ABSTRACT
Background: Vaccines against SARS-CoV-2 virus significantly reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients, which is even higher than in a previously reported cohort of COVID-19 patients. Method(s): 8 patients (4 female and 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): All sera from VITT patients contained antibodies against PF4 [OD 3.0+/-0.68] with the ability to activate platelets (8/8). Sera induced significant increase in procoagulant markers (CD62P and phosphatidylserine externalization) [CD62P/PS positive PLTs: 40.82+/-7.02%] compared to COVID-19 patients [FI CD62P/PS positive PLTs:15.71+/-7.70];p=0.8977. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 antibody as well as IVIG [FI CD62P/PS positive PLTs:1.01+/-0.36];p=0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (8.64+/-0.53, SAC+/-SEM) compared to control (0.72+/-0.0.07, SAC+/-SEM);p=0.001), which was inhibited by IVIG (4.07+/-0.51, p= 0.001). Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT.
ABSTRACT
Background: Vaccines against SARS-CoV- 2 virus reduce morbidity and mortality of the pandemic. But with millions of people vaccinated in a short period of time, even very rare side effects like the clotting disorder vaccine-induced thrombotic thrombocytopenia (VITT) became apparent. We recently identified an increase in procoagulant platelets in these patients. Aim(s): Investigation of the impact of procoagulant platelets in thrombus formation. Method(s): 8 patients (4 female, 4 male) who were hospitalized with suspected thrombotic complications 5 to 16 days after ChAdOx1 nCoV-19 vaccination were included in this study. The blood samples were analyzed by using enzyme immune assays, flow cytometry, ex vivo thrombus formation assay and heparin-induced platelet aggregation assay. Result(s): The median age was 38 years. All patients had thrombocytopenia at admission. Three had a fatal outcome and five were successfully treated. All sera from VITT patients contained high titer antibodies against platelet factor 4 (PF4) [OD: 3.0 +/- 0.68] with the ability to activate platelets in the HIPA assay (8/8). Sera from VITT patients induced significant increase in procoagulant markers (P-selectin [CD62P] and phosphatidylserine externalization) [% CD62P/ PS positive PLTs: 40.82 +/- 7.02] compared to COVID-19 patients [% CD62P/PS positive PLTs: 15.71 +/- 7.70]. The generation of procoagulant platelets was PF4 dependent. The formation of procoagulant platelets could be significantly reduced by use of the monoclonal IV.3 [% CD62P/PS positive PLTs: 1.05 +/- 0.21];p = 0.0001 antibody as well as IVIG [% CD62P/PS positive PLTs: 1.01 +/- 0.36];p = 0.0001. In thrombus formation model, IgGs from VITT patients induced increase platelet surface area (Mean % SAC +/- SEM: 10.38 +/- 1.30) compared to control IgG, which was inhibited by IVIG (4.08 +/- 0.96), p = 0.001. Conclusion(s): Our ex vivo microfluidic thrombus formation model supports the significance of procoagulant platelet in the pathogenesis of VITT. It may offer significant clinical implications and therapeutic options like evaluation of IVIG as a recommended therapy or other drugs for treatment of clinical picture of VITT. (Table Presented).
ABSTRACT
Background: Accumulating evidence indicates an association between Sars-CoV-2 associated coagulopathy and increased platelet activation. The molecular mechanisms was investof thrombus formation in patients affected by COVID-19. Aims: To investigate the role of phosphoinositid-3-kinase/AKT (PI3K/AKT) signaling pathway in platelet activation in patients with COVID-19. Methods: The Activation status of platelets and PI3/AKT signaling in COVID-19 patients or after incubation of washed healthy platelets with patients' sera was analysed by flow cytometry and Western blot. The functionality was tested by platelets adhesion ability on fibrinogen with PI3K and AKT inhibitors was analysed in vitro. Results: Platelets from COVID-19 patients admitted to the intensive care unit (ICU) showed significantly higher expression of P-selectin (CD62). Western blot analysis showed that platelets from COVID-19 patients display increased phosphorylation of the PI3K as well as of the downstream target protein kinase B/AKT at Ser473 residue. More importantly, sera from these patients induced phosphorylation of PI3K and AKT in healthy donor platelets leading to enhanced activation, which was dependent on Fc-gamma-RIIA (FcγRIIA). The inhibition of AKT as well as PI3K prevented the enhanced activation, adhesion to fibrinogen as well as procoagulant platelet formation. Conclusions: Our study shows that pAKT/AKT signaling pathway is significantly associated with platelet activation in severe COVID-19 patients, which is mediated by FcγRIIA. The strong correlations between platelet activation and pAKT/AKT suggest that inhibiting PI3K/AKT phosphorylation might represent a promising strategy to prevent onset of thrombosis in patients with severe COVID-19.
ABSTRACT
Background : The pathophysiology of COVID-19-associated coagulopathy is to be complex and multifactorial. In this study, we hypothesized that the coagulopathy in COVID-19 is accompanied by immune mediated procoagulant platelets with subsequent alteration of the coagulation system. Aims : We aimed to further analyze the role of platelets (PLTs) leading to increased thromboembolic events in patients with severe COVID-19. Methods : Blood samples from COVID-19 intensive care unit (ICU) patients were analyzed for procoagulant markers. Flow cytometry was used to investigate depolarization of mitochondrial inner transmembrane potential (ΔΨm), intracellular Ca2+ concentration, and phosphatidylserine (PS) externalization. Results : PLTs from COVID-19 patients ( n = 21) showed significantly higher ΔΨm depolarization (1.39 ± 0.07 vs. 0.99 ± 0.06, P = 0.0005), Ca2+ concentration (2.73 ± 0.31 vs. 1.00 ± 0.05, P < 0.0001) and PS externalization (2.05 ± 0.48 vs. 0.86 ± 0.11, P = 0.0236), compared to healthy control (HC), respectively. Most importantly, PS exposure was associated with SOFA score (sequential organ failure assessment, r = 0.5635, P = 0.0078) and plasma levels of D-Dimer ( r = 0.4473, P = 0.0420). Finally, patients with thromboembolic events had higher PS externalization compared to those without thrombosis (2.85 ± 0.75 vs. 0.99 ± 0.20, P = 0.0340). Sera from COVID-19 patients also induced significant increase in procoagulant markers (ΔΨm depolarization (1.52 ± 0.117 vs. 0.958 ± 0.082, P = 0.0086), Ca2+ concentration (1.372 ± 0.074 vs. 0.984 ± 0.055, P = 0.0036) and PS externalization (1.624 ± 0.126 vs. 0.969 ± 0.100, P = 0.0051)) compared to sera from HC. Interestingly, immunoglobulin G (IgG) fractions from COVID-19 patients induced an Fc gamma receptor IIA dependent platelet apoptosis (ΔΨm depolarization (2.23 ± 0.25 vs. 1.22 ± 0.12, P = 0.0216), Ca2+ concentration (1.80 ± 0.15 vs. 0.58 ± 0.04) and PS externalization (9.59 ± 1.52 vs. 2.12 ± 0.20, P = 0.0371)). Conclusions : Our study shows that COVID-19 patients had increased IgG-mediated procoagulant platelets. The strong correlations between procoagulant PLTs and increased D-Dimer levels as well as the incidence of thromboembolic complications may indicate that procoagulant platelets potentially contribute to sustained increased thromboembolic risk in COVID-19 ICU patients.