Extracorporeal membrane oxygenation aggravates platelet glycoprotein V shedding and δ-granule deficiency in COVID-19-associated acute respiratory distress syndrome.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a lifesaving therapy in patients with acute respiratory distress syndrome (ARDS). Hemostatic complications are frequently observed in patients on ECMO and limit the success of this therapy. Platelets are key mediators of hemostasis enabling activation, aggregation, and thrombus formation by coming in contact with exposed matrix proteins via their surface receptors such as glycoprotein (GP) VI or GPIb/V/IX. Recent research has elucidated a regulatory role of the GPV subunit. The cleaved soluble GPV (sGPV) ectodomain was identified to spatiotemporally control fibrin formation through complex formation with thrombin. OBJECTIVES: We aimed to decipher the impact of ECMO on platelet phenotype and function, including the role of GPV and plasmatic sGPV. METHODS: We recruited 36 patients with ARDS in the wake of COVID-19 pneumonia and performed a longitudinal comparison of platelet phenotype and function in non-ECMO (n = 23) vs ECMO (n = 13) compared with those of healthy controls. Patients were assessed at up to 3 time points (t1 = days 1-3; t2 = days 4-6; and t3 = days 7-14 after cannulation/study inclusion). RESULTS: Agonist-induced platelet activation was assessed by flow cytometry and revealed decreased GPIIb/IIIa activation and α-granule release in all ARDS patients. During ECMO treatment, agonist-induced δ-granule release continuously decreased, which was independently confirmed by electron microscopy and was associated with a prolonged in vitro bleeding time. GPV expression on the platelet surface markedly decreased in ECMO patients compared with that in non-ECMO patients. Plasma sGPV levels were increased in ECMO patients and were associated with poor outcome. CONCLUSION: Our data demonstrate an ECMO-intrinsic platelet δ-granule deficiency and hemostatic dysfunction beyond the underlying ARDS.

Ontogenesis of functional platelet subpopulations from preterm and term neonates to adulthood: The PLINIUS study.

Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1- pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.

Uncoupling of platelet granule release and integrin activation suggests GPIIb/IIIa as a therapeutic target in COVID-19.

Thromboembolic events are frequent and life-threating complications of COVID-19 but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct but incompletely understood role. Several studies demonstrated altered platelet function in COVID-19 with some controversial findings, while underlying disease-specific mechanisms remain ill defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared with control participants. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission onward, platelets in COVID-19 failed to activate the integrin glycoprotein IIb/IIa (GPIIb/IIIa) in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole-mount electron microscopy. By contrast, α-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of α-granule release was not observed in patients with sepsis, despite a similar disease severity. We found overall unaltered thrombus formation in COVID-19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates, thrombus formation was virtually abrogated in sepsis, whereas we detected overall normal-sized and stable thrombi in blood from patients with COVID-19. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers, eptifibatide, or tirofiban that had only a minor effect in control participants' blood. We provide evidence that low-dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.

Acquired platelet GPVI receptor dysfunction in critically ill patients with sepsis.

Glycoprotein VI (GPVI), the platelet immunoreceptor tyrosine activating motif (ITAM) receptor for collagen, plays a striking role on vascular integrity in animal models of inflammation and sepsis. Understanding ITAM-receptor signaling defects in humans suffering from sepsis may improve our understanding of the pathophysiology, especially during disease onset. In a pilot study, platelets from 15 patients with sepsis were assessed consecutively at day of admission, day 5 to 7, and the day of intensive care unit (ICU) discharge and subjected to comprehensive analyses by flow cytometry, aggregometry, and immunoblotting. Platelet function was markedly reduced in all patients. The defect was most prominent after GPVI stimulation with collagen-related peptide. In 14 of 15 patients, GPVI dysfunction was already present at time of ICU admission, considerably before the critical drop in platelet counts. Sepsis platelets failed to transduce the GPVI-mediated signal to trigger tyrosine phosphorylation of Syk kinase or LAT. GPVI deficiency was partially inducible in platelets of healthy donors through coincubation in whole blood, but not in plasma from patients with sepsis. Platelet aggregation upon GPVI stimulation increased only in those patients whose condition ameliorated. As blunted GPVI signaling occurred early at sepsis onset, this defect could be exploited as an indicator for early sepsis diagnosis, which needs to be confirmed in prospective studies.