Amyloid-Fibrinogen Aggregates Microclots Predict Risks of Disseminated Intravascular Coagulation and Mortality, 2024, Schofield et al.

SNT Gatchaman

SNT Gatchaman

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Amyloid-Fibrinogen Aggregates Microclots Predict Risks of Disseminated Intravascular Coagulation and Mortality
Schofield, Jeremy; Abrams, Simon Timothy; Jenkins, Rosalind; Lane, Steven; Wang, Guozheng; Toh, Cheng-Hock

Microclots have been associated with various conditions, including post-acute sequelae of SARS-CoV2 infection. They have been postulated to be amyloid-fibrin(ogen) aggregates, but their role as a prognostic biomarker remains unclear. To examine for their possible clinical utility, blood samples were collected for the first 96 hours from critically ill patients (n=104) admitted to the intensive care unit (ICU). Detection was by staining platelet-poor plasma samples with Thioflavin T and visualized by fluorescent microscopy. Image J software was trained to identify and quantify microclots, which were detected in 44 [42.3%] patients on ICU admission but not in the remaining 60 [57.7%] or in 20 healthy controls [0.0%]. Microclots on admission to ICU were associated with a primary diagnosis of sepsis (microclots present in sepsis=23/44 [52.3%] vs microclots absent in sepsis=19/60 [31.7%], P=0.044). Multicolour immunofluorescence demonstrated that microclots consisted of amyloid-fibrinogen aggregates, which was supported by proteomic analysis.

Patients with either a high number or larger-sized microclots had a higher likelihood of developing disseminated intravascular coagulation (DIC) (OR=51.4 [95% CI=6.3-6721.1], P<0.001) and had an increased probability of 28-day mortality (OR=5.3 [95% CI=2.0-15.6], P<0.001). This study concludes that microclots, as defined by amyloid-fibrin(ogen) aggregates, are potentially useful in identifying sepsis and predicting adverse coagulopathic and clinical outcomes.

KEY POINTS

Microclots are present in increased number and size in critically ill patients compared to healthy controls and are associated with sepsis.

Microclot levels measured on admission to critical care predict risks of disseminated intravascular coagulation development and mortality.

Link | PDF (Blood Advances) [Open Access]
 
What do we know about this team @SNT Gatchaman? Is it likely that this is independent replication of the finding that microclots exist? Or is the team associated with the original proponents of the phenomenon?
 
A Liverpool Hospital team:

Image J software was trained to identify and quantify microclots, which were detected in 44 [42.3%] patients on ICU admission but not in the remaining 60 [57.7%] or in 20 healthy controls [0.0%]. Microclots on admission to ICU were associated with a primary diagnosis of sepsis (microclots present in sepsis=23/44 [52.3%] vs microclots absent in sepsis=19/60 [31.7%], P=0.044).
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So sounds as though they used software to identify the micro clots, which removes bias. The association with sepsis looks pretty weak, it's only just significant (p=0.44). But the complete absence of micro clots in healthy controls versus being present in 42% of ICU patients is interesting.


Multicolour immunofluorescence demonstrated that microclots consisted of amyloid-fibrinogen aggregates, which was supported by proteomic analysis. Patients with either a high number or larger-sized microclots had a higher likelihood of developing disseminated intravascular coagulation (DIC) (OR=51.4 [95% CI=6.3-6721.1], P<0.001) and had an increased probability of 28-day mortality (OR=5.3 [95% CI=2.0-15.6], P<0.001).
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They did protein analysis of the clots. The 'dose response' in terms of the risk of mortality and DIC looks persuasive.

Blood samples were collected for the first 96 hours (study duration) from patients (n=104) admitted to the intensive care unit (ICU) at the Royal Liverpool University Hospital between June 2009 and June 2013.
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Old ICU samples
Blood samples were collected from 10 healthy controls recruited in September 2023 (Reference Number: 16/NW/0170) and 10 historical healthy controls (Reference Number: 13/NW/0089).
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Some, but not all, of the controls were old samples collected at the same time as the ICU samples.

I have to stop reading for now. Can anyone find problems with the paper that make their findings unlikely to be true?
 
It looks as if these post-centrifugation aggregates occur in plasma from people whose coagulation systems are already partially activated in vivo. I wonder whether the aggregates are any better at telling that than standard coagulation screens.

Since disseminated intravascular coagulation is not reported very often in Long Covid presumably in that situation the aggregates are not indicative of high risk.
 
Hutan said:
A Liverpool Hospital team.
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Doug Kell (and recently Resia Pretorius as honorary professor) are both based in Liverpool, however this team seems to be new and not connected to the "original team clots".

Hutan said:
So sounds as though they used software to identify the micro clots, which removes bias.
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In their earliest publications Pretorius et al in fact used the same objective measurement system using ImageJ, but in later publications then moved away and implemented their grading system which is combination of objective+subjective, which certainly introduces a tremendous amount of bias. Might be useful to see whether this group used 2D or 3D software (my guess is it's the same 2D software Pretorius et al initially used).

Caroline Dalton at Sheffield Hallam Univeristy in her not yet published work also uses a automated device (cytation 5) capable of 3D measurements for clot detection/counting.

I wonder how much correlation there is in this study to standard coagulation markers (VWF etc).
 
Hutan said:
Is it likely that this is independent replication of the finding that microclots exist?
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There is no doubt that aggregates containing fibrin that stain with ThioT exist - you can see them in the micrographs. The question is whether or not anything similar exists in vivo or whether perhaps ThioT acts as a nucleating agent. They are not clots as normally understood.

It would be unsurprising if cases going to ITU had a higher propensity to form aggregates in vitro that correlated with DIC. I read the paper quickly and did not see any standard assays being done (or reported) for DIC - fibrin degradation products etc.

Edit
I wonder how much correlation there is in this study to standard coagulation markers (VWF etc).

Or yes, this.
 
That all makes sense - so still probably an artefact of the in vitro sample preparation, with the different prevalence in sick and healthy people due to the presence of molecules associated with infection or inflammation.

If that wasn't the case, the microclots would presumably be able to be seen directly in fresh blood. Apart from the stories of massive clots in the filtering machines of clinics offering expensive Long Covid treatment, which is something quite different, I don't think we have seen reports of microclots in fresh unprepared blood?

So the idea of the microclots clogging up capillaries is much less likely, unless there is some similar in vivo process, where a biological molecule catalyses microclot formation and the microclots stay hidden in the capillaries? Yes, I don't think we see a lot of thrombosis in ME/CFS or ME/CFS-like Long Covid. We have seen some suggestion of disintegration of microcapillaries though.
 
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Hutan said:
The association with sepsis looks pretty weak, it's only just significant (p=[0.044]).
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It's not a new idea that ME/CFS might be a chronic form of ("low grade") sepsis. Sepsis is the dysregulated immune response to a pathogen and the traditional context was acute / ICU / high mortality. The understanding of sepsis has evolved in the last decade. Sepsis can already be considered "chronic" in the ICU setting, i.e. protracted admission with high mortality rather than succumbing early on. Microthrombi in sepsis is a well-established concept. Perhaps this abnormal coagulation is a common factor between acute and chronic forms of sepsis, where these authors suggest number and size of microclots correlates with severity and mortality acutely.

Impaired energy production is a key mechanism that leads to organ failure and death in the ICU context but whether there could be other hidden ME/CFS-like symptoms is an interesting question. You're not going to be able to see orthostatic intolerance, PEM, cognitive dysfunction, sensory hypersensitivities etc in someone that's intubated/ventilated. Things like gut dysfunction are well recognised though and nutrition can be a major challenge.

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Sepsis and septic shock: endothelial molecular pathogenesis associated with vascular microthrombotic disease (2019, Thrombosis Journal)

Roles of Coagulation Abnormalities and Microthrombosis in Sepsis: Pathophysiology, Diagnosis, and Treatment (2021, Archives of Medical Research)

Endothelial Activation and Microcirculatory Disorders in Sepsis (2022, Frontiers in Medicine)

Coagulation Disorders in Sepsis and COVID-19—Two Sides of the Same Coin? A Review of Inflammation–Coagulation Crosstalk in Bacterial Sepsis and COVID-19 (2023, Journal of Clinical Medicine)

Sepsis Produces Microthrombosis and Not Coagulation: Implications for Sepsis Therapy (2020, Annals of Public Health Reports)
Sepsis and septic shock (2016, Nature Reviews Disease Primers)

Sepsis Immunometabolism: From Defining Sepsis to Understanding How Energy Production Affects Immune Response (2019, Critical Care Explorations)

Sepsis—Pathophysiology and Therapeutic Concepts (2021, Frontiers in Medicine)

Energetic dysfunction in sepsis: a narrative review (2021, Annals of Intensive Care)

Metabolic Reprogramming and Its Regulatory Mechanism in Sepsis-Mediated Inflammation (2023, Journal of Inflammation Research)
The role of the gut microbiota in sepsis (2017, The Lancet Gastroenterology & Hepatology)

The Role of Gut Microbiota in the Clinical Outcome of Septic Patients: State of the Art and Future Perspectives (2023, International Journal of Molecular Sciences)

The leaky gut and the gut microbiome in sepsis – targets in research and treatment (2023, Clinical Science)
 
Hutan said:
That all makes sense - so still probably an artefact of the in vitro sample preparation, with the different prevalence in sick and healthy people due to the presence of molecules associated with infection or inflammation.

If that wasn't the case, the microclots would presumably be able to be seen directly in fresh blood. Apart from the stories of massive clots in the filtering machines of clinics offering expensive Long Covid treatment, which is something quite different, I don't think we have seen reports of microclots in fresh unprepared blood?
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I don't think we've ever seen evaluation where the blood wasn't spun down to platelet poor- or platelet rich- plasma first. However, one aspect of the history that may be lost as we picked up once Covid and LC were in play, is that the microclots were initially seen in blood on scanning electron microscopy. It was only later (2015/16) that they tagged them with ThT to realise that they were amyloid in form. They were initially called "dense matted deposits".

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Comparisons of the fibrin networks during pregnancy, nonpregnancy and pregnancy during dysfibrinogenaemia using the scanning electron microscope (2009, Blood Coagulation & Fibrinolysis)

The changed ultrastructure of fibrin networks during use of oral contraception and hormone replacement (2010, Journal of Thrombosis and Thrombolysis)

Differences in fibrin fiber diameters in healthy individuals and thromboembolic ischemic stroke patients (2011, Blood Coagulation & Fibrinolysis)

A novel method for assessing the role of iron and its functional chelation in fibrin fibril formation: the use of scanning electron microscopy (2013, Toxicology Mechanisms and Methods)

The simultaneous occurrence of both hypercoagulability and hypofibrinolysis in blood and serum during systemic inflammation, and the roles of iron and fibrinogen (2014, Integrative Biology)
 
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