Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19, 2023, Aggarwal et al.

SNT Gatchaman · Feb 9, 2024

Link to post with the abstract of the final published paper here

Preprint
Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19
Anu Aggarwal; Tamanna K. Singh; Michael Pham; Matthew Godwin; Rui Chen; Thomas M. McIntyre; Alliefair Scalise; Mina K. Chung; Courtney Jennings; Mariya Ali; Hiijun Park; Kristin Englund; Alok A. Khorana; Lars G. Svensson; Samir Kapadia; Keith R. McCrae; Scott J. Cameron

Background
Post-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC.

Aims
Our objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC.

Methods
PASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions.

Results
A mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age-and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets – a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals.

Conclusions
PASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

Link | PDF (Preprint: BioRxiv)

SNT Gatchaman · Dec 4, 2023

The most significant finding in our study is that platelet-deplete plasma in PASC patients ‘primes’ and markedly activates healthy, washed platelets through every receptor signaling pathway evaluated. We did not see this when platelet-deplete plasma from healthy controls was incubated with washed platelets before agonist stimulation. The hyper-reactive platelet effect caused by PASC plasma persisted even after heating plasma to 55°C suggesting the complement cascade or circulating antibodies are likely not responsible for augmented platelet activation. Using an antibody array, we could not find antibodies directed against platelet protein targets of coagulation proteins in the blood that could explain the observation in patients with PASC. Therefore, we offer the suggestion that a putative pro-thrombotic factor exists in the blood of patients with PASC, and its presence leads to an adaptive response in the host in which platelet reactivity and thrombosis are paradoxically tipped in the opposite direction, presumably as a protective response from thrombosis.
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In summary, patients who have long recovered from acute SARS-CoV-2 but with persistent symptoms under the constellation of PASC show evidence of dysregulated platelet reactivity, alterations in normal coagulation of platelet-deplete plasma, and diminished clotting in whole blood ex vivo under shear stress conditions. These events are likely a consequence of adapting to a platelet-activating factor in plasma of patients with PASC. The stimulus behind the exhausted platelet phenomenon, particularly through GPVI, in PASC patients, should be investigated and identification of the putative pro-thrombotic mediator should be prioritized given the implications for patients with PASC being considered as donors of blood and plasma products in future.
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SNT Gatchaman · Dec 4, 2023

When platelet-deplete plasma from healthy individuals or PASC patients was added to healthy, twice washed platelets, there was a surprisingly profound activation of platelets following stimulation with all platelet receptor agonists tested caused by PASC patient plasma incubation.
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SNT Gatchaman · Dec 4, 2023

Given that immunothrombosis is common in COVID-19 and the complement system likely contributes to these responses, we heated and inactivated plasma from PASC donors at 55˚C and repeated the experiments. We observed that platelets were persistently activated if spiked with room temperature plasma or heat-inactivated plasma prior to agonist stimulation from PASC patients suggesting that complement activation is unlikely to be a driver in the augmented platelet response observed.
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SNT Gatchaman · Dec 4, 2023

Platelet aggregation in PRP [platelet-rich plasma] was evaluated by LTA [Light Transmission Aggregometry] after stimulating platelets with surface receptor agonists for the P2Y 12 receptor (ADP), the thromboxane receptor (U46619), the GPVI receptor (CRP), and PAR1 (TRAP-6). Overall, platelet aggregation by LTA in PASC was similar to healthy individuals except through the thromboxane receptor which demonstrated increased reactivity following stimulation with the agonist U46619 (87%±3 for PASC vs. 72%±4 for healthy subjects, p=0.03).
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To distinguish platelet function that may be altered by mediators in PRP, these studies were repeated in a dose response manner after isolating and washing platelets twice, then assessing platelet reactivity by alpha granule exocytosis by appearance of P-selectin on the surface of the platelet as reported previously. When washed platelets from healthy volunteers and PASC patients were stimulated with platelet surface receptor agonists, we found similar reactivity to healthy individuals through the following receptors: P2Y12 , thromboxane, PAR 1. Curiously, platelet reactivity through GPVI was markedly decreased in PASC patients compared with healthy controls (Fig. 3D).
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SNT Gatchaman · Feb 9, 2024

Now published in Vascular Medicine —

Dysregulated platelet function in patients with postacute sequelae of COVID-19
Anu Aggarwal; Tamanna K Singh; Michael Pham; Matthew Godwin; Rui Chen; Thomas M McIntyre; Alliefair Scalise; Mina K Chung; Courtney Jennings; Mariya Ali; Hiijun Park; Kristin Englund; Alok A Khorana; Lars G Svensson; Samir Kapadia; Keith R McCrae; Scott J Cameron

BACKGROUND
Postacute sequelae of COVID-19 (PASC), also referred to as “Long COVID”, sometimes follows COVID-19, a disease caused by SARS-CoV-2. Although SARS-CoV-2 is well known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. Our objective was to evaluate platelet function and thrombotic potential in patients following recovery from SARS-CoV-2, but with clear symptoms of patients with PASC.

METHODS
Patients with PASC and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by light transmission aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions.

RESULTS
A mild increase in platelet aggregation in patients with PASC through the thromboxane receptor was observed, and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in patients with PASC compared to age-and sexmatched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in patients with PASC. Plasma from patients with PASC was an extremely potent activator of washed, healthy platelets – a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals.

CONCLUSIONS
Patients with PASC show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exist in patients with PASC to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

Link | Paywall (Vascular Medicine)

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Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19, 2023, Aggarwal et al.

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Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19, 2023, Aggarwal et al.

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