Establishment of an in vitro assay to detect antibody-mediated procoagulant platelet formation on a single cell level in real time

Introduction Procoagulant platelets (PLTs), a subpopulation of PLTs that is characterized by increased externalization of phosphatidylserine (PS), are increasingly identified to promote a prothrombotic environment in different diseases. Recently we observed that procoagulant PLT formation can be induced via engagement of immune receptor Fc-gamma-RIIA by COVID-19, VITT and HIT patient immunoglobulin subclass G (IgG) antibodies (Abs). Here, Fc-gamma- RIIA engagement by patient Abs resulted in significant formation of procoagulant PLTs and loss of mitochondrial potential that was associated with high thrombin formation as well as increased thrombus formation. In the cur- rent study, we aim to establish a PLT adhesion assay that allows investigation of PLT mitochondria during procoagulant PLT formation. Method PLTs were spread on human serum albumin, fibrinogen or collagen precoated glass slides. Adhesion and subsequent shape change of PLTs as well as procoagulant PLT formation were investigated in real time using immune fluorescence microscopy. For the detection of PLT shape change, mitochondrial dynamics and PS externalization, PLTs were double stained with MitoTracker green, a mitochondrial dye that binds to free thiol groups of cysteine residues in the mitochondrial membrane, and Annexin-V, respectively. For the visualization of mitochondrial release from PLTs intracellular compartment, a monoclonal Ab that binds to a subunit of the translocase of the outer membrane (TOM) complex on the mitochondrial membrane, namely TOM22, was used. Results During the observation period, a subgroup of PLTs that was spread on collagen became procoagulant as determined by an increased binding of Annexin- V on the PLT surface. Contrary, these changes were nearly absent in PLTs that adhered to fibrinogen (percentage [ %] of Annexin-V positive cells: 19.80 +/- 3.42 % vs. 1.92 +/- 0.62 %, p value 0.0357). Interestingly, procoagulant PLT formation was associated with a significant loss of MitoTracker green signal in PLTs while it remained constant in non-procoagulant PLTs attached on both extracellular matrix coatings. Loss of MitoTracker green signal was associated with translocation of mitochondrial proteins from intracellular to extracellular, as a higher count of TOM22 Ab-positive labelled structures, most likely extracellular mitochondria were detected on collagen but not on fibrinogen coated glass slides. Conclusion Our findings indicate, that the formation of procoagulant PLTs is associated with dramatic changes of the mitochondrial integrity in PLTs. Further attempts, that investigate the potential pathophysiological role of PLT mitochondrial release in Ab-mediated prothrombotic disorders may contribute to a further understanding of the role of PLT mitochondria in these complex diseases.

Procoagulant platelet activity in VITT induced increased thrombus formation

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.

Increased thrombus formation via procoagulant platelets in vaccine induced thrombotic thrombocytopenia (VITT)

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).

Functional studies on the interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia

Background: With increasing number of vaccinations against SARS-CoV-2, rare but life threatening thrombotic events at unusual sites have been reported, and collectively this phenomenon is termed as vaccine-induced immune thrombotic thrombocytopenia (VITT). Pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT), and associated with platelet-activating antibodies against platelet factor 4 (PF4). Aim(s): Current guidelines for anticoagulation in VITT patients are issued accordingly, with a focus on non-heparin anticoagulants. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux and argatroban with VITT-Ab/ PF4 complexes. Method(s): We utilized an in-house enzyme immunoassays (EIA) to estimate antibody binding, inhibition and dissociation of preformed PF4-VITT complexes. Using biolayer interferometry (BLI), we analyzed binding kinetics and dissociation of complexes in real time. In a flow-based ex vivo model, we assessed the impact of anticoagulants on VITT-mediated thrombus formation. Result(s): We found that heparin and danaparoid not only inhibited VITT IgG binding to PF4 but were also able to effectively dissociate preformed PF4/IgG complexes in EIA. In BLI, binding of PF4 specific antibodies was observed for all VITT samples tested, and we found remarkable changes in their dissociation after addition of various anticoagulants. Furthermore, IgGs from VITT patients induce increased thrombus formation in comparison to the healthy controls (mean % SAC +/- SEM: 11.59 +/- 0.57 vs. 1.99 +/- 0.34 respectively, p < 0.001), which can further be effectively inhibited with danaparoid and heparin (mean % SAC +/- SEM 2.82 +/- 0.50 and 1.85 +/- 0.56. p < 0.001). Fondaparinux and argatroban inhibited thrombus formation;however, they did not affect antibody binding. Conclusion(s): Taken together, our data shed a light on suitability of anticoagulants in VITT, and indicate that negatively charged anticoagulants can disrupt VITT-Ab/ PF4 interactions, which might serve as an approach to reduce antibody-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulation in VITT patients could be made.

Camp prevents antibody induced thrombus formation in COVID-19

Background: COVID-19 antibody (Ab) induced procoagulant platelets (PLTs) were recently identified and associated with a higher risk of thromboembolic events in patients infected with SARS-CoV-2. However, the underlying mechanisms leading to procoagulant PLTs in COVID-19 remain elusive. Aims: In this study the mechanisms of COVID-19 Ab induced procoagulant PLT formation as well as the contribution of this PLT subpopulation in thrombus formation were investigated. Methods: Washed PLTs were incubated with IgG fractions from severe COVID-19 ICU or non-COVID-19 ICU patients and analyzed for changes in the expression of caspase 3 using immunoblot as well as CD62P (P-selectin) and phosphatidylserine (PS) via double staining in flow cytometry. To investigate the impact of procoagulant PLTs on thrombus formation, collagen coated microfluidic channels were perfused with whole blood after incubation with IgG antibodies from COVID-19 or control patients. Results: ICU COVID-19 IgG induced a significant increase in caspase 3 cleavage and formation of CD62P/PS positive procoagulant PLTs compared to the ICU control group (31.63%±3.86 vs. 4.04%±1.16, P = 0.0007). Remarkably, incubation of PLTs with ICU COVID-19 IgG resulted in increased thrombus formation (mean % surface area covered [SAC]±SEM: 13.95±1.55 vs. 2.86±1.10, P = 0.0070) and was significantly inhibited by PLT FcγRIIA blockade (16.49±1.02 vs. 5.84±1.93, P = 0.0090). Most importantly and of potential therapeutic relevance, Ab induced procoagulant PLTs and increased thrombus formation were markedly reduced in the presence of Iloprost, a prostacyclin analogue and elevator of intracellular cAMP (41.36%±3.60 vs. 22.22%±3.92, P = 0.0156 and 14.63±2.31 vs. 3.85±0.95, P = 0.0079, respectively). Conclusions: Our findings indicate that COVID-19 Abs induce procoagulant PLTs and increased thrombus formation in a FcγRIIA dependent pathway. As these changes were inhibited via Iloprost, upregulation of cAMP in PLTs might be a promising future therapeutic approach for the complex coagulopathy observed in critical ill COVID-19 patients. (Figure Presented) .

Platelet activation via PI3K/AKT signaling pathway in COVID-19

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.

COVID-19 infection is associated with antibodymediated procoagulant platelets: Data from an observational study

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.