Defective Platelet Aggregation Research Articles

Glanzmann Thrombasthenia – A Rare Case Report

Glanzmann thrombasthenia is a rare inherited bleeding disorder resulting from mutation in platelet membrane glycoprotein (GP) IIb or IIIa leading to impaired platelet function which is characterized by defective platelet aggregation and diminished clot retraction. Glanzmann thrombasthenia patients commonly visit to physician with features of bleeding. Here we discuss about the case of a 32-years-old young female presented with menorrhagia, ecchymosis and occasional gum bleeding. Her coagulation profile was in favor of Glanzmann thrombasthenia. This rare disease has a good outcome if early diagnosis and proper management can be ensured. Delta Med Col J. Jan 2021;9(2): 101-103

SINE-VNTR-Alu retrotransposon insertion as a novel mutational event underlying Glanzmann thrombasthenia

BackgroundGlanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. ObjectivesWe aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. MethodsA patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αⅡb and β3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. ResultsGT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing β3 protein content. Meanwhile, both the expression and transportation of β3 were damaged by the ITGB3 c.2222G>A. Almost no αⅡb and β3 expressions were detected on the patient's platelets surface. ConclusionNovel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αⅡbβ3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.

Decreased Platelet Aggregation in Patients with Decompensated Liver Cirrhosis and TIPS Implantation.

Transjugular intrahepatic portosystemic shunt (TIPS) implantation is an effective treatment of portal hypertension in patients with decompensated liver cirrhosis. However, some patients develop TIPS thrombosis with recurrence of portal hypertension. The role of platelets in TIPS thrombosis and the necessity of antiplatelet therapy is unclear. Therefore, we aimed to study platelet function in patients with liver cirrhosis prior to and after TIPS implantation. Platelet aggregation was tested in peripheral and portal-vein blood patient samples on the day (D) of TIPS implantation (D0), D4 and D30 following the procedure (platelet count above 100 × 103/µL, aspirin starting on D5) using whole-blood impedance aggregometry (WBIA) and light transmission aggregometry (LTA). In addition, surface platelet activation markers (P-selectin, activated GPIIb/IIIa) and platelet-neutrophil complexes (PNCs) were assessed by flow cytometry. Thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP) and arachidonic acid (AA) were used as agonists. Healthy subjects were included as controls. Agonist-induced platelet aggregation was reduced (WBIA: TRAP-6 p < 0.01, ADP p < 0.01, AA p < 0.001; LTA: TRAP-6 p = 0.13, ADP p = 0.05, AA p < 0.01) in patients (D0, n = 13) compared with healthy subjects (n = 9). While surface activation markers at baseline were negligibly low, the percentage of PNCs was higher in patients than in controls (p < 0.05). ADP-induced P-selectin expression was increased (p < 0.001), whereas TRAP-6-induced GPIIb/IIIa activation was impaired (p < 0.001) in patients versus controls. PNC formation in response to agonists was not different between groups. Results did not differ between peripheral and portal-vein blood of patients (D0, n = 11) and did not change over time (D0, D4, D30) following TIPS implantation (n = 9). In summary, patients with decompensated liver cirrhosis display in vitro platelet aggregation defects in response to various agonists. Defective aggregation persists upon TIPS implantation. Therefore, we conclude that antiplatelet treatment to prevent TIPS thrombosis is questionable.

Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2V617F mutation

BackgroundJAK2V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis. ObjectivesOur main objective was to explore and compare platelet function depending on the MPN molecular marker. MethodsWe analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels. ResultsWe observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN. ConclusionsThese results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR.

Novel Approach to Improve the Identification of the Bleeding Phenotype in Noonan Syndrome and Related RASopathies

To characterize bleeding phenotype in Noonan Syndrome, to test the utility of following national guidelines in detecting this phenotype, to evaluate thromboelastography (TEG) as a diagnostic tool and to evaluate the cohort for genotype-phenotype correlations. Participants with a clinical diagnosis NS or related RASopathy were enrolled in a cohort study. Study procedures included clinical bleeding assessment, coagulation testing per guidelines and hematology consultation. TEG was completed in a subset and genetic testing was conducted for those without a molecular diagnosis. International Society of Haemostasis and Thrombosis Bleeding Assessment Tool (ISTH-BAT) scores were calculated with hematology consultation. Bleeding phenotype was defined as abnormal bleeding score. Twenty participants enrolled; 12completed clinical and laboratory evaluation, five of whom met the definition for bleeding phenotype. Four of the five participants with a bleeding phenotype had platelet aggregation defects and at least one additional coagulation defect. TEG was performed in nine participants, four with bleeding phenotype and five without, and results were normal in all cases. No genotype-phenotype correlation was found. Five of 20 participants had a bleeding phenotype identified. Based on available data we do not recommend incorporating TEG into clinical practice for NS patients. Platelet aggregation defects were the most common abnormalities, which would not be detected on Tier 1 testing of current guidelines, therefore we propose a new algorithm.

Hermansky-Pudlak Syndrome: Spectrum in Oman.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, characterized by oculocutaneous albinism, a hemorrhagic diathesis secondary to storage pool-deficient platelets, and in some patients' pulmonary fibrosis, granulomatous colitis, and immunodeficiency. To date, 11 different types of Hermansky-Pudlak syndrome were identified. HPS type 2 is distinctively characterized by severe neutropenia and recurrent sinopulmonary infections. HPS is more common in Puerto Rico, and this is the first report deciphering the genotypic spectrum of HPS in Oman. Between 2001 and 2021, 8 Omani cases with HPS (3 HPS type 2, 1 HPS type 3, and 4 HPS type 6) had been suspected clinically and confirmed through genetic mutation analysis. Patients had mild hemorrhagic phenotype, and variable platelet aggregation defects with different platelet agonists. All patients had characteristic eye manifestations. In addition, patients with HPS type 2 had severe neutropenia. Novel mutations in AP3B1(c.205-1G>C, c.12_13delTA (p.Asn4Lysfs*6) and HPS6 (c.19_20delCT (p. Leu7Alafs*168) were not reported in population variant databases. Diagnosis of HPS had markedly improved in Oman; however, increased clinician awareness is needed. A high index of suspicion and early referral for diagnosis and initiation of proper treatment might help improve outcomes.

P809: MODULATION OF ARACHIDONIC ACID PATHWAY BY IBUPROFEN RESULTS IN COMPLETE HEMATOLOGICAL RESPONSE IN GHOSAL HEMATODIAPHYSEAL DYSPLASIA WITHOUT NEED FOR CORTICOSTEROID

Background: Ghosal hematodiaphyseal dysplasia (GHDD) is a rare autosomal recessive disorder characterized by pancytopenia and increased bone density of the long bones. First described in families of Indian and Middle Eastern descent, subsequent studies identified the genetic lesion in this disease as due to loss of function mutations in the TBXAS1 gene encoding thromboxane synthase, a key component of the arachidonic acid pathway responsible for the formation of thromboxane A2 from prostaglandin H2. As well as pancytopenia, laboratory features include platelet aggregation defects and increase in osteoblastic activity, the latter being the basis of increased bone density in patients with Ghosal syndrome. Simultaneously, loss of thromboxane synthase activity may be associated with shunting of prostaglandin precursors away from production of thromboxane into production of pro-inflammatory prostaglandins. This may be the mechanism responsible for the observed marrow suppression seen in these patients. We describe a 3 year old female of non-consanguineous parents of Filipino origin who presented with marked pancytopenia. Next Generation Sequencing bone marrow failure gene panel revealed a novel homozygous mutation in TBXAS1. Subsequent DXA scanning revealed increased bone mineral density consistent with the diagnosis (Z -score +2.4 for lumbar spine (L1-4)) although clinical sequelae of bony disease were not apparent. Aims: While multiple case reports document that these patients have hematological response to long term corticosteroid therapy, we hypothesized that inhibition of cyclooxygenase (COX) upstream of thromboxane synthase in the arachidonic acid pathway may be effective in this condition. Methods: Patient was treated with ibuprofen at regular therapeutic doses (approximately 30 mg/kg/24 hours daily initially) and serial full blood counts were monitored. Results: The patient exhibited a rapid and complete response to ibuprofen with normalization of full blood counts. The dose of ibuprofen was reduced to 10 mg/kg/day, and hematologic response is maintained on low dose after one year.The treatment is well tolerated with no side effects. Image:Summary/Conclusion: Treatment of GHDD with ibuprofen is a novel approach that offers clear potential benefits in comparison to the known adverse effects profile of long term corticosteroids. Bone density scan and markers are in process to ascertain the impact of this treatment on bone density, although this may take longer to show beneficial effects. The treatment was easily tolerated and well managed by the patient and family at home with minimum disruption to her quality of life.

Evaluation of novel coagulation and platelet function assays in patients with chronic kidney disease

BackgroundHemostasis evaluation in chronic kidney disease (CKD) is critical for optimal management of thrombotic and bleeding events. Standard coagulation screens are inadequate for predicting coagulopathy in CKD. ObjectiveTo evaluate hemostasis parameters in patients with different stages of CKD using novel coagulation assays. Patients/MethodsCross‐sectional study of 30 healthy controls (HC) and 120 CKD patients (10 Stage 2, 20 Stage 3, 20 Stage 4, 20 Stage 5 not requiring renal replacement therapy, 20 transplant, 10 newly started on hemodialysis [HD], 20 established on HD). Standard laboratory tests were performed in addition to rotational thromboelastometry (ROTEM), multiple electrode aggregometry (MEA), thrombin generation assays, D‐dimer, and markers of thrombogenesis (thrombin‐antithrombin [TAT]), fibrinolysis, and endothelial activation (intercellular adhesion molecule‐1 [ICAM‐1]). ResultsD‐dimer, TAT, and ICAM‐1 concentrations were significantly higher in patients with CKD than HC (P < .01). ROTEM maximum clot firmness was significantly higher in patients than in HC (P < .01). In CKD Stage 5 patients (pre‐HD and started HD) adenosine diphosphate and thrombin receptor activating peptide MEA tests were significantly lower than HC indicating platelet aggregation defect (P < .05). Multivariate analysis confirmed the direct effect of estimated glomerular filtration rate (eGFR) in the variance of ROTEM and MEA tests. Endogenous thrombin potential and peak thrombin were not statistically different between groups, but Stage 5 CKD patients had prolonged lag time (7.91 vs. 6.33, P < .001) and time to thrombin peak (10.8 vs. 9.5, P < .05) compared to HC. ConclusionsPatients with CKD exhibit features of concomitant hypercoagulability measured by ROTEM and platelet dysfunction measured with MEA. eGFR was an independent determinant of platelet dysfunction and hypercoagulability.

Rotational thromboelastometry in critical phase of dengue infection: Association with bleeding

BackgroundThe critical phase of dengue carries a high risk of bleeding. Associations of coagulation test parameters and the risk of bleeding in the critical phase is unclear. This study examines the association of rotational thromboelastometry (ROTEM delta and ROTEM platelet) with bleeding risk of patients with dengue in the critical phase. MethodsA total of 105 patients with confirmed dengue in the critical phase were recruited, with two subsequent prospective time point analyses of ROTEM parameters and platelet count within 24 and 48 hours from the onset of the critical phase. Conventional coagulation tests were performed only at the initial time point. ResultsTwenty of 105 patients developed bleeding after onset of the critical phase. Within the first 24 hours of critical‐phase onset, platelet count, coagulation tests, and ROTEM delta were unable to differentiate patients with bleeding manifestations from those without (P < .05). Area under the curve of thrombin receptor activating peptide‐6 assay of ROTEM platelet (TRAPTEM) discriminated patients with bleeding manifestations from those without, at a cutoff value of <12.5 Ω*min at a sensitivity and specificity of 73.7%, and 60.2%. In patients who developed bleeding, the maximum lysis of extrinsic pathway of ROTEM was significantly lower in patients with severe bleeding compared to those with mild to moderate bleeding. (4.3 ± 3.4% vs 9.4 ± 7.5%; P = .01). ConclusionAn association with bleeding manifestations and TRAPTEM suggest a potential role for defective platelet aggregation in the pathogenesis of bleeding in the critical phase of dengue.

Rare missense variants in Tropomyosin‐4 (TPM4) are associated with platelet dysfunction, cytoskeletal defects, and excessive bleeding

BackgroundA significant challenge is faced for the genetic diagnosis of inherited platelet disorders in which candidate genetic variants can be found in more than 100 bleeding, thrombotic, and platelet disorder genes, especially within families in which there are both normal and low platelet counts. Genetic variants of unknown clinical significance (VUS) are found in a significant proportion of such patients in which functional studies are required to prove pathogenicity. ObjectiveTo identify the genetic cause in patients with a suspected platelet disorder and subsequently perform a detailed functional analysis of the candidate genetic variants found. MethodsGenetic and functional studies were undertaken in three patients in two unrelated families with a suspected platelet disorder and excessive bleeding. A targeted gene panel of previously known bleeding and platelet genes was used to identify plausible genetic variants. Deep platelet phenotyping was performed using platelet spreading analysis, transmission electron microscopy, immunofluorescence, and platelet function testing using lumiaggregometry and flow cytometry. ResultsWe report rare conserved missense variants (p.R182C and p.A183V) in TPM4 encoding tromomyosin‐4 in 3 patients. Deep platelet phenotyping studies revealed similar platelet function defects across the 3 patients including reduced platelet secretion, and aggregation and spreading defects suggesting that TPM4 missense variants impact platelet function and show a disordered pattern of tropomyosin staining. ConclusionsGenetic and functional TPM4 defects are reported making TPM4 a diagnostic grade tier 1 gene and highlights the importance of including TPM4 in diagnostic genetic screening for patients with significant bleeding and undiagnosed platelet disorders, particularly for those with a normal platelet count.

The Y14-p53 regulatory circuit in megakaryocyte differentiation and thrombocytopenia

SummaryThrombocytopenia-absent radius (TAR) syndrome is caused by RBM8A insufficiency. We generated megakaryocyte-specific Rbm8a knockout (Rbm8aKOMK) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, providing evidence that genetic deficiency of Rbm8a causes a disorder of platelet production. Rbm8aKOMK mice accumulated low-ploidy immature megakaryocytes in the bone marrow and exhibited defective platelet activation and aggregation. Accordingly, depletion of Y14 (RBM8A) in human erythroleukemia (HEL) cells compromised phorbol-ester-induced polyploidization. Notably, Y14/RBM8A deficiency induced both p53 and p21 in megakaryocytes and HEL cells. Treatment with a p53 inhibitor restored ex vivo differentiation of Rbm8aKOMK megakaryocytes and unexpectedly activated Y14 expression in HEL cells. Trp53 knockout partially restored megakaryocyte differentiation by reversing cell-cycle arrest and increased platelet counts of Rbm8aKOMK, indicating that excess p53 in part accounts for thrombocytopenia in TAR syndrome. This study provides evidence for the role of the Y14-p53 circuit in platelet production and a potential therapeutic strategy.

Unique case of 19p13 syndrome with storage pool disease

Abstract Introduction/Objective Microdeletion of a region of the short arm of chromosome 19 results in a very rare syndrome called 19p13.3 deletion syndrome, which manifest itself in developmental delay as well as structural abnormalities such as facial dysmorphism and macrocephaly. Methods/Case Report We present a case of 14-month-old patient, born at term and was large for her gestational age. She had dysmorphic facial features including posterior cleft palate for which, she required placement of G-tube. Post-delivery, she experienced respiratory distress as well as hypoglycemic episodes. Over the period of time, her mother also noticed occasional bleeding through her gums with teething. Genetic workup was performed, which revealed 2.4 Mb of microdeletion at chromosome 19 region p13.3, including deletion of PIAS4, MAP2K2, GNA11, TBXA2R, RAX2 genes. TBXA2R mutation is associated with bleeding disorder due to a defect in platelet aggregation. The mutation in TBXA2R can lead to platelet type 13 bleeding disorder. For this purpose, a platelet aggregation study was performed to evaluate platelet function disorders. However, the result of the platelet aggregation study was inconclusive as it showed decrease responses to all agonists including arachidonic acid, epinephrine, ADP, collagen and ristocetin. Further work-up by electron microscopy (EM) of platelets (PL) revealed a significant decrease of delta granules (DG) (0.89 DG/PL, normal 4-6 DG/PL), consistent with delta granule storage pool deficiency (δ-SPD). Other abnormalities observed by EM included occasional gray platelets, platelets with immature and/or decreased numbers of α-granules, and rare giant α-granules. Results (if a Case Study enter NA) NA Conclusion To the best of our knowledge, no other case of 19p13.3 microdeletion syndrome with δ-SPD and associated abnormalities in α-granules has previously been described in the literature. Although it is unclear if there is any relationship between δ-SPD and 19p13.3 deletion syndrome, further investigation is warranted.

Dubin Johnson Syndrome associated with defective ristocetin – induced platelet agglutination

Background: This is the first reported case of platelet aggregation defect in association with Dubin-Johnson Syndrome. Case presentation: We reported a 14 months old female infant with recurrent attacks of mild to moderate ear and nose bleeding and moderate amount of subarachnoid hemorrhage with ristocetin induced platelet agglutination other than Von Willebrand Factor disease and Bernard–Soulier syndrome. The patient also had fluctuating bilirubin level (Maximum of 15 mg/dl) and remaining liver function tests were normal; the patient diagnosed as Dubin Johnson Syndrome by liver biopsy. Conclusions: Dubin-Johnson Syndrome may be associated with platelet aggregation defect.

A Rare Case of Bleeding Disorder: Glanzmann’s Thrombasthenia

Glanzmann’s thrombasthenia is an extremely rare autosomal recessive inherited bleeding disorder characterized by defective platelet aggregation leading to prolonged bleeding time. Patients may present with easy bruising, purpura, epistaxis, menorrhagia and gingival bleeding. Though the disease is rare, the prognosis is usually excellent with supportive care. Here, we report the case of Glanzmann’s thrombasthenia in a young female who presented with complaints of epistaxis and a history of easy bruising. The patient improved with symptomatic and supportive care. The patient got discharged and is doing well under regular follow-up.

The brain-derived neurotrophic factor prompts platelet aggregation and secretion

AbstractBrain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.

GATA1 pathogenic variants disrupt MYH10 silencing during megakaryopoiesis

BackgroundGATA1 is an essential transcription factor for both polyploidization and megakaryocyte (MK) differentiation. The polyploidization defect observed in GATA1 variant carriers is not well understood. ObjectiveTo extensively phenotype two pedigrees displaying different variants in the GATA1 gene and determine if GATA1 controls MYH10 expression levels, a key modulator of MK polyploidization. MethodA total of 146 unrelated propositi with constitutional thrombocytopenia were screened on a multigene panel. We described the genotype‐phenotype correlation in GATA1 variant carriers and investigated the effect of these novel variants on MYH10 transcription using luciferase constructs. ResultsThe clinical profile associated with the p.L268M variant localized in the C terminal zinc finger was unusual in that the patient displayed bleeding and severe platelet aggregation defects without early‐onset thrombocytopenia. p.N206I localized in the N terminal zinc finger was associated, on the other hand, with severe thrombocytopenia (15G/L) in early life. High MYH10 levels were evidenced in platelets of GATA1 variant carriers. Analysis of MKs anti‐GATA1 chromatin immunoprecipitation‐sequencing data revealed two GATA1 binding sites, located in the 3′ untranslated region and in intron 8 of the MYH10 gene. Luciferase reporter assays showed their respective role in the regulation of MYH10 gene expression. Both GATA1 variants significantly alter intron 8 driven MYH10 transcription. ConclusionThe discovery of an association between MYH10 and GATA1 is a novel one. Overall, this study suggests that impaired MYH10 silencing via an intronic regulatory element is the most likely cause of GATA1‐related polyploidization defect.

Quantitative 3D microscopy highlights altered von Willebrand factor α‐granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

BackgroundPlatelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α‐granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD. ObjectiveQuantitative analysis of the platelet α‐granule compartment and VWF storage in healthy individuals and VWD patients. Patients/MethodsStructured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques. ResultsSIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase‐activated receptor 1 (PAR‐1) activating peptide and revealed a large intra‐ and interdonor variability in VWF‐positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α‐granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α‐granules of a patient with homozygous p.C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p.C1190 in comparison to VWF in plasma. ConclusionsOur findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α‐granules and to gain new insights in the etiology of VWD.

International, Practice-Based Recommendations for Prediction of Bleedings and Anti-Platelet/Anti-Coagulant (AP/AC) Co-Medications in CLL Patients Taking Ibrutinib

Introduction: bleeding events (grade 1-2, 50%, grade 3-4 <5%) have been repeatedly reported in patients taking BTK inhibitors, including ibrutinib (IBR) and acalabrutinib. We lack clear recommendations from real-life clinical practice experience, since all existing management decision support systems have been made out from clinical trials (many of which didn't allow the recruitment of patients taking AC). Furthermore in clinical trials, no thorough exploration of platelet aggregation test was available.Methods: we reviewed the in vitro and in vivo evidence collected in 3 different centres from 3 countries (France, Russia, Austria) including: bleeding symptoms with grade, AP/AC co-medications, agonist-induced platelet aggregation tests (ristocetin RIPA, collagen CIPA), platelet counts. In the French series, complete PK evaluation (t0-0.5h-1h-2h-4h-6h) at one month was available for some patients (Cmax, Cmin, AUC).Results: based on our first 121 pts, our aggregation tests monitoring point towards several common considerations:1/ For patients taking IBR alone (without AP/AC):- Half patients' platelets do not demonstrate any collagen-induced platelet aggregation (CIPA) defect with IBR, even sampled at various time points. This 50/50 segregation between IBR-sensitive and IBR-resistant pts is also observed among 47 healthy donors' platelets. Furthermore, acalabrutinib (sparing TEC kinase more than IBR) in vitro induced very similar (in time and size) aggregation defects only in the IBR-sensitive pts, reinforcing the view of a BTK-effect with the two inhibitors.- Among the 50% early bleedings due to ibrutinib, 50% will recover over time (clinical and CIPA test recovery). As a consequence, in our global population 25-30% of patients still present bleeding after 6 months of therapy (100% grade 1-2). No PK parameter was found to explain the discrepancies of bleeding events among patients, suggesting dose reductions of IBR are not mandatory from the start to leverage bleeding risk. The late risk of severe haemorrhage (grade 3-4) seen in 5 patients seems to be linked with AC (not AP) intake.- the CIPA test has a very good sensitivity (85.7%) and a good specificity (62.5%) to discriminate the patients with bleeding diathesis the first 3 months. The specificity (not sensitivity) was however significantly reduced (33.3%) after 3 months, compromising its utility in pts requiring AC therapy while on ibrutinib for >3 months. Yet, the large majority of patients with bleeding syndromes had a weak aggregation response to collagen 3.3 µg/ml. A lower dose of collagen in vitro can be used (2.2 instead of 3.3microg/ml) to increase specificity after 3-6 months of ibrutinib.2/ for patients taking IBR + AP or AC- the percentage of patients with bleeding syndromes before 6 months did not increase significantly compared to patient under IBR treatment alone (the bleeding grade did not increase neither). Conversely, after 6 months, with AP the risk increases a little bit to 40% (all grade 1-2 except 1 grade 3) as compared to 90% bleeding events in the 10 pts with AC (mainly VKA, including 2 grade 3 and 1 grade 4).- the CIPA tests are no longer valid for patients under AP therapy before start of IBR, as their platelet aggregation response is very low, as expected. In vitro, addition of aspirin or clopidogrel to IBR-resistant platelets reduced aggregation to a significantly lesser extent than that observed in IBR-responsive platelets. This is of interest in the investigation of IBR pts requiring a dual AP therapy.- Most patients under AC and IBR with bleedings had also weak CIPA results. This result would suggest that a weak platelet response to collagen could increase the risk of bleeding (inherent to the AC drug), and therefore heparin should be the preferred choice with ibrutinib.- Take home message: our clinical and biological data collectively identify a strong concern with the use of a triplet therapy, IBR+AP+AC, which should be avoided.Conclusion: the monitoring of CIPA testing is not recommended before starting IBR if no AP/AC is added. In clinical situations where AP/AC is mandated, it can predict an “IBR-related” additional risk factor the bleeding risk of the AP drug. Our results are currently being compared with those of RIPA testing (in the Austrian cohort). We will present updated recommendation charts (including direct anticoagulants use) during the meeting. DisclosuresPanteleev:Amgen: Research Funding. Jaeger:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Payrastre:Janssen: Research Funding. Ysebaert:Janssen: Consultancy, Research Funding, Speakers Bureau.

Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

AbstractThe exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

A Pilot Study on Probing of Imatinib Induced Platelet Dysfunction in Patients with Chronic Myeloid Leukemia-Chronic Phase and Absence of Associated Bleeding Manifestation: Trying to Solve an Enigma.

Imatinib, the first Tyrosine Kinase Inhibitor (TKI) used for the treatment of chronic myeloid leukaemia (CML) has revolutionized the management by inhibiting BCR-ABL tyrosine kinase. According to earlier reports there are concerns regarding the adverse effect of imatinib on haemostasis by causing platelet dysfunction. Here we studied platelet function usingplatelet aggregometry, in 19 CML chronic phase (CML-CP) patients on imatinib therapy, in complete haematologic response (CHR). The median duration of imatinib therapy before performing the test was 154days. This study reveals that there are large inter-individual variations in platelet functions among imatinib treated patients and different levels of variability have been seen for different agonists. Most common aggregation abnormality (< 50% aggregation) was seen withlow dose collagen (1μg/ml) in 31.57% patients. Despite in-vitro platelet aggregation defects, none of the patients showed any bleeding symptoms. This enigma can possibly be explained by the fact that platelet specific agonists, epinephrine and collagen act in synergy for platelet aggregation compared against individual low dose agonists, supported by ex-vivo experiments in normal healthy control group (n = 5) (p value < 0.0004 for epinephrine, p value < 0.0001 for collagen). This experiment was also confirmed in a CML-CP patient. In future, more studies are needed to find out the exact mechanism of this inhibition.