SARS-CoV-2 infection increases the risk of thrombosis by different mechanisms not fully characterized. Although still debated, an increase in D-dimer has been proposed as a first-line hemostasis test ...associated with thromboembolic risk and unfavorable prognosis. We aim to systematically and comprehensively evaluate the association between thrombin generation parameters and the inflammatory and hypercoagulable state, as well as their prognostic value in COVID-19 patients. A total of 127 hospitalized patients with confirmed COVID-19, 24 hospitalized patients with SARS-CoV-2-negative pneumonia and 12 healthy subjects were included. Clinical characteristics, thrombin generation triggered by tissue factor with and without soluble thrombomodulin, and also by silica, as well as other biochemical parameters were assessed. Despite the frequent use of heparin, COVID-19 patients had similar thrombin generation to healthy controls. In COVID-19 patients, the thrombin generation lag-time positively correlated with markers of cell lysis (LDH), inflammation (CRP, IL-6) and coagulation (D-dimer), while the endogenous thrombin potential (ETP) inversely correlated with D-dimer and LDH, and positively correlated with fibrinogen levels. Patients with more prolonged lag-time and decreased ETP had higher peak ISTH-DIC scores, and had more severe disease (vascular events and death). The ROC curve and Kaplan Meier estimate indicated that the D-dimer/ETP ratio was associated with in-hospital mortality (HR 2.5; p = 0.006), and with the occurrence of major adverse events (composite end-point of vascular events and death) (HR 2.38; p = 0.004). The thrombin generation ETP and lag-time variables correlate with thromboinflammatory markers, and the D-dimer/ETP ratio can predict major adverse events in COVID-19.
Patients with multirefractory immune thrombocytopenia (ITP) have limited treatment options. Recent data suggest that specific anti-platelet antibodies may cause destruction of platelets by favoring ...platelet loss of sialic acid. In this multicenter study 35 patients with ITP, including 16 with multirefractory disease, were analyzed for antiplatelet-antibodies, thrombopoietin (TPO) levels, and platelet desialylation. In selected cases, responses to a novel treatment strategy using oseltamivir were tested.
We found that antibodies against GPIbα were overrepresented in multirefractory patients compared to responders (n = 19). In contrast to conventional ITP patients, multirefractory patients exhibited a significant increased platelet activation state (granule secretion) and desialylation (RCA-1 binding) (p < 0.05), and a trend toward higher plasma TPO concentrations. The decreased sialic acid content seemed to be restricted to platelet glycoproteins, since other plasma proteins were not hypoglycosylated. A total of 10 patients with multirefractory ITP having remarkable loss of platelet terminal sialic acids were given oseltamivir phosphate. When the antiviral drug was combined with TPO receptor agonists (TPO-RAs) or with immunosuppressant drugs, platelet responses were observed in 66.7% of patients. All responding patients presented with antibodies reactive only against GPIbα.
These findings suggest that desialylation may play a key pathogenic role in some multirefractory ITP patients, and provide diagnostic tools for the identification of such patients. Furthermore, we show that sialidase inhibitor treatment in combination with therapies that help to increase platelet production can induce sustained platelet responses in some patients with anti-GPIbα -mediated thrombocytopenia that have failed previous therapies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Antiphospholipid syndrome (APS) is a thromboinflammatory disorder caused by circulating antiphospholipid autoantibodies (aPL) and characterized by an increased risk of thrombotic events. The ...pathogenic mechanisms of these antibodies are complex and not fully understood, but disturbances in coagulation and fibrinolysis have been proposed to contribute to the thrombophilic state. This study aims to evaluate the role of an emerging hemostatic molecule, FXI, in the thrombotic risk of patients with aPL. Cross-sectional and observational study of 194 consecutive and unrelated cases with aPL recruited in a single center: 82 asymptomatic (AaPL) and 112 with primary antiphospholipid syndrome (APS). Clinical and epidemiological variables were collected. The profile of aPL was determined. Plasma FXI was evaluated by Western blotting and two coagulation assays (FXI:C). In cases with low FXI, molecular analysis of the F11 gene was performed. FXI:C levels were significantly higher in patients with APS than in patients with AaPL (122.8 ± 33.4 vs. 104.5 ± 27.5; p < 0.001). Multivariate analysis showed a significant association between symptomatic patients with aPL (APS) and high FXI (>150%) (OR = 11.57; 95% CI: 1.47–90.96; p = 0.020). In contrast, low FXI (<70%), mostly caused by inhibitors, was less frequent in the group of patients with APS compared to AaPL (OR = 0.17; 95%CI: 0.36–0.86; p = 0.032). This study suggests that FXI levels may play a causal role in the prothrombotic state induced by aPLs and holds the promise of complementary treatments in APS patients by targeting FXI.
Congenital disorders of glycosylation (CDG) are rare diseases with impaired glycosylation and multiorgan disfunction, including hemostatic and inflammatory disorders. Factor XII (FXII), the first ...element of the contact phase, has an emerging role in hemostasia and inflammation. FXII deficiency protects against thrombosis and the p.Thr309Lys variant is involved in hereditary angioedema through the hyperreactivity caused by the associated defective O-glycosylation. We studied FXII in CDG aiming to supply further information of the glycosylation of this molecule, and its functional and clinical effects. Plasma FXII from 46 PMM2-CDG patients was evaluated by coagulometric and by Western Blot in basal conditions, treated with N-glycosydase F or activated by silica or dextran sulfate. A recombinant FXII expression model was used to validate the secretion and glycosylation of wild-type and variants targeting the two described FXII N-glycosylation sites (p.Asn230Lys; p.Asn414Lys) as well as the p.Thr309Lys variant.
PMM2-CDG patients had normal FXII levels (117%) but high proportions of a form lacking N-glycosylation at Asn414. Recombinant FXII p.Asn230Lys, and p.Asn230Lys&p.Asn414Lys had impaired secretion and increased intracellular retention compared to wild-type, p.Thr309Lys and p.Asn414Lys variants. The hypoglycosylated form of PMM2-CDG activated similarly than FXII fully glycosylated. Accordingly, no PMM2-CDG had angioedema. FXII levels did not associate to vascular events, but hypoglycosylated FXII, like hypoglycosylated transferrin, antithrombin and FXI levels did it.
N-glycosylation at Asn230 is essential for FXII secretion. PMM2-CDG have high levels of FXII lacking N-glycosylation at Asn414, but this glycoform displays similar activation than fully glycosylated, explaining the absence of angioedema in CDG.
Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in
in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim ...was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.
Abstract
The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in
SERPINC1
are identified for up to 70% ...of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in
SERPINC1
. Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions.
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