Ulrich J. Sachs

In Caucasians, maternal allo-immunization against human platelet antigen (HPA)-1a is the most common cause for fetal and neonatal alloimmune thrombocytopenia (FNAIT). Intracranial hemorrhage (ICH) is a serious complication. The lack of a consistent correlation between antibody concentration, severity of thrombocytopenia, and ICH makes it difficult to predict thrombocytopenia in subsequent pregnancies. HPA-1a is located on integrin β3, which forms heterodimers with different alpha chains. The αvβ3 heterodimer is present on endothelial cells (ECs), and a specific antibody interaction with fetal ECs may explain the sporadic nature of ICH. Analysis of human sera was consistent with the presence of anti-HPA-1a of the anti-αvβ3 type in ICH positive cases. We show evidence that these sera induce VEGFR2 fixation to the cell surface, lead to VEGFR2 phosphorylation, enhance ROS production, and interfere with endothelial sprouting. Analysis of blinded human FNAIT samples demonstrates that combined biochemical and functional testing correctly identifies 6/6 ICH positive and 4/4 ICH negative cases. In a murine model, female b3 knockout mice develop anti-platelet antibodies after immunization with wild-type platelets. After mating with b3 wildtype males, only pups born to mice with anti-αvβ3 develop ICH. Matching our findings obtained with human material, only murine sera containing anti-αvβ3 induce VEGFR2 fixation on the cell surface, lead to VEGFR2 phosphorylation, induce ROS production, and hamper endothelial loop formation. In conclusion, we find evidence that ICH in FNAIT is based on vascular impairment and substantiate previous reports that impaired angiogenesis could represent a mechanism of ICH in FNAIT.

Introduction: The GTH‐AHA‐EMI study showed that emicizumab reduces bleeding in patients with acquired haemophilia A (AHA). However, 22 clinically relevant new bleeds (CRNB) occurred in 14 of the 47 study patients, most of which required haemostatic treatment. Aim: To describe characteristics, treatment, and outcome of CRNB that occurred during the 12 weeks of haemostatic prophylaxis with emicizumab. Methods: Data were extracted from the GTH‐AHA‐EMI study data base. Further information was collected retrospectively using a standardized questionnaire answered by study investigators. Results: Of the 22 CRNB, 9 and 13 were classified as major and non‐major, respectively. 15 bleeds occurred spontaneously, and 20 bleed required treatment. Recombinant factor VIIa was used for treatment in 16 of 20 treated bleeds, recombinant porcine factor VIII was used in three events. Eight patients were admitted to the hospital because of CRNB; extension of hospitalization was reported in 13 patients; 9 were admitted to an intensive care unit; invasive interventions were required in nine patients. Most bleeds were treated successfully; permanent harm from bleeding was reported in two patients. No thromboembolic events or other adverse events were reported. Conclusion: Although emicizumab reduces bleeding in AHA, breakthrough bleeding can still occur, was often severe and required haemostatic treatment. Haemostatic treatment was safe and usually effective. Trial Registration: NCT04188639

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs when maternal anti-fetal platelet antibodies, most frequently against HPA-1a, cause fetal platelet destruction. The precise pathogenic mechanisms remain incompletely understood. Here, we examined potential mechanisms underlying anti-HPA-1a-mediated platelet clearance, focusing on phagocytosis, complement fixation, platelet activation, and desialylation. Anti-HPA-1a antibodies in pooled plasma and in 9 of 10 individual FNAIT patient samples induced FcγR-dependent platelet phagocytosis in vitro, indicating this as the major contributor to platelet destruction. In contrast, complement fixation, platelet activation (CD62P expression), and desialylation (RCA-I binding) were observed in only a minority of patients. To model anti-HPA-1a-mediated platelet clearance in vivo, we developed a dual-humanized mouse model combining HPA-1a-expressing platelets (from APLDQ transgenic mice) with mice engineered to express human FcγRs in place of murine FcγRs. Platelets from APLDQ mice sensitized with anti-HPA-1a sera underwent rapid clearance in these FcγR-humanized recipients. Pre-treatment with an FcγRIII-blocking monovalent antibody significantly reduced clearance, confirming FcγRIII's central role in mediating platelet destruction. These results demonstrate that FcγRIII-dependent phagocytosis is a dominant mechanism in anti-HPA-1a-driven platelet clearance in this model. Although complement activation and platelet desialylation were observed in a minority of samples, their contribution to platelet destruction remains speculative. Together, these findings highlight the complexity of FNAIT pathophysiology yet identify FcγRIII blockade as a promising strategy to prevent platelet clearance in this disease.

Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results.

Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody’s Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum–sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3–5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent “CD177-null” phenotype.