrell, Dr. Leo Nicolai, and Dr. Larry Frelinger. And each of them has contributed an article that focuses on different aspect of the idea of platelet heterogeneity. I'd like to kick it off by asking Dr. Morel to share with us his concept of the idea of different subtypes of platelets.
- [01:17]
A
Thanks for allowing me to be a part of this. I think we have a lot of new emerging concepts about what platelets do and what their functional roles are and understanding that they do, of course, have their important role in forming a thrombus and stopping bleeding, but. But they also have these other more diverse roles in recruiting and activating white blood cells and directing the immune responses. And I think now we're even starting to get a better understanding of even the different types of megakaryocytes that platelets are coming from, both in the bone marrow, where there's emerging literature that it's a diverse population of megakaryocytes with the ability to produce platelets which have different phenotypes defined by their RNA expression. And we also are starting to understand that there's other locations outside the bone marrow that are capable of producing platelets, including the lung and the spleen. Whether the platelets coming from these different locations are therefore phenotypically and functionally different, I think is something that we're a little limited by technology right now. But I think there are concepts that a lot of us are very interested in further exploring that relate to heterogeneity.
- [02:22]
B
Thanks so much, Dr. Nikolai. There are classifications for different types of known subpopulations. Would you like to orient the audience a little bit about those different subpopulations that we already know exist and then later on we could perhaps address a little bit of what we know about their role in disease.
- [02:43]
C
Thank you so much for allowing me to join this podcast. Yeah. What has been known for quite some time is that upon activation and activation signals, you can get very diverse platelet subtypes. And that includes classically aggregating platelets that are known to form a clot, but also procobal platelets that you get if you have a very strong activation, mostly via two different pathways that then allow the activate or the binding of coagulation factors and then coagulation to take place on these platelets. It's also known that platelets can be seen secretory. Of course, these functions are also partly overlapping. And apart from these clear functional differences that are triggered by different activation pathways, it has also long been known that there's a subpopulation of young platelets in the blood, and they are also called reticulated platelets because they have more RNA in them. That's also how you detect them mostly. But Larry can, I think, say more about this, because this was part of his review to help classify and quantify these subpopulations. And these cells have been known to be associated with cardiovascular events, thrombosis risk, and they seem to be more active, more reactive to stimuli. And there I think you get pre clinical research that paints a clear picture, but also clinical data that kind of highlights that this platelet heterogeneity actually is important for the patient.
- [04:15]
B
Thank you. And with that lead in, Dr. Frelinger, would you like to talk to us a little bit about how the age of platelets may actually influence this idea of platelet subpopulations?
- [04:25]
D
Sure. When platelets are born, whether it's in the bone marrow or in the lung, they come out as a larger size. And as they circulate, they lose bits of membrane, they lose certain surface proteins in small amounts, and. And gradually over time, become smaller. So the first level of distinguishing platelets based on their age is their size. Now, as they lose that membrane, there's also intracellular changes. The platelets, when they're initially formed, have higher amounts of RNA, and that RNA degrades fairly quickly. Within about 24 hours, most of it will be degraded. All although there is some RNA that's in a stasis sort of situation and will only be translated when the platelet is later activated. So RNA levels change over time, size changes over time, quantity of surface marker can change over time. But even within an individual, at any one point in time, there's high variation in platelet size and there's high variation in platelet surface marker density. So based on the combination of all of these markers, we can distinguish different.
- [05:44]
B
Populations of platelets thanks so much, Dr. Frelinger. Dr. Nicolai, could you tell us a little bit about distinct data or distinct research that has showed a role of. We can just mention one type, such as pro coagulant platelets. In what type of disease do you see that you see more of or one type of platelet and what is the contribution of that to the disease? And the last part of my question on when you're thinking about the role of pro coagulant platelets in disease would be how do you deal with comments that you know this is only a small population of platelets and that other platelets are not pro coagulant and it's all yet in circulation at the same time?
- [06:28]
C
I think that is a very exciting question. And the field has really turn towards procrocant platelets for research interest, but also because this might be a new therapeutic avenue to follow. I have to say that most of the data we have is derived from mouse studies, but there we see that we can specifically block prochloro function by knocking out specific proteins and platelets, for example in the mitochondrion, but also a scram blase that is called TMEM16F that is important to explore phosphatylserin and then allow the correlation factors to bind. And using these mouse models, we could see that this really affects certain diseases. So on the one hand, knocking out prochloromal function leads to more bleeding in the context of inflammation, because platelets use this function to seal very small injuries. You get inflammatory conditions, but at the same time it protects against stroke, it can protect against arterio thrombosis. And recently our group has also shown that it can protect against venous thrombosis. And this is quite exciting because there is patients that lack this TMM 16F and it's called Scott syndrome and they actually only have a very mild bleeding disorder. And also the data derived from the mouse model shows that actually bleeding is not a major issue in these mice leading from injuries or brain bleeding, for example. So for that reason it might be very interesting to block this function and it could really have a beneficial effect in patients. Although definitely, as you mentioned, only a small subset of platelets actually undergoes this transformation. But it is the crucial link between thrombosis and platelet mediated thrombosis and coagulation. And we now also see that it's a link to inflammation. And so these platelets really sit at this interface. And for this reason it could be very beneficial to understand this function better and maybe also target it very specifically with pharmacological inhibitors.
- [08:41]
B
Great. Dr. Morrell, could you address what we know about what shifts the location of where these megakaryocytes are making platelets? Could you maybe talk to us a little bit about anything that could stimulate lung megakaryocytes over bone marrow megakaryocyte? And then how do you deal with the fact that there are these different types of megakaryocytes making platelets, yet they're all in circulation? So how do we account for the different sources all leading to the platelets that we see in circulation?
- [09:09]
A
You know, one of the challenges right now we have to rely on our mouse models.
- [09:12]
C
Right.
- [09:12]
A
And you have to keep that in context, that we have to use those models to tease out some of the pathophysiology and then hopefully develop some markers, so then we can study this better in humans. And that's kind of where the interface we're at right now, I think. So from our studies, what we found is that a baseline. I mean, I think there's some controversy around these areas, depending on technical aspects of how you try to, you know, come up with a number of how many platelets are coming from different sources. Okay. Now, in our mind, using a model we've developed to kind of label the lung megakaryocytes with a dye, we find a baseline. They're making about 10% of the circulating platelets. Okay. That's with our methods that we've developed. What we've also found is that if you make a mouse thrombocytopenic, however, in particular, if you use a method of making the most thrombocytopenic, so you reduce the bone marrow megakaryocytes, the lung megakaryocytes then are contributing relatively more platelets in those circumstances. We've also used a mouse malaria model, which the mice become thrombocytopenic over a course of a couple weeks. Weeks. And in that model system, we also find that there's an increase in the contribution of the lung megachary to the circulating platelets. So in that way, in those kind of model systems, that the lung might be a reservoir to kind of help make that gap and to bridge that gap between the bone marrow making platelets and other sources making platelets. I mean, there's this other interesting kind of thing we found with the lung megakaryocytes that they. There's at least a subpopulation that lives a long time. Like most of the megakaryocytes in the bone marrow have about a five to seven day life of they've defined themselves now as a megakaryocyte. They make their platelets and then been engulfed because they've shed off their platelets. There's at least a 20% subpopulation in the lung that kind of hangs out for a longer period of time and perhaps prides that reservoir. You know, what gives them that ability to be long lived, we have no idea yet. We have kind of developed some better techniques to really define those. But, you know, I think that's kind of the limitations right now is having the techniques to really define the observations, and that's where we're going in the future. You know, I think the spleen's kind of similar, right. I mean, there's been some work showing that in the spleen, in the context of sepsis, is making more platelets that also express more CD40 ligand, and that might be beneficial in terms of clearing bacteria. So I think in some of these disease settings, these extramedullary sources might be more important. And perhaps that's because the platelets they're making have different immunophenotypes. So, for example, the lung megakaryocytes are very immune differentiated. And we think that's in part because the lung tissue environment is very different than the bone marrow, and it pushes their immune differentiation just like the tissue environment does any other immune cell. Right. And when, when a neutrophil or a monocyte or a macrophage are in different tissues, they get pushed to a different immune phenotype depending on that environment. And it seems like the megakaryocyte has that ability as well. You can, can take a bone marrow megakaryocyte, give it immune molecules, and it upregulates its immune differentiation as well. So I think they're on this continuum of spectrum, and that's where these extra medullary megakaryocytes might have a role in producing platelets that have some of these more immune regulatory phenotypes. And what was the evolutionary pressure that came to develop these? We can only speculate, but there's lots of potential reasons why, you know, looks like all organisms have these extra medullary megakaryocytes.
- [12:39]
B
Thank you so much. And my last question before our summary, you mentioned a lot about techniques, and Dr. Frelinger, you have developed some really wonderful techniques for being able to characterize these different subpopulations. I wonder if you could just speak briefly about what you see as the future for that and how we may actually be able to bring this to the clinic.
- [12:59]
D
The main technique that we have Explored is spectral flow cytometry or high dimensional flow cytometry, using multiple markers to characterize each individual platelet. With a panel that has 16 different probes to look at the quantity of individual markers on an individual platelet, you get a much better idea of what that platelet is all about. When we started off the work on this, we were imagining, because we saw that platelets had all these different functions, we thought that maybe like T cells, there were subsets of platelets that, that only did one job or only did the other. I think what we realize more from what we've seen with looking at platelets with these multiple markers is that the platelets can be modified by their environment and different stimuli to do different jobs. And so we don't have a clear view, or at least clear markers that distinguish platelets that only do one thing or, or another. But it seems like they do end up becoming heterogeneous as a result of their environmental stimuli. Now, the inclusion of these markers in clinical studies is an interesting question, and I'd really like to see that move forward. We're fortunate enough to be able to look at individual patients on a case by case basis for research purposes. And we saw firsthand the power of being able to look at multiple markers at the same same time to distinguish abnormalities that one couldn't see by standard aggregation techniques and other flow cytometry techniques. But getting these into the clinic is going to require a lot of effort and standardization. And at this point, the frequency of these diseases appears to be low. And I'm not sure how long it's going to take before they're actually adopted. The techniques were adopted.
- [15:03]
B
Thank you. I do have one last question and I'll leave it open to anyone to kind of jump in on. And that is, given that this seems to be environmentally based and that the platelets kind of emerge into these different subpopulations. Putting aside the idea of reticulated or young platelets based on age and also origin, I'm wondering if anyone would like to speak a little bit about how that could be a barrier to actually targeting these populations. Because, you know, in circulation then you've got various types of subpopulations and yet you don't even know that this is going to emerge into a population of pro coagulant or a population of aggregatory platelets. So how are we going to target that? I know this is a bit controversial, but how are we going to target a population that's emerging? I don't know if anyone has an answer. It's supposed to be conversational.
- [15:52]
D
I'll take a stab at it. One of the things that we've seen is that it's the strength of signaling pathways that lead to the emergence of different markers. So if there is a way to tone down one pathway versus another in a particular disease setting, then I think that would allow you to guide the platelets more towards, say, maybe an immune phenotype versus a hemostatic phenotype. But that's just speculation on my part.
- [16:23]
A
I agree, Larry. I think a lot of it will come down to understanding the drivers and then targeting the drivers versus the product, because it's going to be so hard in this whole mix of all these large number of platelets to target a particular type, but rather the drivers themselves. And I think some of this could be really interesting if we think about the ideas of making platelets in culture as well. So by understanding some of these drivers, can we make platelets in culture that are less inflammatory, more hemostatic, depending on the needs of the patients as well, which is another, you know, kind of long term area where some of these concepts can be applied.
- [17:01]
C
I also think that it can actually be beneficial that all these platelets can do many things and there may be only just some flavors, because if you think about it, the goal is to inhibit a certain function at the site where they're active. And so if you are specific enough and you just inhibit this one function that doesn't play a role, for example, in bleeding so much or in other homeostatic functions, then it may be not so important where these platelets originally came from or how their megakaryocytes looked and how they were influenced in the circulation. But you really do the targeting at the site of the disease, for example, at a ruptured coronary plaque or similar. So I think this could actually help bring this to the clinic.
- [17:45]
B
Well, thank you all for joining today. We have explored a number of different topics in this field, including how we could identify the platelets of populations, how we could discuss how they could change in location or in age based on disease or inflammatory pathways, and other things that could be going on that could be upregulating one certain subtype in exchange for another. And we've also talked about some of the controversies in this area, including how we can target those platelets, how we could use this as a technique for producing as almost like a designer type of platelet, and how we can use this as a biomarker to explore ways to actually quantify these platelets populations. In individual patients. There is much I think we need to learn about platelets. And this just gives us another idea that actually, platelets are indeed quite heterogeneous, not only in their function, but also in their characterization. Thank you all for joining today.
- [18:45]
A
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.