Platelet Defects Research Articles

Characteristics and Financial Impact of Potentially Inappropriate Platelet Transfusion in the Inpatient Hospital Setting

Introduction Platelets are transfused prophylactically to prevent hemorrhage in a variety of patient populations. However, guidelines indicate that prophylactic platelet transfusions in patients with platelet counts above 50k/uL are usually not indicated, with notable exceptions including those undergoing neurological or cardiac bypass surgery. Common minor procedures such as paracentesis, central line placement, and lumbar puncture have been safely performed at platelet counts below 50k/uL. Despite this evidence, our institution incurred approximately 10 million dollars (USD) in direct platelet costs in 2017, with nearly 40% of platelet transfusions are occurring when the patient's platelet count exceeded 50k/uL. Given the significant financial impact of, and potential adverse effects associated with inappropriate platelet transfusion, we implemented a best practice advisory (BPA) in our electronic medical record (EMR) in order to better characterize patterns of platelet transfusion orders in patients with platelet counts >50k/uL. Methods An EMR-embedded BPA was activated in the inpatient hospital setting of a large, tertiary care academic medical center on May 1, 2019, and triggered whenever a platelet transfusion order was placed on an admitted patient whose most recent documented platelet count was >50k/ul. To inform the comparative impact of BPA alerts on provider behavior, alerts were randomized at the patient level to trigger either in standard or silent fashion. For standard alerts, the BPA appeared on-screen, informing the provider that their platelet transfusion order was potentially inappropriate and citing supportive evidence. Providers had the option of following or overriding the alert (Figure 1). In case of alert override, a pre-specified or free text justification was requested. Pre-specified options included upcoming neurosurgery, cardiac bypass surgery, known qualitative platelet defects, or patients taking antiplatelet drugs. Charge data were based on charges for platelet transfusion orders as listed in the hospital charge master. Results From May 1, 2019 to July 30, 2019, the alert fired 181 times (Figure 2). Alerts were silently triggered in 64 (35%) cases. Of the 117 active alerts, 23 (20%) were followed and 94 (80%) were overridden. The most common reasons for alert override included prophylactic transfusions ahead of non-cardiac and non-neurosurgical operations (18%), upcoming cardiac bypass surgery (18%), qualitative platelet defects (12%), active central nervous system (CNS) bleeding (12%), and active non-CNS bleeding (7%). The estimated cost savings associated with followed alerts was $18,170 USD. Discussion Our BPA was effective in reducing instances of platelet transfusion orders by 20% over a three-month period, translating to an estimated annual savings of nearly $70,000 USD in hospital charges. Conversely, the 80% alert override rate indicates that platelet transfusion in patients with platelet counts >50k/uL remains common, occurring in a variety of contexts. Potentially appropriate reasons for platelet transfusions included orders in the setting of cardiovascular bypass surgery, active CNS bleeding, or qualitative platelet defects, representing circumstances in which platelet thresholds are often set higher than 50k/uL. Alternatively, 25% of alert overrides occurred in potentially inappropriate contexts, including patients undergoing non-cardiovascular/non-neurosurgical procedures and patients with non-CNS active bleeding, settings where routinely targeting a platelet threshold >50k/uL is not supported by evidence. As a result of our study's randomized design, future directions include comparative analyses between patient care encounters in which alerts were silently versus visibly triggered, allowing for rigorous determination as to whether providers' interaction with our BPA influences subsequent rates of potentially inappropriate platelet utilization as compared to a control group. Overall, our findings show that platelets are frequently ordered in potentially inappropriate settings, and that reducing these orders imparts significant financial savings. These results provide an impetus for interventions directed at educating providers on appropriate platelet ordering practices, in order to further reduce unnecessary expenditures and optimize patient care. Disclosures No relevant conflicts of interest to declare.

A New Molecular Variant in the PTGS1 Gene That Abrogates Generation of Thromboxane A2 Synthesis and Associates with Platelet Dysfunction and Bleeding

Introduction: Thromboxane A2 [TxA2] is generated from arachidonic acid by cyclooxigenase-1 (COX-1) (prostaglandin H synthase-1) and thromboxane synthase. Aspirin, which irreversibly inhibits COX-1, is a widely used antiplatelet therapy with proven clinical efficacy. Inherited platelet disorders (IPD) are rare diseases caused by alterations of relevant genes in platelet formation and/or function. Despite the relevance of the TxA2 pathway in platelet physiology, few patients with mutations in PTGS1, the gene encoding COX-1, have been identified (<5 cases worldwide). Objective: Characterization of a patient with aspirin-like platelet defect and moderate bleeding, enrolled in the Spanish multicentric project "Functional and molecular characterization of patients with IPD". Methods: The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding (BAT-ISTH=6) and an aspirin-like platelet dysfunction. No coagulation defect or other relevant clinical symptoms were present. Platelet phenotyping included: blood count, PFA-100; platelet aggregation [LTA], glycoproteins (GP), activation and secretion of granules by flow cytometry (FC), TxA2 synthesis by enzyme-immunoassay, synthesis of eicosanoids by tandem gas chromatography with mass spectrometry (LC-MS), western-blot (WB) of platelet lysates, and immunofluorescence (IF) assays. The patient's DNA was analyzed with a HTS-gene panel (Bastida et al, Haematologica 2018). A HEK 293T cell transfection model was established to further assess the pathogenicity of the candidate variant found in the patient. Results: The index case has normal platelet size and count (206x109/L; 11.4 fL). PFA-100 times were normal for COL-ADP and prolonged for COL-EPI (>300s). The FC analysis showed normal expression of GPs (Ib/IX, IIb/IIIa, Ia, GPVI) and reduced fibrinogen*488 binding (20-30%) in response to ADP, TRAP and low dose CRP (2ug/mL). P-selectin and CD63 secretion with agonists was comparable to those of controls. LTA was normal with ristocetin (1.25mg/mL) and TRAP (25uM), reduced by 40-50% with ADP (10uM) and collagen (3ug/mL) and absent with epinephrine (10uM), low dose collagen (1ug/mL) and arachidonic acid (1.6mM). LTA with U46619 (5uM), a direct agonist of the TxA2 receptor, was normal, suggesting a defect in TxA2 synthesis. Indeed, TxA2 levels in LTA supernatants in the patient were very low (5ng/mL; <10% vs. two controls). A significant reduction (50-90%) in TxA2 production was confirmed in the patient whole blood stimulated with collagen or TRAP, as measured by LC-MS. HTS analysis revealed that the patient is a heterozygous carrier of the variant c.428A>G, [p.Asn143Ser] in PTGS1. This variant, not previously described, affects a conserved residue in the catalytic domain of COX-1, which is one of the three N-glycosylation sites in the enzyme. The variant was not associated with reduced COX-1 expression as evaluated by WB in platelet lysates, and by IF in spread washed platelets and leukocytes. HEK 293T cells transfected with wild-type COX-1 construct (validated by RT-PCR and WB), displayed substantial TxA2 synthesis (500ng/mL; 2.5x105 transfected cells) in response to arachidonic acid. In contrast, similar transfection of p.143Ser COX-1 mutant almost abrogated this TxA2 production (≈50-75ng/mL in 2.5x105 transfected cells). Conclusion: We have identified a novel autosomal dominant COX-1 variant, p.Asn143Ser, associated with functional haploinsufficiency of the enzyme and platelet aggregation defects. To our knowledge, this case represents the third description of variants in PTGS1 (Nance, JTH 2016; Sivapalaratnam, Blood 2018), which cause platelet dysfunction and bleeding. Disclosures Almarza: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Therapeutic Doses of Recombinant FVIIa in Hemophilia Generates Thrombin in Platelet-Dependent and -Independent Manner

Recombinant factor FVIIa (rFVIIa) is used successfully in the treatment of hemophilia patients with inhibitors and other bleeding disorders for more than two decades. However, its mechanism of action is still not completely understood. Most of the data in the literature indicate that platelet phospholipid-dependent and tissue factor (TF)-independent activation of factor X by pharmacological concentrations of rFVIIa is responsible to the hemostatic effect of FVIIa in hemophilia treatment. The successful use of rFVIIa in correcting bleeding associated with platelet defects suggest a platelet-independent mechanism may also contribute to the hemostatic effect of rFVIIa in clinical settings. A recent study of Ivanciu et al. (Blood 124:1705-1714, 2014) suggested that the activated endothelium plays an unexpectedly important and major role in supporting prothrombinase assembly and function at the site of damage. Our studies showed that FVIIa binds endothelial protein C receptor (EPCR) on the endothelium and downregulates EPCR-mediated protein C activation, which indirectly contributes to the hemostatic effect of rFVIIa in the treatment of hemophilia. Recently, it has been reported that platelets express EPCR, and suggested that FVIIa binding to EPCR on platelets could enhance the hemostatic activity of FVIIa (Fager et al., J Thromb Haemost 16:1817-1829, 2018). The present study is conducted to evaluate the contribution of platelets and EPCR to the hemostatic effect of rFVIIa in hemophilia treatment in a mouse model system. In this model, platelets in wild-type and hemophilic (acquired and genetic) mice were depleted using anti-murine CD42b antibodies, subjected to the saphenous vein injury, and the hemostatic effect of pharmacological concentrations of rFVIIa administered to mice was evaluated by measuring the amount of thrombin: antithrombin (TAT) complexes generated in the blood. In initial experiments, we found that TAT levels measured in the blood collected at the injury site and not from the blood collected via sub-mandibular site provide a better correlation between TAT levels and the hemostatic effect (clot formation). Therefore, in all studies, blood was collected from the injury site to measure TAT levels. Administration of anti-CD42b antibodies depleted platelets about 95% within 2 to 4 h after the administration of the antibody. Depletion of platelets in wild-type (WT) C57BL/6J mice suppressed TAT levels significantly (51 ± 18 ng/ml) (p<0.01) compared to mice that received no antibody or isotype-specific control antibodies (250 ± 41 ng/ml and 217 ± 31 ng/ml respectively). Induction of hemophilic condition in WT mice by administration of FVIII neutralizing antibodies reduced TAT levels (17 ± 4.0 ng/ml) following the injury. Platelet depletion in these mice further reduced TAT levels (4.7 ± 0.6 ng/ml). Administration of rFVIIa, 1 or 4 mg/kg, to the antibody-induced hemophilic mice, increased the generation of TAT significantly, 75 ± 13 ng/ml and 134 ± 27 ng/ml, respectively. In platelet-depleted, antibody-induced hemophilic mice, administration of the same doses of rFVIIa increased TAT levels to 25 ± 4.0 ng/ml and 58 ± 5.4 ng/ml, respectively. Although TAT levels in rFVIIa-treated platelet-depleted hemophilic mice were lower than that was observed in rFVIIa-treated hemophilic mice (administered with control IgG), still, it was significantly higher compared to TAT levels in hemophilic mice not treated with rFVIIa. Next, to assess the role of platelets and EPCR to the hemostatic effect of rFVIIa, we depleted platelets in FVIII-/-, EPCR-deficient FVIII-/-, and EPCR-overexpressing FVIII-/-mice, subjected them to the saphenous vein injury and administration of rFVIIa (4 mg/kg). The TAT levels in platelet-depleted mice were - 11 ± 1.6 ng/ml in FVIII-/-, 9 ± 0.6 ng/ml in EPCR-deficient FVIII-/-, and 8 ± 1.0 ng/ml in EPCR-over expressing FVIII-/- mice. Administration of rFVIIa to platelet-depleted mice increased TAT levels to 54 ± 10 ng/ml in FVIII-/-, 53 ± 8 ng/ml in EPCR-deficient FVIII-/-, and 42 ± 3.2 ng/ml in EPCR-overexpressing FVIII-/-mice. The differences among them are not statistically significant. Overall, our data indicate that cellular sources other than platelets contribute to the hemostatic effect of rFVIIa. There is no evidence, at least in the murine model, platelet EPCR, if at all present, contributes to the hemostatic effect by augmenting FVIIa binding to platelets. Disclosures Rao: Takeda: Research Funding.

Spectrum of Abnormalities and Clonal Transformation in Germline RUNX1 Familial Platelet Disorder and a Comparative Analysis with Somatic RUNX1 Mutations in Myeloid Neoplasms

Introduction: Germline RUNX1 mutations/deletions result in a Familial Platelet Disorder with propensity to Myeloid Malignancy (FPDMM); an autosomal dominant condition characterized by thrombocytopenia, qualitative platelet defects, and myeloid clonal evolution (MDS and AML). The advent of next generation sequencing (NGS) has allowed detection of clonal cytopenias of unclear significance (CCUS) prior to bone marrow (BM) morphological changes. Somatic RUNX1 mutations (RUNX1MT) can be seen in myeloid malignances, including MDS/MPN overlap syndromes, with an indeterminate prognostic impact. We carried out this study to define the landscape of clonal evolution in FPDMM patients and to compare germline RUNX1MT with somatic mutations. Methods After IRB approval, families with RUNX1MT FPDMM (detected by germline sequencing and deletion/duplication assays) and patients with 2016, WHO-defined MDS/MPN overlap syndromes (except JMML) were included in the study. Index patients with FPDMM underwent BM biopsies and in select cases NGS at diagnosis and then as indicated for follow up, or at disease progression. All MDS/MPN overlap syndrome patients underwent a 29-gene panel NGS assay on BM samples obtained at diagnosis by previously described methods (Patnaik et al., BCJ 2016). Results Eight index patients with FPDMM were included; median age at diagnosis 58 (range, 0-71) years, 3 (38%) males (Figure A). Four (50%) had intragenic deletions involving multiple exons in 3 cases and 2 nucleotides in 1 case, 1 (13%) each had missense, frameshift and splice site mutations, while 1 (13%) patient had a whole gene deletion (Figure C). Notably, in 4 (100%) of 4 patients with large deletions, NGS testing was negative for RUNX1 abnormalities. Four (50%) index patients presented with a myeloid neoplasm; JAK2, SRSF2, ASXL1 mutant CMML, MDS/MPN-U with trisomy 8, AML with del7q and normal karyotype AML (negative NGS). Three (38%) index patients had CCUS at diagnosis with TET2 (67%) being the most common acquired mutation, and others including SRSF2, EZH2, and SF3B1. Cumulatively, myeloid clonal evolution was seen in 7 (88%) patients. Three (38%) index patients (AML-2, CMML-1) underwent allogeneic HCT, and at last follow up were in remission. Median age at myeloid clonal evolution was 61 (range, 4-71) years; median overall survival (OS) from date of myeloid clonal evolution was 12 (range, 7-53) months. For the comparative analysis, 499 patients with MDS/MPN overlap syndromes were included; 382 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T (MDS/MPN with ring sideroblasts and thrombocytosis), 17 (3%) with aCML (atypical CML), and 52 (10%) with MDS-MPN-U (unclassifiable). Median age at diagnosis was 71 (range, 18-99) years, and 329 (66%) were male. RUNX1MT were detected in 57 (11%) patients; 43 (11%) with CMML, 5 (30%) with aCML, and 9 (17%) with MDS-MPN-U. No RUNX1MT were found in MDS-MPN-RS-T. Overall, 62 RUNX1MT were identified in 57 patients, with 52 (39 CMML, 4 aCML, 9 MDS-MPN-U) and 5 (4 CMML, 1 aCML) patients harboring 1 and 2 RUNX1MT, respectively. RUNX1MT spanned the entire coding sequence without specific hotspots (Figure B): 27 (44%) were missense, 2 (3%) involved the splicing sites, 5 (8%) were nonsense and 28 (45%) were frameshift. There was only one RUNX1MT (R273K), seen in both germline and somatic patients (Figures B and C). Compared to wild type patients, RUNX1MT patients with MDS/MPN overlap syndromes had significantly lower platelet counts (median 72 vs 117 x109/L, p<0.0001), without any other phenotypic differences. In the somatic RUNX1MT cohort, co-occurring mutations included ASXL1 (54%), SRSF2 (54%), TET2 (49%), and NRAS (28%), with NRAS (28% vs 13%, p=0.0028), and SRSF2 (54% vs 39%, p=0.0218) mutations clustering with RUNX1MT. Median OS of the entire cohort was 29 (range, 0-170) months. On univariate analysis, while there was a trend for an inferior OS in RUNX1MTvs wild type patients (20 vs 29 months, p=0.0523), the leukemia-free survival was clearly inferior (p=0.0175, Figure D). Conclusion RUNX1 MT FPDMM is a germline predisposition syndrome with a high rate of myeloid clonal evolution (88%), including clonal cytopenias (38%). Germline RUNX1MT have minimal overlap with somatic mutations seen in myeloid malignancies, with NGS testing often missing RUNX1 deletions. Somatic RUNX1MT in myeloid malignancies are significantly associated with thrombocytopenia and an inferior leukemia-free survival. Figure Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

The limitation of genetic testing in diagnosing patients suspected for congenital platelet defects.

The limitation of genetic testing in diagnosing patients suspected for congenital platelet defects.

Defective Platelet Thromboxane A2 Signaling and Serotonin Release in the Pathogenesis of Bleeding during Viral Infection

Viral hemorrhagic fever (VHF) refers to a group of diverse acute human diseases caused by single stranded RNA viruses typically associated with fever, vascular damage and abnormal vascular barrier function. These infections are a continuing threat to public health due to the high case-fatality rate and potential bioterrorism threats. Critically infected VHF patients with unfavorable prognosis often present bleeding symptoms and shock. Thrombocytopenia associated with endothelial, coagulation and platelet dysfunction has been reported during these infections, but their relationship to bleeding is still poorly defined. We have previously established that infection by lymphocytic choriomeningitis virus (LCMV) in extremely thrombocytopenic mice causes severe mucocutaneous bleeding and death mediated by interferon α/β receptor (IFNAR) signaling. We now show in vivo that IFNAR is expressed on megakaryocytes but not on circulating platelets, contrary to previously reported evidence. To explain how increased IFN-I in LCMV-infected mice alters the function of platelets lacking IFNAR, we cross-transplanted WT or IFNAR-/- bone marrow (BM) cells into IFNAR-/- and WT mice. Upon platelet depletion and LCMV infection, only chimeras with WT bone marrow died, suggest that IFN-I-sensitive bone marrow-derived cells are responsible for the mucocutaneous bleeding observed in conditions of extreme thrombocytopenia. Moreover, the results of BM transplantation studies prove that endothelial cell alterations and infection-related inflammatory conditions are not alone sufficient to cause hemorrhage. Circulating platelets during LCMV infection show a generalized aggregation disfunction that persists longer for the arachidonic acid/thromboxane A2 (AA/TBXA2) activation pathway. These aggregation defects are associated, and possibly linked to an impaired secretory ability of both α- and δ-granule. Since circulating platelets are unresponsive to IFN-I, these findings are consistent with the possibility that bone marrow-resident megakaryocytes are targets of IFN-I stimulation during LCMV infection and are the origin of structural and functional platelet abnormalities observed in the circulation. Indeed, megakaryocytes from LCMV-infected mice exhibited fewer granules limited to the perinuclear zone rather than normally diffused throughout the demarcation membrane system; the latter was also underdeveloped. Moreover, platelet transcriptome evaluation of genes involved in platelet production and function showed that IFN-I downregulates bone marrow expression of NF-E2 - a key transcription factor for thrombocytopoiesis - as well as of cyclooxygenase-1 and thromboxane-A synthase. The latter enzymes are required for TBXA2 synthesis from AA amplifying platelet activation. Thus, in addition to being decreased in number, circulating platelets of LCMV-infected animals have impaired activation-induced cargo release from storage granules supporting hemostasis. Experimentally, we observed that bleeding during LCMV infection is increased following aspirin, but not clopidogrel, administration. This suggested that serotonin secretion may be the final effector required to control erythrocyte extravasation. Accordingly, bleeding associated with LCMV infection was markedly enhanced in mice lacking platelet serotonin (TPH1-/-mice), and this was ameliorated by transfusion of normal platelets. In conclusion, we have identified a IFN-I targeting of megakaryocytes leading to reduced production of dysfunctional platelets with abnormal serotonin release as the cause of potentially lethal bleeding in a mouse model of arenavirus infection. Our demonstration that infusion of viable platelets can prevent lethal anemia in LCMV-infected susceptible mouse strains indicates that the same therapeutic intervention monitored by platelet response to AA stimulation may aid in treating severe VHF cases in human patients. Disclosures Aiolfi: MERU-VasImmune, Inc: Other: Stock option. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

Reliability and Feasibility of the Self-Administered ISTH-Bleeding Assessment Tool.

Introduction Standardized bleeding assessment tools (BATs), such as the International Society for Thrombosis and Hemostasis (ISTH)-BAT, are screening instruments used during the diagnostic workup of suspected bleeding disorders. A self-administered ISTH-BAT (self-BAT) would enhance screening and save time during an outpatient clinic visit. Aim This study was aimed to investigate the reliability and feasibility of the self-BAT. Methods The electronic self-BAT was created from the ISTH-BAT and paper-version of self-BAT and optimized by patients and physicians. Patients with a (suspected) congenital platelet defect (CPD), who had previously undergone physician-administered ISTH-BAT assessment, were invited to complete the self-BAT. Optimal self-BAT cut-off values to detect a bleeding tendency, as defined by the ISTH-BAT, were evaluated by receiver operator characteristic (ROC) curve analysis to reach a sensitivity ≥95%. Reliability was tested by assessing sensitivity, specificity, and intraclass correlation (ICC). Feasibility was evaluated on comprehension and length of self-BAT. Results Both versions of the BAT were completed by 156 patients. Optimal cut-off values for self-BAT to define a bleeding tendency were found to be identical to those of the ISTH-BAT. Normal/abnormal scores of the ISTH-BAT and self-BAT were agreed in 88.5% (138/156, 95% confidence interval [CI]: 0.83–0.93) of patients. The sensitivity and specificity of the self-BAT to detect a bleeding tendency were 96.9 and 48.1%, respectively. The ICC was 0.73. Self-BAT questions were graded by 96.8% (151/156) as “very easy,” “easy,” and “satisfactory” and questionnaire length as “exactly right” by 91% (142/156) of patients. Conclusion In patients with a (suspected) CPD, the self-BAT is sufficiently reliable and feasible to detect a bleeding tendency, which supports its use as a screening tool.

Recent advances in inherited platelet disorders.

The increasing use of high throughput sequencing and genomic analysis has facilitated the discovery of new causes of inherited platelet disorders. Studies of these disorders and their respective mouse models have been central to understanding their biology, and also in revealing new aspects of platelet function and production. This review covers recent contributions to the identification of genes, proteins and variants associated with inherited platelet defects, and highlights how these studies have provided insights into platelet development and function. Novel genes recently implicated in human platelet dysfunction include the galactose metabolism enzyme UDP-galactose-4-epimerase in macrothrombocytopenia, and erythropoietin-producing hepatoma-amplified sequence receptor transmembrane tyrosine kinase EPHB2 in a severe bleeding disorder with deficiencies in platelet agonist response and granule secretion. Recent studies of disease-associated variants established or clarified roles in platelet function and/or production for the membrane receptor G6b-B, the FYN-binding protein FYB1/ADAP, the RAS guanyl-releasing protein RASGRP2/CalDAG-GEFI and the receptor-like protein tyrosine phosphatase PTPRJ/CD148. Studies of genes associated with platelet disorders advanced understanding of the cellular roles of neurobeachin-like 2, as well as several genes influenced by the transcription regulator RUNT-related transcription factor 1 (RUNX1), including NOTCH4. The molecular bases of many hereditary platelet disorders have been elucidated by the application of recent advances in cell imaging and manipulation, genomics and protein function analysis. These techniques have also aided the detection of new disorders, and enabled studies of disease-associated genes and variants to enhance understanding of platelet development and function.

Сравнение двух методов лабораторной оценки агрегации тромбоцитов и резистентности к антиагрегантной терапии

Сравнение двух методов лабораторной оценки агрегации тромбоцитов и резистентности к антиагрегантной терапии

Quercetin improves platelet function and ultrastructure in cholestatic liver injury in rats: Role of ORAI1 gene expression

Platelet dysfunction in liver diseases and especially in cholestatic liver diseases is not fully understood. The aim of the present work is to investigate the functional changes of platelets in bile duct ligated (BDL) rat model of cholestasis. The work was conducted on 30 male rats divided into 3 groups: Sham, BDL and quercetin-treated BDL (BDL-Q). Results: Rats from the BDL group showed prolonged bleeding time and diminished platelet aggregation in response to ADP (50 μmol/L), however, changes in platelet count were insignificant. Electron microscopic (EM) photos of platelets from BDL group showed signs of activation. vWF levels were decreased in the plasma of BDL group. ORAI1 gene expression was significantly reduced in their platelets after being exposed to ADP. In BDL-Q rats, bleeding time and platelet aggregation defects were improved. EM demonstrated a picture that is midway between sham and BDL platelets. vWF levels and ORAI1 gene expression increased significantly. ORAI1 expression in platelets was positively correlated to vWF levels. The study concluded that defective platelet function in BDL rat model of cholestasis is due to decrease ORAI1 gene expression and that quercetin administration enhances its expression and improves platelet function.

Hematopoietic transcription factor gene mutations and inherited platelet defects

Hematopoietic transcription factors (TF) are proteins that bind to specific DNA sequences and regulate expression of genes. Hematopoietic TF act in a combinatorial manner to bind sequence-specific DNA within promoter regions to regulate specific gene expression, either as activators or repressors. Hematopoietic TF gene mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these hematopoietic TF affect diverse aspects of megakaryocyte biological function, and platelet production and function, culminating in thrombocytopenia and platelet dysfunction. Some hematopoietic TF mutations are associated with predisposition to hematologic malignancies. The molecular and genetic mechanisms in inherited platelet defects (IPD) are unknown. A growing number of evidences suggest that hematopoietic TF gene mutations are important underlying causes for defects in platelet production, morphology and function. The review summarizes the current scientific progress of hematopoietic TF gene mutations in IPD. Key words: Transcription factors; Hematopoietic system; Mutation; Blood platelet disorders; Thrombocytopenia; Inherited platelet defects

Blutgerinnungsstörungen unter ECMO- und LVAD-Therapie

Extracorporal membrane oxygenation (ECMO) is used to stabilize severe cardiocirculatory and/or respiratory failure in emergency situations. Left ventricular assist devices (LVAD) are used for the treatment of severe chronic heart failure. ECMO and LVAD systems are increasingly employed and provide substantial benefit for respective patients. However, the use of ECMO and LVAD systems is associated with a multifactorial coagulopathy, which is characterized by thromboembolic and hemorrhagic complications. ECMO- and LVAD-induced thromboembolic events are caused by contact activation of plasmatic coagulation and platelets at the artificial surfaces of the respective system. Shear forces inside ECMO and LVADs further contribute to prothrombotic platelet activation. To prevent thrombotic occlusion of ECMO and LVAD systems anticoagulants are routinely administered. For this purpose heparin is primarily used. This may however result in heparin-induced thrombocytopenia, which can further complicate ECMO- and LVAD-associated coagulatory dysfunction. Bleeding complications during ECMO and LVAD therapy can be related to systemic anticoagulation. Qualitative and quantitative platelet defects as well as shear force induced acquired von Willebrand disease further contribute to hemorrhagic events. In conclusion, the management of the ECMO- and LVAD-associated coagulopathy is based on the understanding of its contributing factors. Respective causes for thrombotic and/or hemorrhagic complications should be identified with coagulation assays including viscoelastic point of care tests and platelet aggregometry. Once the underlying reason for the observed coagulopathy has been identified further treatment measures should be individually tailored.

Platelet Function in Experimental Models of Liver Cirrhosis

(1) Background: Platelet function is commonly altered in liver cirrhosis. In experimenta models of liver cirrhosis we have analyzed the mechanisms of this defective function. (2) Methods: Experiments were performed in a model of liver cirrhosis by bile duct-ligation in washed platelets by means of fluorescence spectroscopy with fura-2. (3) Results: We have found several alterations compatible with the existence of a hyperaggregatory state. These alterations are related to a defective platelet calcium handling, specifically to an enhanced intracellular calcium release evoked by thrombin and an increased amount of calcium stored in the intracellular organelles and are present before the appearance of ascites. Homocysteine plays a role in this enhanced platelet aggregation response, probably through an enhanced formation of reactive oxygen species, which is prevented by Folic acid pretreatment. Bile acids show a tendency to reduce calcium movements across platelet membranes which would reduce this hyperaggregatory state characterisitic of the non-ascitic phase of the disease. (4) Conclusions: In experimental models of liver cirrhosis, we have found platelet function alterations related to an increased calcium release secondary to an increased amount of calcium stored in the intracellular organelles. These alterations are related to a higher homocysteine sensitivity that induces an enhanced production of reactive oxygen species. Chronic treatment with folic acid eliminates those alterations, and it can be of interest to minimize risks associated with thrombotic events in cirrhosis.

Update on diagnostic testing for platelet function disorders: What is practical and useful?

Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis. This review summarizes the authors' experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD. Blood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high-quality evidence, limit their ability to offer an expanded test menu for PFD. PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome

BackgroundThrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients.ObjectiveWe carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction.MethodsWe analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis.ResultsWe show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up.ConclusionsOur study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

Leucocyte adhesion deficiency-A multicentre national experience.

Leucocyte adhesion deficiency (LAD) is a rare, innate autosomal recessive immunodeficiency with three subtypes. Twenty-nine patients with LADs were diagnosed and treated in Israeli Medical Centers and in the Palestinian Authority. We discuss the phenotypic, genotypic and biochemical features of LAD-I, LAD-II and LAD-III diagnosed during the neonatal period and early infancy in 18, 6 and 5 patients, respectively. Consanguinity was frequent. Common features were severe infections of variable aetiology, excessive leukocytosis and delayed umbilical cord detachment. In LAD-I, the integrin CD18 expression varied from negligible to normal. However, CD11a expression was negligible in all tested patients, suggesting both CD11a and CD18 should be used to assess this subtype. LAD-II patients showed distinctive facial features, physical malformations, short stature and developmental delay. These patients show defective expression of SLeX (CD15a) on cell surface glycoproteins and lack of H antigen on erythroid cell surfaces resulting in Bombay blood group (hh). LAD-III showed intact but inactive β2 integrins associated with severe infections and significant bleeding disorders caused by defective platelet aggregation and thrombocytopenia. We report four patients with two new unpublished mutations: two LAD-I patients with c.1099delG in ITGB2 and two LAD-III patients with c.1069C>T in FERMT3. LAD-I patients harbouring the c.119_128 deletion in ITGB2 seemed to have better outcomes as compared to other LAD-I patients. Eight patients with LAD-I and -III underwent successful haematopoietic stem cell transplantation. Cumulative survival was 75%, 50% and 40% for LAD-I, LAD-II and LAD-III, with a median follow-up of 4 (0.08-19), 3.25 (1-32) and 6 (0.08-8) years, respectively. Prenatal diagnosis is recommended in families with LAD syndromes.

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.

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

Prospective Diagnosis of VWD in a Large Cohort of Patients with Bleeding Symptoms through the Zimmerman Program

Congenital Aspirin-like Defect As a Result of Autosomal Recessive Variants in PTGS1

Inherited defects of platelet function disorders are rare and difficult to diagnose due to lack of standardized platelet tests. An aspirin-like platelet defect is characterised by reduced thromboxane A2 (TXA2) signalling due to a defect in the arachidonic acid (AA) pathway in platelets. Patients with aspirin-like defect present with mild to moderate bleeding symptoms and impaired platelet aggregation responses to AA and ADP. This is similar to the irreversible effect of aspirin on platelets, which is mediated through inhibition of prostaglandin H synthase-1 also known as cyclooxygenase-1 (PTGS1/COX1). We for the first time report platelet function disorders due to autosomal recessive inheritance of variants in PTGS1.In a total of 3563 cases with bleeding disorders, comprising 1169 whole genome sequenced probands of the BRIDGE-BPD study and 2394 panel sequenced index cases of the ThromboGenomics cohort, we identified 15 unrelated cases of each cohort with an aspirin-like platelet function defect. Two of these cases had rare a variants in PTGS1, the gene encoding COX-1, which catalyses the conversion of arachidonic acid to prostaglandin H2. The first case presented with epistaxis and peri-operative bleeding. She had reduced platelet aggregation responses to arachidonic acid, ADP, collagen and epinephrine. Incubation of control blood with collagen resulted in enhanced levels of thromboxane B2, PGD2, PGE2, 11-HETE and 15-HETE which was absent in the index case.We identified a homozygous missense variant in PTGS1, p.Trp322Ser with a Combined Annotation Dependant Depletion Score (CADD) of 31.0. This variant was absent from GnomAD. The variant co-segregated in an autosomal recessive inheritance mode, with aspirin-like defect phenotype in the seven family members who were investigated. PTGS1 was not expressed on the platelets by western blot. Immunophenotyping demonstrated absence on the platelet surface but presence on neutrophils. The second case of the presented with menorrhagia, nosebleeds, easy bleeding and bruising. She had reduced aggregation responses to arachidonic acid, ADP, collagen and epinephrine. We identified two variants in cis: a splice-donor variant (g. 125133553 T>A), CADD 24.3; and an upstream non-coding variant (g. 125132069 C>G), CADD 16.63. The frequency of these variants were respectively; 1.7 x 10-5 and 8 x 10-3 in GnomAD. Platelet RNA and protein expression studies in the propositus revealed alternative splicing with the generation of a smaller protein due the splice variant. In contrast, the non coding variant had no effect on promoter or enhancer activity and therefore, is likely benign. In this case, the mode of inheritance is autosomal dominant with a dominant negative effect, which has been reported previously. For the other 13 cases of the Bridge-BPD study we also interrogated the non-coding space and interactors in the arachidonic acid pathway, none of which had genetic variants explaining the phenotype. For the 15 ThromboGenomics cohort cases because they were sequenced on targeted platform similar investigations could not take place. These cases could have a non inherited cause for the platelet defect or it is also permissible that variation in a hitherto undefined pathways unique to individual cases might be causal.In conclusion, we for the first time report autosomal recessive inheritance of variants in PTGS1 as cause for a rare inherited bleeding disorder. The effect of the mutation are selective loss of expression of PTGS1 within platelets and decreased enzyme function. Two previous reports demonstrated autosomal dominant inheritance. The first demonstrated autosomal dominant inheritance of variants in PTGS1 as modifier in a well characterized family with haemophilia A and platelet function disorder (Nance et al JTH 2016). The second reported rare heterozygous variants in PTGS1 in two cases with a bleeding tendency which was not further specified in the report (Bastida et al Haematologica 2018). DisclosuresLaffan:Pfizer: Honoraria; Roche: Consultancy, Speakers Bureau.

Chronic Lymphocytic Leukaemia Is Associated with a Broad Platelet-Function Defect Which Is Exacerbated By Ibrutinib and Acalabrutinib

INTRODUCTIONBleeding from thrombocytopathy is a common complication of advanced chronic lymphocytic leukaemia (CLL). In addition to disease-related thrombocytopenia, the presence of the CLL clone and/or therapeutic interventions may further impair platelet function. In particular, the BTK inhibitors ibrutinib and acalabrutinib are known to inhibit platelet glycoprotein VI (GPVI)-mediated platelet aggregation. We compared platelet function and markers of GPVI activation between untreated CLL patients, ibrutinib-treated CLL patients and healthy controls, and studied the in vitro effects of ibrutinib and acalabrutinib on clinically utilised platelet function assays to assess their impact on GPVI-mediated as well as non-GPVI-mediated platelet activation pathways.METHODSBlood samples from 17 healthy volunteers and 8 untreated CLL patients were spiked with vehicle or comparable plasma concentrations of ibrutinib (0.3µM, 1.0µM) and acalabrutinib (1.8µM, 6.0µM) attainable during the treatment of CLL. Additional samples were obtained from 5 CLL patients undergoing ibrutinib treatment. Platelet function was evaluated using whole blood multiple electrode aggregometry (MEA - Multiplate®) and light transmission aggregometry (LTA - AggRAM®) in response to varying concentrations of aggregation-inducing reagents (collagen, CRP-XL, ADP, TRAP, ristocetin, arachidonic acid, and adrenaline). Shear-induced platelet adhesion was assessed using PFA-100®. Soluble GPVI plasma levels were assessed by ELISA.RESULTSIn the absence of treatment, CLL patients exhibited significant platelet defects on whole-blood platelet function analyses in response to various agonists including ADP, ristocetin, TRAP and collagen (MEA) and prolongation of PFA-100® collagen/epinephrine closure time. This impairment was not replicated in assays using platelet-rich plasma (LTA). Ibrutinib-treated CLL patients demonstrated an additive impairment of platelet function, especially in regards to collagen-mediated activation by MEA or PFA-100®. There was no significant difference in soluble GPVI levels between normal, untreated or ibrutinib treated CLL patients.Addition of clinically-attainable concentrations of ibrutinib and acalabrutinib in vitro produced similar concentration-dependent inhibition of platelet function in healthy controls, with inhibition of aggregation evident in response to various agonists including collagen, CRP-XL, ristocetin and ADP but not arachidonic acid or TRAP. Ibrutinib also impaired aggregation in response to epinephrine, and caused selective prolongation of the PFA-100® collagen/epinephrine closure time, an effect not observed with acalabrutinib. MEA appears more sensitive and reproducible than LTA to describe the various inhibitory effects on platelet aggregation. Similar concentration-dependent inhibition of platelet function was observed by adding ibrutinib and acalabrutinib in vitro to blood samples from untreated CLL patients.CONCLUSIONSCLL is associated with a broad platelet function defect, which can be exacerbated by BTK inhibitors. Acalabrutinib induces a platelet function defect similar but less potent to that observed with ibrutinib, with the exception of shear-induced platelet adhesion (PFA-100®) which was only abnormal with ibrutinib. Routine platelet function assays are capable of quantifying BTK inhibitor-induced platelet dysfunction in CLL patients, with the most sensitive and reproducible measure being collagen-induced aggregation by MEA. There was no evidence for BTK-dependent platelet GPVI cleavage. Whole-blood platelet function assays may have utility in managing CLL patients presenting with bleeding or requiring urgent surgery during therapy with BTK inhibitors. DisclosuresMcGregor:Pfizer: Other: Conference travel support; Bristol-Myers Squibb: Other: Conference travel support; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria. Baker:CSL Behring: Research Funding; Biogen Idec: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support, Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Conference travel support; Roche: Other: Conference travel support; Novo Nordisk: Other: Conference travel support.