Prolonged platelet hyperactivity after COVID-19 infection, 2023, Nara et al.

[SNT Gatchaman]

Prolonged platelet hyperactivity after COVID-19 infection
Noriko Nara; Mie Shimizu; Masahiro Yamamoto; Tomoki Nakamizo; Azusa Hayakawa; Ken Johkura

Platelet hyperactivity often occurs in patients with coronavirus disease 2019 (COVID-19). However, it remains unclear how long platelet hyperactivity lasts after the acute phase, owing to a lack of follow-up studies. To elucidate the course of platelet hyperactivity, we serially measured platelet activity in patients with COVID-19 up to 40 days after hospital admission using an easily assessable haematology analyser that semi-quantitates platelet clumps on a scattergram.

Our results showed that platelet hyperactivity persisted for at least 40 days even after acute inflammation subsided in most patients with COVID-19, regardless of disease severity. Persistent platelet hyperactivity may contribute to thromboembolic complications in post-COVID-19 patients.

Link | Paywall (British Journal of Haematology)

 

[SNT Gatchaman]

Liberal selected quotes —

In the acute phase of COVID-19, several studies have reported platelet hyperactivity [...] the course of platelet hyperactivity after the acute phase remains unclear because of the lack of follow-up studies.

conventional measurements of platelet activity are complicated and expensive. In addition, previous methods used to study platelet activity in patients with COVID-19, such as light transmission aggregometry, flow cytometry and massive single-cell image-based profiling, require centrifugation for analysis during platelet-rich plasma preparation from citrated blood. [...] may disturb the accurate measurement of platelet activity, especially when the platelets are hyperactive, because large activated or preaggregated platelets may be excluded from the measurement.

We previously established a rapid and simple method for detecting platelet hyperactivity in whole blood using a conventional haematology analyser. [...] does not involve a platelet separation process and is suitable for routine clinical testing because it is automatic and inexpensive.

This analyser can detect platelet clumping with high specificity and sensitivity. Active platelets in peripheral blood form clumps with each other and leukocytes through surface proteins in citrated blood tubes because sodium citrate is a weaker inhibitor of platelet aggregation than EDTA-2K. EDTA blood served as a negative control.

Platelet clumps were deemed ‘positive’ when detected in citrated but not in EDTA blood.

Platelet–neutrophil aggregates, identified on the scattergram as a large size neutrophil fraction, were considered positive when the large size neutrophil fraction accounted for more than 5% of the total neutrophils.

 

[SNT Gatchaman]

prospectively included consecutive patients with COVID-19 admitted[...] between April 2021 and September 2021. Exclusion criteria were age < 18 years, use of oral anti-thrombotic drugs and pro-thrombotic states, such as any known pre-existing infectious disease, active cancer or major trauma. [...] Sixty-three patients were included

For comparative data, we referred to scattergrams created similarly for 67 healthy subjects in our previous study to avoid confounding data from asymptomatic COVID-19-infected subjects.

mean patient age was 52.6 years (15.3). Of the patients, 61.9% were male. Hypertension, dyslipidaemia and diabetes mellitus were present in 23.8%, 54.0% and 30.2% of patients respectively. The mean BMI was 25.6 (5.0).

During hospitalization, patients on high-flow nasal cannula oxygen therapy or mechanical ventilators [...] Two ventilated patients experienced venous thrombosis despite heparin therapy.

The prevalence of dyslipidaemia, diabetes and BMI were significantly higher in the infected population than in the reference population

Blood samples were collected from each patient on the day of hospital admission (Day 1) and Days 5, 10 and 40.

 

[SNT Gatchaman]

increased [CRP and IL-6] levels on Day 1 (median [IQR] was 7.0 [4.7–12.8] mg/L and 53.4 [35.3–76.4] mg/L, respectively), were decreased rapidly within a few days.

In contrast, the platelet clump appearance rate in patients with COVID-19 on Day 1 (42.6%) (compared to that in the uninfected reference group [25.3%, p = 0.10]) increased further on Day 5 (72.9%) (p < 0.01) and remained high on Days 10 (72.7%) (p < 0.01) and 40 (66.7%) (p < 0.01)

Multiple regression analysis showed that disease severity (oxygen demand >6 L/ min) was associated with the occurrence of platelet clumps in the chronic phase (coefficient = 0.41, confidence interval: 0.07– 0.75). In contrast, age, sex, BMI and comorbidities showed no significant association with chronic phase platelet clumps

Platelet–neutrophil aggregates, which did not occur in the uninfected reference group, also appeared on Day 1 (32.8%) and more frequently on Days 5 (81.4%) and 10 (83.6%). However, the appearance rate markedly decreased on Day 40 (9.5%), unlike the platelet clump appearance rate, which remained high.

 

[SNT Gatchaman]

platelet hyperactivity persisted for at least 40 days in most patients with COVID-19, even after the resolution of acute inflammation. Furthermore, this trend was also observed in mildly ill patients.

the precise mechanism underlying prolonged platelet hyperactivity remains unknown.[...] suggested that sequentially generated neutrophil extracellular traps (NETs) continuously activate platelets, our data do not support this mechanism because platelet–neutrophil aggregate formation, a key pathway in the stimulation of NET formation had already subsided by Day 40, when the platelet clump appearance rate was still high. In addition, serum IL-6, a potent stimulator of NETs activation, returned to normal.

Possible alternative pathways include the activation of vascular endothelial cells. [...] Although we have not measured markers of vascular endothelial damage, it is possible that platelets in the bloodstream are continuously stimulated and activated by friction with the damaged vascular endothelium and maintain their aggregability.

 

[EndME]

Thank you for sharing and summarising @SNT Gatchaman. Do you have details on the sample size?

 

[SNT Gatchaman]

Their prior refs are —

Simple, rapid, and automated method for detection of hyperaggregability of platelets using a hematology analyzer (2003, American Journal of Hematology)

Simple, rapid and automated method for detection of hyperaggregability of platelets in sleep apnea syndrome (2006, Journal of Thrombosis and Haemostasis)

 

[SNT Gatchaman]

Do you have details on the sample size?

63 vs 67 HC, see post #3.

 

[EndME]

63 vs 67 HC, see post #3.

Thank you! I saw the number of healthy controls but couldn't find the number of patients with Covid.

 

[SNT Gatchaman]

Although FWIW the reference for their previous HC data is Pitfall of Light Transmission Aggregometry-Based Assessment of Platelet Function in Acute Ischemic Stroke Patients (2020, Journal of Stroke and Cerebrovascular Diseases)

which said 65 HCs rather than 67.

This study included 22 patients with acute ischemic stroke (within 7 days after onset) classified as noncardioembolic and 65 healthy volunteers.

 

[Hutan]

Interesting , thank you SNT.

They say 2/3 of the post-Covid people had the platelet clumps at day 40. What percentage of the healthy controls had platelet clumps at day 40? They also say that the likelihood of having the platelet clumps at day 40 was aligned with acute disease severity (but not other things like co-morbidities).

They also say that the platelet neutrophil aggregates (which didn't occur at all in the healthy control reference group) had decreased a lot by day 40. But a rate of 9.5% for these platelet neutrophil aggregates actually is in the ball park that could be interesting to us.

It is an interesting idea that the standard blood preparation techniques might be removing the clumps and aggregates.

There is the issue of low ESRs (Erythrocyte Sedimentation Rates) possibly being more common in people with ME/CFS and yet there is also the idea of sticky, clumping blood. A low ESR means that it takes a long time for the red blood cells to settle out of blood. While acknowledging that the idea of low ESRs in ME/CFS may not have good data behind it, how do we reconcile even normal ESRs (i.e. no evidence of high ESRs being an ME/CFS feature) with a hypothesis of platelet hyperactivity or these platelet neutrophil aggregates being important? Does an ESR test involve the addition of so much anti-coagulation additive that it stops any possibility of platelet clumps or platelet neutrophil aggregates?

 

[SNT Gatchaman]

What percentage of the healthy controls had platelet clumps at day 40?

HCs (uninfected) didn't have a day 40, instead they were a historical group with a single normal baseline data set.

 

[Hutan]

HCs (uninfected) didn't have a day 40, instead they were a historical group with a single normal baseline data set.

Ah, of course. Thanks for the pictures.

I understand why the investigators used the historical group, so as to avoid confounding with asymptomatic infections, but it does increase the possibility of some confounding with different experimental techniques. It will be great to see some replication of this, especially in mild-acute Long covid cases.

Are the methods for measuring the platelet hyperactivity and neutrophil aggregates something that other researchers can easily copy?

A Japanese team.

 

[SNT Gatchaman]

Broadly I would expect yes. There's a bit of subjectivity in the assessment —

Both EDTA-2K (2 mL) and citrated blood (1.8 mL) samples were analysed by an automated haematology analyser (CELL-DYN Sapphire Hematology System, Abbott Diagnosis, Abbott Park). This analyser can detect platelet clumping with high specificity and sensitivity.

When platelet clumps were observed, they were divided into 1+, 2+ and 3+ groups according to the length of the platelet clump fraction as an indicator of platelet hyperactivity, as shown in Figure 1. Platelet–neutrophil aggregates, identified on the scattergram as a large size neutrophil fraction (Figure 1; Figures S1 and S2), were considered positive when the large size neutrophil fraction accounted for more than 5% of the total neutrophils.

Figure 1 is in my post above, S1 and S2 below. I think you can download the supplementary material, non-paywalled here.

 

[SNT Gatchaman]

Open access commentary on this paper in Br J Haematology —

Post-COVID-19 thrombotic sequelae: The potential role of persistent platelet hyperactivity (2023)

A longer follow-up, at least 12 months, detecting all possible clinical post-COVID-19 sequelae and monitoring platelet clump formation would be useful to assess the pathophysiological role of platelet hyperactivation in long COVID-19. Such study would be facilitated by the automated assay available to the authors, which is simple, inexpensive and rapid, and, as such, suitable for outpatient survey. Moreover, long COVID-19 is often an exclusion diagnosis, and diagnostic options including imaging to detect microclots, corneal microscopy to identify small fibre neuropathy, very sensitive electrocardiograms to assess cardiac injury, or biomarkers, such as the levels of EVs, are under development.

Although many key findings critical to understanding long COVID-19 have been identified, current treatment options are limited and under investigation. While personalized rehabilitation, low-dose naltrexone and intravenous immunoglobulin may help in specific long COVID-19 cases, there is increasing evidence that early prophylactic anticoagulation can be effective in improving outcomes; sulodexide is also being explored for its anticoagulant properties and low bleeding risk. More insights on the role of platelet hyperactivity in long COVID-19 would be of great help for stratifying patients at higher risk and would offer a ground to explore the efficacy of anti-aggregant therapy to prevent and/or treat postacute COVID-19 complications.