Plasma from patients with pulmonary embolism show aggregates that reduce after anticoagulation, 2023, Baker et al.

[SNT Gatchaman]

Plasma from patients with pulmonary embolism show aggregates that reduce after anticoagulation
Baker, Stephen R.; Halliday, Georgia; Ząbczyk, Michal; Alkarithi, Ghadir; Macrae, Fraser L.; Undas, Anetta; Hunt, Beverley J.; Ariëns, Robert A. S.

Background
Microclots, a term also used for amyloid fibrin(ogen) particles and henceforth named aggregates, have recently been reported in the plasma of patients with COVID-19 and long COVID. These aggregates have been implicated in the thrombotic complications of these diseases.

Methods
Plasma samples from 35 patients with acute pulmonary embolism were collected and analysed by laser scanning confocal microscopy and scanning electron microscopy before and after clotting.

Results
Here we confirm the presence of aggregates and show that they also occur in the plasma of patients with pulmonary embolism, both before and after clotting. Aggregates vary in size and consist of fibrin and platelets. We show that treatment with low-molecular weight heparin reduces aggregates in the samples of patients with pulmonary embolism. Double centrifugation of plasma does not eliminate the aggregates.

Conclusions
These data corroborate the existence of microclots or aggregates in diseases associated with venous thromboembolism. Important questions are raised regarding their pathophysiological relevance and further studies are warranted to investigate whether they represent cause or consequence of clinical thrombosis.

Plain Language Summary
When blood turns from liquid to solid, a protein called fibrin and cells called platelets aggregate to form a blood clot. Small aggregates have been found in the blood of people with COVID-19 and long COVID. Here, we show that small aggregates also occur in the blood of patients with pulmonary embolism, a disorder in which blood clots are trapped in an artery in the lung, preventing blood flow. We confirm that aggregates consist of fibrin and platelets, and show that the number of aggregates is lower when patients are treated with blood thinning drugs. These results suggest other disorders of the blood should also be investigated to see whether aggregates are present and whether they have an impact on the outcome for the patient. This could help us understand the cause of diseases associated with blood clotting, which might offer new approaches for diagnosis and treatment.

Link | PDF (Nature Communications Medicine)

 

[SNT Gatchaman]

We collected plasma samples (pre-pandemic; 2018) from 35 patients with acute PE (aged 63.1 ± 15.8 years, 34% male) on admission to hospital and after 24 h of treatment with enoxaparin, a low-molecular weight heparin (LMWH) at a dose of 1.0 mg/kg s.c. every 12 h. Patients with known cancer, sepsis, autoimmune diseases, hypotension and pregnant women were excluded.

Venous blood was drawn without stasis onto 3.2% sodium citrate tubes (1 part of citrate to 9 parts of blood [...]), and blood samples were centrifuged at 2500×g for 10 min within 30 min to prepare platelet-poor plasma. In a follow-up study, plasma was taken from a patient with PE prior to treatment with LMWH, which was subsequently centrifuged twice at 2500 g for 10 min each time. All plasma samples were aliquoted, immediately frozen, stored at −80 °C, and shipped to the laboratory in Leeds on dry ice. Plasma samples were thawed in a 37 °C water bath for 10 min.

 

[SNT Gatchaman]

For laser scanning confocal microscopy, plasma was diluted 1/6 in tris-buffered saline, mixed with 50 µ g/ml AlexaFluor488-labelled human fibrinogen, 0.1 U/ ml human alpha thrombin and 10 mM CaCl 2 (final concentrations), and loaded into the channel of an uncoated µ -slide. Plasma clots were allowed to form for 2 h in dark humidity chambers followed by imaging using a Zeiss LSM880 with 40× and 63× oil immersion objectives. To determine if the aggregates contained cell-membrane or other hydrophobic structures, DiOC6(3) (3,3′Dihexyloxacarbocyanine Iodide, 1.2 µ g/ml) was added prior to the addition of thrombin. To determine if aggregates were present prior to clotting, the same protocol was followed without adding thrombin.

Fig. 1 Laser scanning confocal microscopy images of aggregates in plasma and plasma clots from patients with PE. Presence of aggregates in PE patient plasma prior to clot initiation with thrombin (a–c). Representative images of aggregates in PE patient plasma samples following the addition of thrombin showing platelet rich (d–f) and platelet poor (g, h) aggregates. Alexa Fluor 488 labelled fibrinogen is shown green (a, d, g) and DiOC6(3) staining is shown in red (b, e, h). An overlay of the two channels is also shown (c, f, i). Scale bars are 50 µ m.

 

[SNT Gatchaman]

Fig. 2 High-resolution scanning electron micrographs of an aggregate in a PE patient plasma clot. The presence of aggregates was observed in clots using SEM (a). Small fibres can be seen within the aggregate (white arrows; b). Scale bars are 5 µ m (a) and 2 µ m (b) respectively.

 

[SNT Gatchaman]

Aggregates were found in 8 (23%) out of 35 plasma clots from patients with acute PE. There were no differences in demographic and clinical variables, along with plasma fibrinogen, when the 8 patients were compared with the remainder. Strikingly, the presence of aggregates was substantially reduced in patients with acute PE receiving therapeutic-dose LMWH. After treatment, aggregates were found in only 1 (3%) out of 35 plasma clots.

When we first observed aggregates in plasma from patients with acute PE, we were sceptical about our finding. We questioned whether aggregates were an artefact of blood sampling or plasma preparation procedures. However, our phlebotomists and laboratory staff preparing the samples are highly skilled, and aggregates remained present after double centrifugation of the plasma.

Other key questions for future studies are elicited by the size of the aggregates. If the aggregates were present in the blood before the draw, why do they not precipitate into the pellet alongside the red blood cells (~50 µm2 surface area) and platelets (~3–5 µm2 ) during centrifugation for plasma preparation? Are they smaller to begin with, and then grow in the plasma or the clotting sample, acting as a nucleus for fibrin deposition?

Or are they just much less dense?

If present in the blood, any aggregate >50 µm2 may in principle block small arterioles in tissues like the lung for example.

From Persistent 129Xe MRI Pulmonary and CT Vascular Abnormalities in Symptomatic Individuals with Post-acute COVID-19 Syndrome (2022, Radiology) —

RBC:TP is a composite of the ratio of two tissue volumes (the pulmonary capillary (plus potentially some pulmonary venous) blood volume to the alveolar membrane volume), gas transfer, and pulmonary blood flow - measured using Hp-XeMRI. A lower figure suggesting that infection with Sars-CoV-2 may have induced some microstructural abnormality to either one or both volumes, causing a reduction in blood volume for example due to widespread microclots, changes in pulmonary blood flow, and/or a thickening of the alveolar membrane, both of which would be expected to cause a reduction in diffusing capacity.

 

[Michelle]

"Patients with known cancer, sepsis, autoimmune diseases, hypotension and pregnant women were excluded."

So I take it these were patients with provoked PE, like surgery or air travel or something? Or did they include patients with unprovoked PE? Would that matter?

 

[EndME]

I still feel like this discussion is largely going in circles and all the research doesn't add too much to the discussion. Pretorius et al. have been studying these "microclots" way before Covid came around and argue that these Long-Covid microclots are somehow slightly different (resistant to single trypsinisation, entrap inflammatory molecules), without being able to provide in vivo proof or some standardised unbiased detection method with controls to show this.

In either case studies such as these are rather a confirmation of Long-Covid being a somatic condition than the opposite.