Pseudohomozygous dysfibrinogenemia

Abstract Hypodysfibrinogenemia (HD) is a heterogeneous disorder in which plasma fibrinogen antigen and function are both reduced but discordant. This report addresses the key clinical question of whether genetic analysis enables clinically useful subclassification of patients with HD. We report a new case and identify a further eight previously documented cases that have the laboratory features of HD but biallelic inheritance of quantitative and qualitative fibrinogen gene variants. The cases displayed both bleeding and thrombosis and sometimes had undetectable fibrinogen activity. In all cases, the predicted effect of the coinherited variants is reduced levels of circulating fibrinogen that is all dysfunctional. We propose the term pseudohomozygous dysfibrinogenemia for this subtype of recessively inherited HD that is distinct from the more commonly recognized monoallelic HD caused by a single fibrinogen gene variant

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy

Thrombomodulin-associated coagulopathy (TM-AC) is a newly recognised dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor-induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared to controls, but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with rFVIIa or aPCC did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognised fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of haemostasis

Pitfalls in laboratory monitoring of treatment in people with Haemophilia.

A series of cases and scenarios are described to highlight pitfalls in the interpretation of laboratory results in people with haemophilia receiving treatment. This includes assays which grossly exaggerate levels due to in vitro phenomenon and how results which over or under-estimate true values could lead to under or over treatment, unnecessary clinical concern and impact on EQA performance

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide

Background: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies. Objectives: A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing. Methods: Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays. Results: A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods. Conclusion: ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies

Partial deletion of the αC-domain in the Fibrinogen Perth variant is associated with thrombosis, increased clot strength and delayed fibrinolysis

Genetic fibrinogen (FGN) variants that are associated with bleeding or thrombosis may be informative about fibrin polymerisation, structure and fibrinolysis. We report a four generation family with thrombosis and heritable dysfibrinogenaemia segregating with a c.[1541delC];[=] variation in FGA (FGN-Perth). This deletion predicts a truncated FGN αC-domain with an unpaired terminal Cys at residue 517 of FGN-Aα. In keeping with this, SDS-PAGE of purified FGN-Perth identified a truncated FGN-Aα chain with increased co-purification of albumin, consistent with disulphide bonding to the terminal Cys of the variant FGN-Aα. Clot visco-elastic strength in whole blood containing FGN-Perth was greater than controls and tPA-mediated fibrinolysis was delayed. In FGN-Perth plasma and in purified FGN-Perth, there was markedly reduced final turbidity after thrombin-mediated clot generation. Consistent with this, FGN-Perth formed tighter, thinner fibrin fibres than controls indicating defective lateral aggregation of protofibrils. Clots generated with thrombin in FGN-Perth plasma were resistant to tPA-mediated fibrinolysis. FGN-Perth clot also displayed impaired tPA-mediated plasmin generation but incorporated α2-antiplasmin at a similar rate to control. Impaired fibrinolysis because of defective plasmin generation potentially explains the FGN-Perth clinical phenotype. These findings highlight the importance of the FGN αC-domain in the regulation of clot formation and fibrinolysis

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.</p