Background Thrombomodulin‐associated coagulopathy (TM‐AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. Objectives To report the characteristics of pedigree with a novel THBD variant causing TM‐AC, and co‐inherited deficiency of thrombin‐activatable fibrinolysis inhibitor (TAFI). Patients/methods Identification of pathogenic variants in hemostasis genes by next‐generation sequencing and case recall for deep phenotyping. Results Pedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop‐gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM‐AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. Conclusions These data extend the reported genetic repertoire of TM‐AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra‐cellular domain. The data further confirm that the delay in fibrinolysis associated with TM‐AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin‐TM system and its regulation of TAFI.

BackgroundFor most physicians, quantification of drug-specific immunoglobulin E (drug-sIgE) antibodies constitutes the primary in vitro measure to document immediate drug hypersensitivity reactions (IDHR). Unfortunately, this is often insufficient to correctly identify patients with IgE-mediated IDHR and impossible for non-IgE-mediated IDHR that result from alternative routes of basophil and mast cell activation. In these difficult cases, diagnosis might benefit from cellular tests such as basophil activation tests (BAT).AimThe aim was to review the potential and limitations of quantification of sIgE and BAT in diagnosing IDHR. The utility of quantification of serum tryptase is discussed.MethodsA literature search was conducted using the key words allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry, specific IgE antibodies; this was complemented by the authors’ own experience.ResultsThe drugs that have been most studied with both techniques are β-lactam antibiotics and curarizing neuromuscular blocking agents (NMBA). For sIgE morphine, data are available on the value of this test as a biomarker for sensitization to substituted ammonium structures that constitute the major epitope of NMBA, especially rocuronium and suxamethonium. For the BAT, there are also data on non-steroidal anti-inflammatory drugs (NSAIDs) and iodinated radiocontrast media. For β-lactam antibiotics, sensitivity and specificity of sIgE varies between 0 and 85% and 52 and 100%, respectively. For NMBA, sensitivity and specificity varies between 38.5 and 92% and 85.7 and 100%, respectively. Specific IgE to morphine should not be used in isolation to diagnose IDHR to NMBA nor opiates. For the BAT, sensitivity generally varies between 50 and 60%, whereas specificity attains 80%, except for quinolones and NSAIDs.ConclusionsAlthough drug-sIgE assays and BAT can provide useful information in the diagnosis of IDHR, their predictive value is not absolute. Large-scale collaborative studies are mandatory to harmonize and optimize test protocols and to establish drug-specific decision thresholds.

RATIONALE: The MRGPRX2-receptor was shown to be involved in IgE/FcεRI independent activation of both murine mast cells and a human mast cell line (LAD2). We sought to investigate the expression of the MRGPRX2 receptor on human basophils. METHODS: Peripheral blood basophils of 16 atopic patients with birch pollen allergy and 4 controls were studied for intracellular and surface MRGPRX2-expression before and after stimulation with fMLP, anti-IgE and recombinant birch pollen (rBet v 1) using basophil activation technique. Briefly, basophils were gated as low SSc IgE + CD203c + positive cells. MRGPRX2 was measured intracellularly and on the membrane of resting (CD203c + CD63 -) and degranulating (CD203cc ++ CD63 + ) basophils. Data are expressed as median (range). RESULTS: All human basophils constitutively express intracellular MRGPRX2. In contrast, only a minority of resting basophils express MRGPRX2 on their surface membrane. Actually, MRGPRX2 surface expression in patients was found in 4% (0-14%) of the resting cells, and was comparable to expression in controls (6%[4-20%]). In patients, anti-IgE stimulation resulted in a significantly higher percentages of CD203c ++ CD63 + MRGPRX2 + cells (21%[1-58%]) than CD203c + CD63 -MRGPRX2 + (4%[0-14%])(P50.001). Similar observations were done in patients for allergen-specific stimulation and for fMLP (patients 13% [4-28%]; controls 11%[3-33%]). Moreover, in patients anti-IgE stimulation resulted in a significantly higher percentage of CD203c ++ CD63 + MRGPRX2 + cells than fMLP stimulation (21% [1-58%] vs 13% [4-28%]; P50.04). CONCLUSIONS: MRGPRX2 is expressed on human peripheral blood basophils and up-regulated upon cross-linking of IgE/FcεRI and stimulation with fMLP. These results show that basophils could potentially be activated in an IgE-independent pathway through engagement of MRGPRX2. RATIONALE: Basophils are mature circulating granulocytic cells that play an important role in IgE-dependent immune responses. The objective of this study was to prospectively examine, in very early life, basophils for FcεRIa and activation markers, which are involved in a Th2 response. METHODS: Cord blood and venous blood at 6 months of age were obtained from 65 infants enrolled in the MAAP (Microbes, Allergy, Asthma and Pets) birth cohort. Basophil percentages were determined by manual differential count of 200 cells in whole blood smears. The enriched granulocyte fraction collected after Ficoll separation of peripheral blood mononuclear cells was examined by flow cytometry. Basophils were defined as low side scatter/Class II 2/CD123+ cells. Cells expressing FcεRIa or the activation markers CD203c or CD63 were examined and were reported as % of basophils expressing these markers. Paired t-tests were used to assess change over time. RESULTS: Basophils represented a mean 0.78% (range 0 to 2.5%) and 0.88% (range 0 to 4.0%) of the differential among cord and 6 month blood samples, respectively. There was a statistically significant increase from birth to 6 months of age in the proportio...

Procarboxypeptidase U (proCPU, TAFI, proCPB2) is a basic carboxypeptidase zymogen that is converted by thrombin(-thrombomodulin) or plasmin into the active carboxypeptidase U (CPU, TAFIa, CPB2), a potent attenuator of fibrinolysis. As CPU forms a molecular link between coagulation and fibrinolysis, the development of CPU inhibitors as profibrinolytic agents constitutes an attractive new concept to improve endogenous fibrinolysis or to increase the efficacy of thrombolytic therapy in thromboembolic diseases. Furthermore, extensive research has been conducted on the in vivo role of CPU in (the acute phase of) thromboembolic disease, as well as on the hypothesis that high proCPU levels and the Thr/Ile325 polymorphism may cause a thrombotic predisposition. In this paper, an overview is given of the methods available for measuring proCPU, CPU, and inactivated CPU (CPUi), together with a summary of the clinical data generated so far, ranging from the current knowledge on proCPU concentrations and polymorphisms as potential thromboembolic risk factors to the positioning of different CPU forms (proCPU, CPU, and CPUi) as diagnostic markers for thromboembolic disease, and the potential benefit of pharmacological inhibition of the CPU pathway.