The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia.

Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT-Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation; however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made.

Antibody-induced procoagulant platelets in severe COVID-19 infection.

The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.

Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Cerebral venous sinus thrombosis and thrombocytopenia due to heparin-independent anti-PF4 antibodies after adenovirus infection.

Not available.

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan.

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet life span in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from immune thrombocytopenic patients which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleave of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment in production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

Blood donor-derived buffy coat to produce platelets in vitro.

BACKGROUND AND OBJECTIVES: Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor-derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution. MATERIALS AND METHODS: CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested. RESULTS: Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5·3 x 106 vs. 3·0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coat-derived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7·2 vs. 2·7, respectively). Importantly, platelets produced from buffy coat-derived cells could be activated by agonists. CONCLUSION: Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood-derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production.

Diagnostic value of antibody-induced procoagulant platelets in heparin-induced thrombocytopenia: communication from the ISTH SSC Subcommittee on Platelet Immunology.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated prothrombotic disorder characterized by a drop in platelet count and an increased risk of thromboembolic events. The accurate diagnosis of HIT involves clinical assessment and laboratory testing with well-characterized functional tests. Recent research has shown the potential of investigating procoagulant platelet formation induced by HIT antibodies. To successfully implement these assays in clinical laboratories, careful consideration of technical and preanalytical factors is crucial. In this communication from the SSC Platelet Immunology, we provide a consensus from experts on the use of flow cytometry in HIT diagnosis, highlighting the importance of standardized protocols.

Heparin-activated procoagulant platelet assay: a flow cytometry-based functional test for heparin-induced thrombocytopenia.

BACKGROUND: Functional platelet activation assays are required for the diagnosis of heparin-induced thrombocytopenia (HIT). Due to their sophisticated methodology, they are only available in reference centers. OBJECTIVES: To evaluate the diagnostic accuracy of the flow cytometry-based heparin-activated procoagulant platelet (HAPP) assay in the laboratory diagnosis of HIT. METHODS: Procoagulant platelets (PCP), defined by the expression of phosphatidylserine and CD62-P, were evaluated by flow cytometry in platelet-rich plasma from healthy donors after incubation with patient sera in the absence and presence of heparin. A sample was considered positive in HAPP assay, if the following 3 criteria were met: 1) the percentage of PCPs was ≥10.3% after incubation with 0.2 IU/mL heparin, 2) the fold increase in presence of 0.2 IU/mL heparin compared with buffer was ≥1.5, and 3) 100 IU/mL of heparin resulted in ≥50% inhibition of PCP. HAPP assay was validated in a prospective cohort (n = 202) of consecutive specimens submitted to our laboratory for serologic diagnosis of HIT. Heparin-induced platelet activation (HIPA) assay was used as the reference standard. RESULTS: HIT-positive sera induced PCPs in the presence of 0.2 IU/mL heparin, which was inhibited with 100 IU/mL of heparin. In the prospective validation cohort, there were 15 HIPA+ and 187 HIPA- sera. HAPP was positive in 20 samples in this cohort. Using optimized cut-offs, HAPP assay had a sensitivity of 93.3% and specificity of 96.8%. CONCLUSION: HAPP assay is promising as a simple and reliable functional assay for HIT; however, further studies are needed to confirm our results in larger cohorts.

Platelet-neutrophil interaction in COVID-19 and vaccine-induced thrombotic thrombocytopenia.

Coronavirus disease 2019 (COVID-19) is known to commonly induce a thrombotic diathesis, particularly in severely affected individuals. So far, this COVID-19-associated coagulopathy (CAC) has been partially explained by hyperactivated platelets as well as by the prothrombotic effects of neutrophil extracellular traps (NETs) released from neutrophils. However, precise insight into the bidirectional relationship between platelets and neutrophils in the pathophysiology of CAC still lags behind. Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare autoimmune disorder caused by auto-antibody formation in response to immunization with adenoviral vector vaccines. VITT is associated with life-threatening thromboembolic events and thus, high fatality rates. Our concept of the thrombophilia observed in VITT is relatively new, hence a better understanding could help in the management of such patients with the potential to also prevent VITT. In this review we aim to summarize the current knowledge on platelet-neutrophil interplay in COVID-19 and VITT.

Fomes fomentarius and F. inzengae-A Comparison of Their Decay Patterns on Beech Wood.

Wood-decaying fungi are responsible for the degradation of wood and the alteration in its material properties. Fomes fomentarius (L.) Fr. is one of the most common white-rot fungi colonising coarse wood and standing trees. In recent years, according to their genetic, physiological, and morphological differences, Fomes inzengae (Ces. and De Not.) Lécuru was identified as an independent species. This article aimed to compare the impact of the degradation caused by both species on the anatomical, physical, and mechanical properties of beech wood. When comparing the degradation caused by different strains of both species, no statistically significant difference was found in mass loss (ML) or moisture content (MC). A relevant correlation between ML and MC was confirmed for both species. Variabilities in the density distribution of the degraded and intact bending samples were found to be statistically different. No relevant difference was observed in the modulus of rupture (MOR) between the two species after each exposure period. A strong linear relationship between the MOR and the dynamic modulus of elasticity was revealed for both species. Both species showed decay patterns typical for simultaneous white rot and soft rot. According to the presented results, the impact of both species on the investigated material properties of wood cannot be considered significantly different.

Antibody-mediated procoagulant platelet formation in COVID-19 is AKT dependent.

BACKGROUND: Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2. Recently, we observed that platelets from patients with severe COVID-19 infection express procoagulant phenotype. The molecular mechanisms that induce the generation of procoagulant platelets in COVID-19 patients are not completely understood. OBJECTIVES: In this study, we investigated the role of AKT (also known as Protein Kinase B), which is the major downstream effector of PI3K (phosphoinositid-3-kinase) (PI3K/AKT) signaling pathway in platelets from patients with COVID-19. PATIENTS AND METHODS: Platelets, Sera and IgG from COVID-19 patients who were admitted to the intensive care unit (ICU) were analyzed by flow cytometry as well as western blot and adhesion assays. RESULTS: Platelets from COVID-19 patients showed significantly higher levels of phosphorylated AKT, which was correlated with CD62p expression and phosphatidylserine (PS) externalization. In addition, healthy platelets incubated with sera or IgGs from ICU COVID-19 patients induced phosphorylation of PI3K and AKT and were dependent on Fc-gamma-RIIA (FcγRIIA). In contrast, ICU COVID-19 sera mediated generation of procoagulant platelets was not dependent on GPIIb/IIIa. Interestingly, the inhibition of phosphorylation of both proteins AKT and PI3K prevented the generation of procoagulant platelets. CONCLUSIONS: Our study shows that pAKT/AKT signaling pathway is associated with the formation of procoagulant platelets in severe COVID-19 patients without integrin GPIIb/IIIa engagement. The inhibition of PI3K/AKT phosphorylation might represent a promising strategy to reduce the risk for thrombosis in patients with severe COVID-19.

Upregulation of cAMP prevents antibody-mediated thrombus formation in COVID-19.

Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. The exact mechanisms of COVID-19-associated hypercoagulopathy, however, remain elusive. Recently, we observed that platelets (PLTs) from patients with severe COVID-19 infection express high levels of procoagulant markers, which were found to be associated with increased risk for thrombosis. In the current study, we investigated the time course as well as the mechanisms leading to procoagulant PLTs in COVID-19. Our study demonstrates the presence of PLT-reactive IgG antibodies that induce marked changes in PLTs in terms of increased inner-mitochondrial transmembrane potential (Δψ) depolarization, phosphatidylserine (PS) externalization, and P-selectin expression. The IgG-induced procoagulant PLTs and increased thrombus formation were mediated by ligation of PLT Fc-γ RIIA (FcγRIIA). In addition, contents of calcium and cyclic-adenosine-monophosphate (cAMP) in PLTs were identified to play a central role in antibody-induced procoagulant PLT formation. Most importantly, antibody-induced procoagulant events, as well as increased thrombus formation in severe COVID-19, were inhibited by Iloprost, a clinically approved therapeutic agent that increases the intracellular cAMP levels in PLTs. Our data indicate that upregulation of cAMP could be a potential therapeutic target to prevent antibody-mediated coagulopathy in COVID-19 disease.

[Acquired immune thrombocytopenia: An update on aspects of diagnosis and management relevant for intensive care medicine]. / Erworbene immunvermittelte Thrombozytopenie in der Intensivmedizin: Ein Update über relevante Aspekte der Diagnostik und Therapie.

Acquired thrombocytopenias represent a group of bleeding diseases, which can be mediated by immune or non-immune factors. Acquired immune thrombocytopenia (AITP) leads to an accelerated decrease in platelet count by platelet reactive antibodies arising from several mechanisms. In AITP, autoantibodies, alloantibodies or drug-dependent antibodies are usually targeting platelet surface glycoproteins. The consequence of this is a significant decrease in the number of circulating platelets, leading to clinic pathological disorders including immune thrombocytopenia, heparin-induced thrombocytopenia or drug-induced thrombocytopenia, respectively. The aforementioned disorders are characterized by a severe reduction in platelet count (< 20 × 109/l), which is, with the exception of HIT, associated with high bleeding risk. In this review we provide current insight into recent achievements regarding diagnosis and management of AITP.