and Y.S.; resources, K.J.; writingoriginal draft preparation, K.J. hemostatic capacity and specific defects of platelets with reduced number are addressed. or mutation E1841K in the C-terminal exon 38 encoding for the tail part of non-muscle myosin heavy chain-IIA, showed normal mepacrine uptake but impaired release in response to increasing concentrations of thrombin and the GPVI agonist convulxin. These data were confirmed by the impaired surface expression of the -granule/lysosome membrane marker CD63 (Figure 1 and Figure 4a,b, Table S1). A similar release defect was observed for the -granules expressed by impaired P-selectin surface expression, whereas the thrombin- and convulxin-induced activation of the integrin IIb3 was normal [23]. Open in a separate window Figure 4 Platelet function analysis of a patient with the MayCHegglin anomaly (pathogenic variant E1841K) by flow cytometry and calibrated automated thrombography. (a) Methacycline HCl (Physiomycine) Flow cytometric analysis of mepacrine uptake in resting patient platelets and thrombin- or convulxin (GPVI-agonist)-induced mepacrine release compared to a day control. (b) Flow cytometric analysis -granule/lysosome exocytosis expressed as CD63 surface expression of patient and day control platelets in response to increasing concentrations of thrombin and convulxin, respectively. (c) Thrombogram (calibrated automated thrombography) and quantitation of the endogenous thrombin potential (ETP) of tissue factor (TF) or thrombin-stimulated patient and control platelets in platelet-rich plasma; three replicates; n = 3; * 0.05 versus corresponding controls. AU: arbitrary units. This granule release defect may explain the moderated bleeding symptoms of the patient since birth, as his platelet mass was found to be relatively normal and only distributed to a smaller number of giant platelets (35C60 109/L, variable MPV 12 fl). Thus, the flow cytometric application of a Methacycline HCl (Physiomycine) panel of activation markers offers a comprehensive evaluation of platelet function defects in inherited and acquired thrombocytopenias. 5.4. Platelet-Based Thrombin Generation Tests (Platelet-Rich Plasma) Activated platelets significantly contribute to the amplification of thrombin generation, which is essential for thrombus stabilization and for crosstalk between platelets, leukocytes, and endothelial cells [105,106] (Figure 1 and Figure 2). Platelet-dependent thrombin generation tests are offered as commercially available techniques such as the frequently used calibrated automated thrombography (CAT). Active recombinant tissue factor (TF) serves as trigger in the presence of a high Ca2+ concentration to induce a strong activation response of platelets in PRP (platelet count adjusted to 150 109/L with PPP), leading to the exposure of anionic phospholipids and the subsequent formation of the tenase and prothrombinase complex. This assay allows for the continuous monitoring of fluorescence traces (which are generated through the cleavage of a fluorogenic peptide substrate by thrombin and which directly reflect the in vitro capacity of thrombin generation by platelets) over time in a 96-well plate format by a fluorescence reader [107]. This device complements flow cytometric analysis of platelet procoagulant activity for the identification of causative platelet dysfunction in platelet-based coagulation and secondary hemostasis. A recent review by Panova-Noeva, van der Meijden, and Ten Cate gave a comprehensive overview about clinical applications, pitfalls, and uncertainties Methacycline HCl (Physiomycine) of thrombin generation tests performed with PRP [108]. The knowledge about exclusive defects of platelet procoagulant activity is limited, as observed for the very rare inherited platelet function disorder Scott syndrome caused by pathogenic variants in em ANO6 /em , which is not usually associated with thrombocytopenia [109]. Indeed, impaired thrombin generation can be also affected by defective primary platelet responses, e.g., deficiency or decreased levels of integrin IIb3 or ADP signaling [110,111]. Using thrombin as direct platelet agonist in the CAT assay, we recently confirmed impaired thrombin generation, expressed by decreased thrombin peak and endogenous thrombin potential (ETP), in PRP from a patient with -SPD characterized by decreased release of the feedback agonists ADP and ATP [112]. Similar results were observed for platelets from a biallelic BBS patient, when the platelet mass was adjusted in a control platelet sample from a healthy donor [112] (Figure 3c). This phenomenon can be explained by the impaired binding of thrombin to the GPIb/V/IX complex, which serves as co-receptor for PAR-mediated thrombin signaling [113]. Interestingly, we detected a decreased ETP in PRP from the already described patient with a MayCHegglin anomaly (macrothrombocytopenia, but normal platelet mass) who showed a primary secretion defect of platelet – and -granules (Figure 4c). It is likely that the impaired thrombin generation resulted from a secondary granule secretion defect. Therefore, this test is very helpful to evaluate the impact of primary platelet Rabbit Polyclonal to HNRPLL function defects on thrombin generation and.