Transcription
[Music] So, uh, it's a pleasure to be here today with you and to discuss COVID-19 coagulopathy, uh, and also focus on, uh, vaccine-induced thrombotic thrombocytopenia, VITT. Um, I'm hoping this will serve as a broad-level summary of these two topics and really help to give some clinical pearls about how to maybe differentiate these two in the clinical setting and what really underlies the differences from a pathobiology perspective. So, I have no financial conflicts to disclose. And really, today, I'm hoping to start with a case, uh, just to highlight the complexity of these two presentations in the clinical setting. Then we'll review COVID-19 related coagulopathy, which is a unique viral infection associated entity, uh, how it presents and what we know so far about its management. And then we'll move into vaccine-induced immune thrombotic thrombocytopenia, which is a separate, unrelated entity to COVID-19 infection, but has been linked to adenovirus vaccine-related events. And finally, we'll summarize both of these and return to our case. Um, just a quick note that a lot of the studies we'll be discussing today in COVID-19 were published very early on during the pandemic with the initial waves, as well as with the Delta wave. And so we have seen multiple changes both in clinical presentation and thrombotic rates as we've had increasing vaccination rates and virus mutations. So, some of the epidemiology we discuss may not hold as true today, but it still highlights this unique disorder. All right, and feel free to interrupt at any time. So, we'll start with our case. We have a 52-year-old male who is presenting to hospital in the middle of the COVID-19 pandemic with hypoxic respiratory failure and an altered level of consciousness. He's initially intubated and transferred to the ICU with a CTPE confirming a pulmonary embolism. On initial CT head, there is no evidence of any acute ischemic infarcts, but there is evidence of bilateral parenchymal hemorrhaging and a question of cerebral venous sinus involvement. A follow-up MRI venogram confirmed CVST. On initial investigations, uh, you find an elevated D-dimer at 3,000 fibrinogen equivalent units, hypofibrinogenemia 0.8, and thrombocytopenia at 30. The question then is, what are your next steps for diagnosis and how would you manage this patient?
All right, so just to briefly review COVID-19 itself, as I'm sure many of you know, it's caused by the severe acute respiratory syndrome virus, or SARS-CoV-2. And it primarily manifests as a respiratory disease, but early on in the pandemic, what we really saw was a high degree of thrombotic events in these patients who presented to hospital, particularly both venous and arterial origin, which is quite unique. This included DVT and PE, but also evidence of myocardial infarction, stroke, and even limb ischemia. And so, what we know is that the SARS-CoV-2 virus generally enters the respiratory tract and is able to bind to the ACE receptor on alveolar epithelial cells. In patients who have a milder infection, generally, you have a very limited immune response, and so your alveolar macrophages and your resident dendritic cells are able to secrete antiviral cytokines such as type 1 and type 3 interferon, which really limits the virus's replication and activation. And although we have mild activation of macrophages and neutrophils, it's a very subdued response compared to what we see in critically ill COVID-19 patients. What we learned very early on in the COVID-19 pandemic was that this excess immune response was really what appeared to trigger such an acute presentation. And so, instead of having a very well-regulated, uh, response, you have excess neutrophil activation leading to NETosis and formation of reactive oxygen species. You have exocytokine secretion leading to what's called a cytokine storm, and this really serves not only to cause intense immune cell activation but also damage to surrounding tissue. And so, we saw that with fibrin deposition and destruction of the alveolar walls, as well as activation of endothelial cells and other immune cells such as neutrophils and platelets. And so, all this to say, that's what we noticed was that, uh, there was quite an increase in the events of venous thrombotic events in these critically ill patients. The studies were quite variable in, uh, the exact incidence, but it ranged anywhere from 20 to 65% of critically ill patients having evidence of a VTE. Generally, the studies that showed a higher incidence rate were those that employed screening techniques like a screening ultrasound, which we know is not generally advised in the clinical setting. There was a meta-analysis including 66 studies that showed an estimate of about 14% VTE. And we know that from general hospitalized internal medicine patients, the rate of thrombotic events is about 0.5%. So, this is quite an, uh, increase in incidence, uh, with COVID-19 patients. What we also saw was that a lot of these patients had arterial events, about 3 to 4%, uh, and generally this occurred in those with additional comorbidities, such as underlying cardiovascular disease or metabolic abnormalities. And this is just a nice figure, uh, from a study in The Lancet looking at various databases in Europe, and this is from, I believe, a Spanish database of hospitalized patients. And what we see is that not only is there this increased incidence of VTE and arterial thrombosis, but it's very, uh, related to age. And so, in males and females over 65, this represents the highest risk group for having such events, and of course, associated mortality with that. The evidence for pro-thrombotic phenotype was also seen clinically in these patients. So, when they presented, they often had an elevated D-dimer in hospital, generally greater than two times the upper limit of normal. And many studies actually showed that you could risk-stratify patients based on the degree of D-dimer elevation, and this predicted a poor prognosis. The higher the degree of elevation, we also saw that these patients had increased von Willebrand factor circulating, as well as increased fibrinogen, again suggesting that hyperinflammatory state. Many also presented with a mild thrombocytopenia, reduced ADAMTS13 levels, as well as reduced prothrombin and antithrombin. And all together, these really pointed to a pro-thrombotic disorder related to the COVID-19 infection that goes above and beyond any regular viral infection. And so, the three main, uh, mechanisms that we understand today related to COVID-19 coagulopathy include this hyperinflammation, endothelial cell activation, and platelet cell activation. And so, we'll go, uh, through just a few of these, uh, today. So, starting with the hyperinflammation.
[Music] Uh, one component of the immune system is obviously, uh, neutrophils, who are generally first-line responders to any pathogen infection. And neutrophils are quite unique in that they can undergo a process called NETosis, which stands for neutrophil extracellular trap formation. This is a very regulated form of apoptosis that involves decondensed chromatin and histones that are released from the neutrophil. And they not only serve to physically trap microbes and pathogens, but also contain bactericidal and other antimicrobial proteins on their surface. Unfortunately, this can contribute to thrombosis both through activating platelets, and some studies have even suggested releasing tissue factor during this process. And so, again, early studies in hospitalized patients, this is a cohort of 33 COVID-19 patients, and what the researchers did is they looked at the formation of myeloperoxidase DNA complexes in circulation, which are a marker of NETosis. And in those who were admitted to hospital, there was a significant increase in the circulating marker compared to healthy controls. Um, and particularly those who were admitted to the ICU or required intubation. Uh, this was not seen in convalescent COVID-19 patients, suggesting that it's the acute phase of the reaction. And they also looked at the level of circulating platelet and neutrophil complexes, because again, these NETs can serve to activate and attract platelets. And again, they found that in the hospitalized COVID-19 patients, there was a significant increase in these aggregates. They confirmed that on, uh, three patients in autopsy, there was evidence of pulmonary microthrombies, while tying this link that neutrophils can activate to form these microclots in circulation. And these, uh, findings were all confirmed in a subsequent study of 50 hospitalized patients, again, in the early waves of the pandemic. So, we have not really seen newer studies investigating this. Another key component of the innate immune system is the complement system. And so, this is an evolutionarily conserved, [Music] that relies on protein activation to eventually form what's known as a MAC attack complex that's able to either opsonize pathogens or directly cause lysis of the pathogen. And there are three main ways that we can activate the complement pathway. Regularly, one is the classical immune pathway. So, if you can see here, it's primarily triggered by immune complex activation and C1q protein. There's a lectin pathway, which involves mannose-binding lectin. And both of these pathways ultimately lead to activation of what's known as complement 3 or C3 protein, and this triggers the rest of the cascade, ultimately leading to C5 to C9. And lastly, we have the alternative pathway. And this is an interesting pathway in that it's a spontaneous hydrolysis of the C3 protein leading to formation of its components. And generally, this is regulated by inhibitory factors such as Factor H that prevent this from occurring in the absence of an inflammatory signal. And as you can likely imagine, COVID has been shown to activate all three of these pathways in various studies, again, supporting that hyperinflammatory state. And so, in a small cohort of COVID-19 patients, it was shown that circulating levels of the C4d protein, or complement 4D, was elevated compared to controls. And this is in relation to the classical pathway, suggesting that spike-specific antibodies, uh, may activate the classical pathway of complement. Again, that's fairly indirect evidence, but it does raise a question as to whether spike, uh, dependent antibodies can have an immune, uh, hyperactivation role. The SARS-CoV-2 virus can also directly bind to the mannose-binding lectin through its proteins to activate the lectin-based pathway. And finally, the virus can also bind to heparin sulfate that's exposed on cells, and in doing so, it can inhibit Factor H, which is an inhibitor of complement. And so, through the double negative, it's actually promoting activation of the alternative complement pathway. This is a very complex figure, but, uh, really, if you can see, there are many different, uh, therapeutics that have been developed to inhibit various aspects of the complement cascade, including complement-directed antibodies. There have been several clinical trials, generally small and underpowered, that have failed to really show any clinical benefit, but it would really take a larger base trial to, I think, investigate for any true effect. As you can see here, the complement cascade can also ultimately lead to formation of NETosis, as we've discussed previously, and platelet activation, which is how it's related to the formation of thrombi in COVID-19.
So, the next pathway we'll discuss is endothelial cell activation, and we'll just begin with a brief review of general endothelial cell physiology. Um, and so, as you know, uh, endothelial cells generally regulate vascular tone and permeability as their main role, but they've also been shown to regulate megakaryocyte development in the bone marrow, as well as platelet cell maturation through various, um, factor signaling and trophogens. They're also the main synthesizers and, uh, regulators of von Willebrand factor in the body, and have also been implicated in the immune system via toll-like receptors. So, the role for endothelial cells was quite prominent again early in the pandemic when it was shown that there was increased von Willebrand factor expressed in these patients. And on autopsy, what we could see is that when you look in the lung and the heart, in control, this is von Willebrand factor staining in pink, there's generally baseline staining present in endothelial cells, but with COVID-19 infection, you have a marked increase in the staining and release of this factor, suggesting marked endothelial cell activation. And just to briefly review our von Willebrand factor physiology, as we know, von Willebrand factor is housed in endothelial cells as an ultra-large multimer. Once it's released, it's often quickly cleaved by ADAMTS13 to prevent its pro-thrombotic, uh, state to activate platelets and cause aggregation. But if uncleaved, it's quite potent in, um, triggering this thrombus formation and associated coagulopathy. We've seen this in TTP. And interestingly, several studies have shown that in COVID-19 patients, not only do you have increased, uh, multimers of von Willebrand factor, but you also have a moderately reduced level of ADAMTS13 activity. It's generally not as severe as what we would see in a TTP case where it's less than 10%, but generally hovering around the 30 to 50 mark, suggesting there's some reduction with COVID-19 infection. How that happens, it's not quite sure, but I'm sure there are studies underway currently investigating that. And lastly, we'll focus on platelet cell activation, uh, because I've seen quite a number of studies published in this regard for COVID-19. And again, platelets are not only related to thrombosis, but also have been shown to have various immune roles, both through secreting inflammatory cytokines like interleukin-1 beta, but also through opsonization of bacteria and various pathogens. And really, this is mediated through their, uh, cell surface receptors. And the one we'll be focusing on today will be the Fc gamma R2A receptor, which is known to bind to immunoglobulins of the IgG family. And so, uh, this was a large study of over 100 COVID-19 patients, again, during the initial wave of the pandemic. And what it showed was that, uh, platelets from COVID-19 patients secreted increased levels of inflammatory cytokines and soluble CD40 ligand when, uh, triggered with stimulus such as thrombin. As you can see here, other studies confirmed that markers of platelet activation such as P-selectin and CD63 were also significantly elevated in critically ill patients. And when genetic studies were done to investigate transcriptional changes, uh, there was evidence of increased, uh, markers of platelet cell activation as well, really highlighting that COVID-19 triggers this pro-inflammatory, uh, hyper-activated platelet phenotype. How exactly this occurs is not quite clear. Some studies have hypothesized that there is potentially direct viral infection of the platelets, but this has not been proven yet in vitro. But another interesting concept is the potential of antibody-mediated platelet cell activation. And so, as we discussed, the Fc gamma receptor 2A on platelet surfaces is able to bind to various immunoglobulins, and through this can activate the platelets into a pro-thrombotic phenotype. And so, uh, when you take serum from critically ill COVID-19 patients, it's able to increase platelet apoptosis in vitro through activation, and this is mediated through this Fc receptor 2A. When we add an inhibitor such as 4-IgG, this reaction is blocked, thus confirming it's IgG related. Similarly, if you take the IgG fraction alone from COVID-19 patients, uh, there is some evidence that this can promote thrombus formation in animal models. And so, what is this antibody that's able to activate platelets? Again, it's not quite clear. Some have hypothesized that antiphospholipid antibodies may be the culprit. The initial reports from COVID-19 patients showed that up to 50% could have, uh, evidence of an antiphospholipid antibody, either lupus anticoagulant or beta-2 glycoprotein or cardiolipin. However, we know that viral infections can commonly trigger transient APLAs, and it's not clear if these are truly functional. In one study, uh, published in Science, two years ago, they looked and found that in their patient samples from COVID-19, if they took the IgG fraction and differentiated their cohort between those with high titer and low titer antiphospholipid antibodies, there was a slight trend towards increased thrombus formation when the serum was injected into a mouse model compared to those with a lower titer. And interestingly, the degree of thrombus formation was higher compared to catastrophic antiphospholipid antibody patients, and certainly higher than their control patients. That being said, the IgG fraction is not specific for antiphospholipid antibodies, and so any, uh, other specific antibody could be related to this reaction, including the spike-dependent COVID-19 antibodies. So, it's quite unclear what antibody is the culprit, but it does highlight that something in the immunoglobulin fraction is contributing to this platelet cell activation.
And so, in terms of management for COVID-19 coagulopathy, there have not been, uh, many targeted therapies developed. Generally, it involves supportive management for the COVID-19 infection itself, and really targeting that hyperinflammatory state with dexamethasone, remdesivir, tocilizumab, and many of the other valid therapies that we have. There were, uh, several large studies investigating therapeutic anticoagulation with heparin in equivalent teen patients. These were published in the New England Journal last year, and what they showed was that in, uh, hospitalized patients who were non-critically ill, there was potentially some benefit from therapeutic anticoagulation, and that it reduced or it started increasing survival from hospital with organ-free support days. However, in critically ill COVID-19 patients, there was a trend towards increased bleeding and no evidence of benefit. And so, this suggests that increasing from prophylactic dose anticoagulation to intermediate or therapeutic might have benefit in those with a milder COVID-19 infection. However, there's not really taken a great clinical hold again, secondary to mutations in the virus and increased vaccination internationally, uh, it's unclear whether this is still beneficial in these mild COVID-19 cases. So, that's currently our understanding of COVID-19 coagulopathy. Just to summarize, the highest risk patients appear to be those who are critically ill, admitted to the ICU, or requiring organ support, and we can risk-stratify them based on their age and their D-dimer level. The three main mechanisms appear to be hyperinflammation from NETosis and neutrophil activation, as well as complement endothelial cell activation involving the von Willebrand factor and ADAMTS13 axis, and finally, platelet cell activation, including platelets and a potential immune complex-mediated activation. Management is generally supportive with COVID-19 specific treatments and potentially a role for therapeutic anticoagulation empirically in non-critically ill patients. This is a nice figure by Conway Dal that was recently published in Nature Reviews, and it really just highlights all of these factors from the initial viral infection to how it elicits a hyperactive innate immune response, as well as the relationship to thrombosis and endothelial cell activation. So, I'd highly recommend that review if interested. And before we finish off this topic, I just wanted to discuss a proposed definition of COVID-19 coagulopathy. Again, it's a very non-specific, uh, pro-thrombotic state, so there's no one diagnostic test. But this was, uh, an initial proposed definition by Iba et al during the initial waves of COVID-19, and really looking at these factors such as thrombocytopenia, elevated D-dimer, and evidence of macro/microthrombosis, and using this in conjunction with other findings such as fibrinogen and increased von Willebrand factor to really confirm the diagnosis of coagulopathy.
Okay, so now, if there's any questions or comments, please feel free to interrupt, but we'll move on to vaccine-induced immune thrombotic thrombocytopenia, VITT. And this is a separate prothrombotic disorder, so it's unrelated to COVID-19 infection, um, but is associated with COVID-19 vaccination. And so, as many of you know, I'm sure, uh, this was discovered initially during the mass vaccination campaigns with, uh, adenovirus COVID-19 vaccines, particularly AstraZeneca and Johnson & Johnson. And it's a very interesting presentation, as it initially presented to hospital with severe thrombocytopenia, catastrophic thrombotic events, and very much like a heparin-induced thrombocytopenia, or HIT-type reaction. And so, uh, really, that's what facilitated our understanding of it and what's led to our quick understanding of diagnosis and management. This is a nice figure summary here showing an adenovirus vaccine, and when a patient is exposed to this, for reasons that are still being understood, uh, certain patients develop a pro-thrombotic antibody against platelet factor 4. This can then activate platelets and lead to this pro-coagulant state that we'll discuss shortly. And so, just to quickly differentiate, VITT is really on a spectrum of what we might call anti-PF4 syndromes, which include other conditions such as heparin-induced thrombocytopenia, or HIT, autoimmune HIT, and spontaneous HIT. The key, uh, feature between all these conditions is that they present with thrombosis and thrombocytopenia, often concurrently, but sometimes with delayed onset. And one way to help differentiate these is that they have unique presenting features. And so, autoimmune HIT, unlike classic HIT, may have a more prolonged course. It may be associated with fondaparinux as opposed to a heparin product, and also may have a more severe DIC-type picture. Spontaneously, in VITT, are unique in that they can present in unusual sites of thrombosis, including splenic and CVST. And the patient population is also a key differentiating factor between these disorders. So, as we've discussed, VITT is commonly seen post-adenoviral vaccination. Spontaneous HIT is uniquely associated with, uh, total knee arthroplasty and orthopedic surgery, as well as potentially viral infection. And then HIT is also associated generally post-cardiac surgery or with exposure to unfractionated IV heparin. And autoimmune HIT is really very variable, and so it should always be on the back of your mind when you have, uh, this type of presentation in a clinical patient. The culprit, uh, is platelet factor 4, as we've discussed. This is the antigen target for all of these syndromes. As you know, it's a chemokine that's housed in platelets in the alpha granules and is secreted with activation, as well as, uh, by stimuli such as surgery and infection. And its exact roles are unclear, but it appears to have roles to inhibit platelet cell maturation, as well as angiogenesis. This is a very nice figure from a New England Journal paper by Greinacher many years ago, really showing how when you have bacteria or potentially some infection or a surgical trigger, you can have this platelet factor 4 release. And in the body, it can bind to naturally forming heparin complexes or to exogenously administered heparin, and this can lead to the antigen complex formation required for an anti-PF4 syndrome. So, again, that pathophysiology is primarily based on platelet-activating antibodies, and these are able to bind platelet factor 4, either by itself or when it's complexed to heparin or some other scaffold. And once these pathogenic antibodies bind, they're able to form complexes leading to platelet cell activation and activation through that Fc gamma receptor 2A. The unique thing about VITT, as opposed to the other anti-PF4 syndromes, is that it does not require heparin at all for its function. And this is a really elegant study, uh, published actually from McMaster when at all in Nature. And this is the PF4 molecule, as you can see. And what they did was they used a technique called alanine scanning mutagenesis, where they mutated subsequent amino acids in the platelet factor 4 molecule to identify where exactly the pathogenic antibodies bind to. And so, we can see here in classic HIT samples, what you have is, uh, binding to this blue highlighted region on the PF4 molecule, and this is a HIT-dependent region. And normally, what you have is heparin will bind to this purple region here, and that will facilitate antibody binding in this blue region. Interestingly, some HIT patients who may present with more autoimmune-type features or spontaneous HIT will also have binding of antibodies directly into this heparin groove. And so, these heparin groove binding antibodies will actually compete with heparin for this binding site, and so do not need heparin in order to activate platelets. And interestingly, that's exactly where antibodies seem to bind to. They bind directly into this heparin binding groove on the platelet factor 4 molecule. And so, in the presence of heparin, they actually compete for this binding, and so are inhibited by heparin, as opposed to facilitated by it. As with any anti-PF4 syndrome, there are two diagnostic questions to answer: one, is there an antibody present, and two, does it activate platelets? And so, there are several, uh, tests for, uh, antibodies that we use. One are the enzyme immunoassays. And as we know, they generally function by having, uh, patient antibodies bind to a predefined antigen target such as, uh, platelet factor 4 conjugated with, uh, polyvinyl sulfate or heparin. Once the patient serum is added with the HIT antibodies, a secondary antibody, either with a fluorescent tag or a chemical tag, is added, and then the absorbance is directly measured, which is proportional to the degree of pathogenic antibody in these samples. The other test that we have, uh, is are the rapid assays. And so, these function somewhat differently. Um, what you have generally, uh, is again, your platelet factor 4 and heparin complex. And what you'll have in this example is a latex particle coated with the antibody. And so, once the latex particle is present with the antigen, they will agglutinate and have increased absorbance, being to reduce the light transmission. Subsequently, if you add a patient serum or plasma with a HIT antibody, it will compete with the latex particle for that platelet factor 4 complex. And in doing so, it will inhibit the aggregation and allow increased light transmission. And so, the degree of light transmission is directly proportional to the degree of, uh, HIT antibody in the patient plasma. Interestingly, several studies have looked at comparing rapid assays to enzyme immunoassays in VITT, and what they've shown is that rapid assays are quite poor in identifying anti-PF4 antibodies. Um, so generally, the sensitivity is much less than 50%, and some are even lower than 10%. And so, that's really led to understanding that rapid assays should not be used in the context of VITT setting. So, if you have a patient who's suspected of VITT, particularly, it's important to speak with your lab to understand what type of assay you use and whether an enzyme immunoassay or a more sensitive assay is available. Generally, once, uh, you've identified an antibody, if the clinical suspicion is high enough, there's often no need to proceed to functional testing. But if there's any, uh, question or for completeness' sake, you can proceed to a functional platelet assay. And the one we'll discuss here is a serotonin release assay, which is a gold standard. And the way this works is, uh, platelet-rich plasma, generally from healthy donors, is incubated with a radioactive label such as serotonin. These platelets are then added with heat-inactivated patient serum or plasma that contains a pathogenic antibody, and then, uh, platelet activation and release of that radiolabeled serotonin is done in the absence and presence of heparin. In the case of VITT, we've modified the assay to use platelet factor 4, because we know that that is actually the pro-thrombotic antigen in that condition. And generally, what you'll see in VITT patients, so this is the platelet activation, the percent release of serotonin, the cutoff is generally 20% for this assay. And so, in a VITT patient, when you have buffer and no other stimulus, there may be some degree of activation, but nothing, uh, too remarkable. Heparin will actually inhibit the VITT reaction again, because it competes for that binding site with the antibody, whereas platelet factor 4 will have a marked activation in the serotonin release assay.
Okay, and so, one of the key aspects of VITT is that anticoagulation is mandatory. And this is true of all the anti-PF4 syndromes because they are so highly pro-thrombotic. Anticoagulation is required to prevent any thrombosis, even if the patient presents with thrombocytopenia initially. The approved ones generally are argatroban, has been approved for HIT. Fondaparinux, uh, it has a very low rate of cross-reactivity in about one to two percent, I believe. Um, and so it's often still used if there's no suspicion of autoimmune HIT, and does have increasingly been shown to have benefit in HIT, even in the acute phase, although the evidence is still accumulating for VITT. And so, generally, these are not as favored so far. The important thing to note about anticoagulation is that warfarin is contraindicated in the acute, uh, phase of VITT and any of the anti-PF4 syndromes. And the reason for this is that it can precipitate a DIC and pro-coagulant phenotype due to the imbalance of your protein C and S with your other coagulation factors. And so, although it can be used chronically after the acute phase of an anti-PF4 syndrome or long-term maintenance, it is generally contraindicated acutely. And the second prong for management of VITT, along with anticoagulation, is really immunosuppression. Uh, because we know that it is an antibody-mediated process, uh, steroids to help dampen that, uh, antibody production, uh, is necessary. Um, and if contraindicated, then intravenous immunoglobulin is recommended. There are multiple case reports and studies from autoimmune HIT that have shown IVIG can, uh, improve the degree of platelet count much more rapidly than anticoagulation alone. And because if it is such a severe reaction, IVIG is strongly recommended and has been shown to be beneficial. The mechanism is thought to be secondary to inhibiting that immune complex activation through the Fc receptor, but also potentially through downregulation of B cells and that antibody production.
All right, so just to summarize, VITT is an anti-PF4 syndrome. It's a highly pro-thrombotic disorder. It's uniquely been associated with adenoviral vaccination, particularly with COVID-19. Diagnostic testing should include anti-PF4 enzyme immunoassays and should not include a rapid assay, and platelet activation testing is also recommended but not necessary. Management must include anticoagulation and immunosuppression with potential IVIG as necessary. And so, back to our case. So, we, as you recall, had a 52-year-old who had two thrombotic events identified, a CTPE as well as, uh, CVST. Interestingly, he was found to have a hemorrhaging in the brain as well. Um, and he had evidence of a significant coagulopathy, uh, on his initial investigations. Oh, interesting. Um, and so, the question is, how, what would you do for diagnosis and management? So, I don't know if anyone would like to comment or share before we, we answer the case. I don't think we've had any comments as yet, Stefan, so perfect. So, um, generally, this, this patient, obviously, it's hard to tell at this point, but given the atypical presence of a CVST and multiple thrombi and severe thrombocytopenia, that's generally not what we see with COVID-19 related coagulopathy. It's usually milder thrombocytopenia unless they've gone into a DIC. And so, an antiphospholipid syndrome would probably be at the top of the differential. Um, and so, uh, on additional history, actually, you uncovered that they had received the AstraZeneca vaccine about three weeks ago. Their COVID-19 test is negative, and when you send for an anti-PF4 immunoassay, the optical density comes back at three, which is quite high, and there's evidence of strong platelet activation in the PF4-enhanced SRA. So, this is a case of VITT. A key point, oh, sorry, with the VITT, although this patient does have hemorrhaging, parenchymal hemorrhage as shown in his CT, the reason is likely because of venous congestion from the cerebral venous sinus thrombosis. And so, even in these patients, if, uh, VITT or an anti-PF4 syndrome is confirmed, anticoagulation is still recommended because it will actually improve that venous congestion and improve that flow, uh, to actually help limit the bleeding. But obviously, they should be in a monitored setting to make sure there's no neurologic deterioration. [Music]