Therapeutic strategies in FcγIIA receptor-dependent thrombosis and thromboinflammation as seen in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT)

Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.</p

Magnetic Nanoparticle Labeling of Human Platelets from Platelet Concentrates for Recovery and Survival Studies

Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation

Stability of DNA- and RNA-oligonucleotides in human plasma.

<p>(A) Integrity of 21mer-L1 and (B) 21mer-H1 DNA- and RNA-oligonucleotides was confirmed by polyacrylamide gel electrophoresis without or after preincubation in pooled human plasma for 1 or 5 min, respectively. Each panel represents one representative experiment out of three independent ones.</p

Binding of biotinylated DNA-oligonucleotides to different coagulation factors of the intrinsic coagulation pathway.

<p>Microtiter plate wells were coated with 10 µg/mL each of (A) kininogen or (B) prekallikrein and binding of increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles), 21mer-L1 (closed squares) was assessed. All data represent mean ± SD (n = 3; *p<0.05; 21mer-L1 and 21mer-H3 vs. 21mer-H1) of one representative experiment out of three independent ones. (C) Microtiter plate wells were coated with 10 µg/mL kininogen, factor XI (FXI) or factor XII (FXII) each and incubated with 25 µg/mL each of different biotinylated DNA-oligonucleotides: 21mer-H1 (black bars), 21mer-H3 (white bars) or 21mer-L (hatched bars). All data represent mean ± SD (n = 3) of one representative experiment out of three independent ones. (D) Increasing concentrations of the biotinylated DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H3 (open triangles) or 21mer-L1 (closed squares) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3).</p

Sequences and secondary structures of DNA- and RNA-oligonucleotides.

<p>Described are the secondary structures of (A) DNA- and (B) RNA-oligonucleotides as predicted by the mFold DNA or RNA database. Delta G (ΔG) values represent changes in free enthalpy representative for the stability of the compounds with negative (exergonic) or positive values (endergonic).</p

Procoagulant activity of snRNAs.

<p>(A) Increasing concentrations of U6snRNA (closed triangles, black line) or poly (I:C) (closed diamonds, dotted line) were analyzed for prekallikrein auto-activation. All data represent mean ± SEM (n = 3). (B) Integrity of U6snRNA was confirmed by agarose gelelectrophoresis.</p

Procoagulant activity of different linear and hairpin-forming DNA-oligonucleotides.

<p>Increasing concentrations of the linear DNA-oligonucleotides 21mer-L1 (closed squares), 21mer-L2 (closed triangels) or 21mer-L3 (closed circles) were analyzed for (A) prekallikrein auto-activation or (B) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3). Increasing concentrations of the hairpin-forming DNA-oligonucleotides 21mer-H1 (closed circles), 21mer-H2 (open squares) or 21mer-H3 (open triangles) were analyzed for (C) prekallikrein auto-activation or (D) procoagulant activity in a turbidity clot-lysis assay. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n≥3; *p<0.05; 21mer-H1 vs. 21mer-H3; #p<0.05; 21mer-H2 vs. 21mer-H3). (E) The sizes of DNA-oligonucleotides were analyzed by polyacrylamide gel electrophoresis. Shown is one representative experiment out of three independent ones.</p

Influences of DNA-aptamers on the intrinsic coagulation pathway.

<p>(A) The activation of prekallikrein was followed in the presence of increasing doses of the DNA-aptamers 15mer-thrombin (open circles, interrupted line), 44mer-APC (closed squares), 26mer-AS1411 (closed circles) or 25mer-VEGF (closed triangles, dotted line). (B) Turbidity clot-lysis assays were performed in the absence (black bars) or presence of 1.25 µg/mL (white bars) and 10 µg/mL (striped bars) of the DNA-aptamers 15mer-thrombin, 44mer-APC, 26mer-AS1411 or 25mer-VEGF, respectively. Coagulation was initiated by recalcification, clotting times were defined as respective time points of maximal absorbance. The clotting time of untreated plasma was defined as 100%. All data represent mean ± SEM (n = 3; *p<0.05; 1.25 µg/mL or 10 µg/mL vs. control). (C) The activation of prekallikrein was followed in the presence of increasing doses of the oligonucleotide 21mer-H1 (closed circles) and 21mer-H1-HEG (closed squares). All data represent mean ± SEM (n = 6).</p

Platelet-activating anti-PF4 antibodies mimic VITT antibodies in an unvaccinated patient with monoclonal gammopathy

Transient prothrombotic disorders caused by plateletactivating antibodies against platelet factor 4 (PF4) include heparin-induced thrombocytopenia (HIT), spontaneous HIT syndrome,1  and, most recently, vaccine-induced immune thrombotic thrombocytopenia (VITT).2 Here, we identified prothrombotic, platelet-activating anti-PF4 antibodies, not associated with heparin treatment, in a patient with monoclonal gammopathy that resulted in a chronic hypercoagulability state. </p

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease

MYH9-related disease patients with mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in MYH9 -related disease patients, and treatment with tranexamic acid can improve the hemostatic function. Teaser Impaired hemostasis in Myh9 mutant mice due to reduced platelet forces can be improved by tranexamic acid