Identification and functional investigation of genes involved in inherited bleeding disorders

Inherited bleeding disorders are a heterogeneous group of conditions that reflect abnormalities of the vascular endothelium, coagulation factor proteins and platelets. Clinical diagnosis is often challenging despite patients having extensive bleeding histories and 60% of patients recruited to the Genotyping and Phenotyping of Platelets (GAPP) study remain undiagnosed. A novel assay was trialled in 71 affected patients to investigate platelet spreading ability. This identified patients with abnormal platelet area and circularity who were negative for defects in aggregation and revealed that this assay could identify platelet morphological defects that might be missed by lumi-aggregometry alone. In addition, whole exome sequencing was employed in 10 unrelated families in this undiagnosed patient cohort. The definitive genetic cause of disease was identified in 1 of these families, a missense variant in THBD, with several plausible candidate variants remaining for the other families. In 2 of these families, and a further family from the GAPP study cohort, novel and rare genetic variants in TTLL10 were identified. The protein tubulin tyrosine ligase-like 10 (TTLL10) shows a potential role in the post translational modification of tubulin microtubules in platelets thereby affecting normal platelet physiological processes. In conclusion, genetic variants identified in genes not previously known to cause bleeding could lead to further understanding of haemostasis and thrombosis following subsequent functional studies

Inherited Platelet Disorders - Insight from Platelet Genomics using Next Generation Sequencing

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders associated with normal or reduced platelet counts and bleeding diatheses of varying severities. The identification of the underlying cause of IPDs is clinically challenging due to the absence of a gold-standard platelet test, and is often based on a clinical presentation and normal values in other hematology assays. As a consequence, a DNA-based approach has a potentially important role in the investigation of these patients. Next-generation sequencing (NGS) technologies are allowing the rapid analysis of genes that have been previously implicated in IPDs or that are known to have a key role in platelet regulation, as well as novel genes that have not been previously implicated in platelet dysfunction. The potential limitations of NGS arise with the interpretation of the sheer volume of genetic information obtained from whole exome sequencing (WES) or whole genome sequencing (WGS) in order to identify function-disrupting variants. Following on from bioinformatic analysis, a number of candidate genetic variants usually remain, therefore adding to the difficulty of phenotype–genotype segregation verification. Linking genetic changes to an underlying bleeding disorder is an ongoing challenge and may not always be feasible due to the multifactorial nature of IPDs. Nevertheless, NGS will play a key role in our understanding of the mechanisms of platelet function and the genetics involved

Whole exome sequencing identifies a mutation in thrombomodulin as the genetic cause of a suspected platelet disorder in a family with normal platelet function

Here, we describe a mother and son with a lifelong bleeding tendency and posttraumatic bleeding who were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study with a suspected platelet function disorder. However, despite a clinically significant bleeding score, both had normal platelet counts and normal platelet function. The patients’ blood was analyzed by light transmission aggregometry and genotyping by whole exome sequencing, as outlined by the GAPP study. Approximately 25 000 genetic variants were found for each patient as a result of sequencing and were filtered using a specialized bioinformatics pipeline. A heterozygous variant displaying autosomal dominant inheritance (c.1611 C>A) was found in the gene THBD which encodes the glycoprotein thrombomodulin. This sequence change results in a stop codon (p.Cys537Stop) and truncation of the protein and has been previously described in two other families with bleeding events which suggests it may be a recurrent mutation. In summary, this study shows that patients with a suspected platelet disorder but who present with a normal pattern of platelet aggregation should be investigated for defects in nonplatelet genes

Evaluation of the Total Thrombus-Formation System (T-TAS): application to human and mouse blood analysis

The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyze platelet (PL) or fibrin-rich thrombus formation, respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N = 22) increased with shear within PL (4:40 ± 1.11, 3:25 ± 0.43 and 3:12 ± 0.48 mins [1000, 1500 and 2000s−1]) and AR chips (3:55 ± 0.42 and 1:49 ± 0.19 [240s−1 and 600s−1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s−1 compared to 1500/2000s−1 (260 ± 51.7, 317 ± 55.4 and 301 ± 66.2, respectively). In contrast, no differences in the AUC between 240s−1 and 600s−1 were observed in the AR chip (1593 ± 122 and 1591 ± 158). The intra-assay coefficient of variation (CV) (n = 10) in the PL chip (1000s−1) and AR chip (240s−1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1 µM), but not with ticagrelor (10 µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n = 30) with normal PL counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a PL function defect. The onset (T10) of thrombus formation in WT mice (N = 4) was shorter when compared to humans e.g. PL chip (1000s−1) T10 were 02:02 ± 00:23 and 03:30 ± 0:45, respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected PL function defects and monitoring PL function within mice

Post-translational polymodification of β1-tubulin regulates motor protein localisation in platelet production and function

In specialized cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviors. The mechanisms by which b1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell derived MK, and healthy human donor platelets. We find distinct patterns of polymodification in MK and platelets, mediated by the antagonistic activities of the cell specific expression of tubulin tyrosine ligase like enzymes and cytosolic carboxypeptidase enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganization required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved

Investigation of the contribution of an underlying platelet defect in women with unexplained heavy menstrual bleeding

Heavy menstrual bleeding (HMB) is often undiagnosed in women and can cause discomfort and distress. A haemostatic cause for excessive bleeding is often not routinely investigated and can lead to hysterectomy at an early age. A prospective cohort study was carried out to determine whether certain patients with unexplained HMB have an underlying platelet function defect (PFD). The Genotyping and Phenotyping of Platelets (GAPP) study recruited 175 women with HMB and 44 unrelated volunteers from 25 Haemophilia Centres across the UK, and a tertiary gynaecology service. Bleeding history was assessed using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH-BAT). Platelet count, platelet size, haemoglobin and mean corpuscular volume were measured in whole blood using the Sysmex XN-1000 Haematology Analyzer. Platelet function testing using lumiaggregometry and flow cytometry was performed in patients included in this study. A PFD was identified in 47% (82/175) of patients with HMB. Cutaneous bleeding was the most frequent additional bleeding symptom (89% in PFD and 83% with no PFD). Whole blood platelet count was significantly lower (P < 0.0001) between the PFD group and no PFD group. The prevalence of anaemia did not differ between patients and healthy volunteers. Clinical evaluation alone is insufficient to determine presence of an underlying PFD in patients with HMB. Platelet function tests may be considered and clinical guidelines may include them in their algorithms. An appropriate diagnosis and subsequent tailored management of HMB may prevent unnecessary surgery and help manage future haemostatic challenges