Abnormal Platelet Aggregation Research Articles

Zastosowanie laseroterapii i rVIIa w leczeniu krwawienia do tkanek miękkich w trombastenii Glanzmanna

Glanzmann’s thrombasthenia (GT) is a rare thrombocytopathy. It is an autosomal recessive disorder. This disease is caused by the quantitative or qualitative defect of the αIIbβ3 membrane integrin, which is the receptor binding the adhesion proteins to the platelet surface in the initial stage of the coagulation process. Abnormal platelet aggregation associated with the αIIbβ3 mutation is the cause of serious and challenging bleeding. The first-line treatment for severe bleeding is transfusion of platelet concentrate or recombinant factor VIIa (rFVIIa) in case of refractoriness to transfusion. The primary objective of this study was to describe clinical effects observed in laser treatment of muscle bleeding in patients with coagulation disorders. We present the case of a 13-year-old boy with TG who was admitted to the hospital with quadriceps muscle hematoma after falling from the bicycle. Due to the lack of response to a platlet concentrate we used rFVIIa with a good effect. Due to the presence of a large painful muscle hematoma, we started laser therapy. Significant pain reduction and rapid hematoma regression were observed.

A Retrospective Study of Clinical and Laboratory Characteristics in Patients Diagnosed with Platelet Storage Pool Deficiency

Introduction:Platelet storage pool deficiency (PSPD) is a disorder of platelet dysfunction with a varying degree of bleeding severity that can range from no symptoms to severe bleeding with hemostatic challenges. The true incidence of the disorder is unknown; however, some believe that it could be as common as von Willebrand disease (vWD). The current standard for diagnosis of PSPD is platelet electron microscopy (EM). However, there is a lack of standardization of techniques and reference ranges. In addition, some experts debate the clinical significance of the diagnosis based on the abnormal platelet EM finding alone. Available literature on adult patients with PSPD is scarce. Therefore, we conducted this study with the aim to describe the clinical and laboratory characteristics of patients diagnosed with PSPD based on specified laboratory criteria. We also intended to evaluate if platelet EM testing can be used to discern patients under evaluation for bleeding symptoms.Methods:We performed a retrospective cohort study of all adult patients referred for hemostatic workup who had platelet EM testing performed at The Ohio State Medical Center from 1/1/2004 to 10/26/2016. Patients with confirmed hemophilia or vWD were excluded. We collected data including patient demographics, symptoms, laboratory data, management plans, and outcomes. The reference range in our laboratory is 3.68-6.24 delta granules per platelet. Patients with less than 3.68 granules per platelet were diagnosed with PSPD (cases), while patients with at least 3.68 granules per platelet were designated as the comparison group. Characteristics in the two groups were compared by Fisher's exact test for categorical variables and the non-parametric Wilcoxon rank sum test for continuous variables since some of the continuous variables had highly skewed distributions.Results:A total of 291 patients were included in the study, 207 patients with PSPD based on EM results (cases) and 84 in the comparison group. Of the 207 cases, median age was 39 (range 12-84) years old and 81.2% were female. Among the cases, 38.7% had <2 delta granules per platelet, 42.0% had 2-2.99, and 19.3% had 3-3.68. The most common presenting symptom was cutaneous bruising (42.5% of cases and 38.1 % in the comparison group). Other common presenting symptoms included menorrhagia (29.9% of cases and 26.2 % of comparison), post-op bleeding (23.7% of cases and 38.1% of comparison), and epistaxis (14.0% of cases and 10.7% of comparison). At presentation, 4.8% of cases were asymptomatic. With the exception of higher incidence of post-op bleeding as a presenting symptom in the comparison group (p=0.015), there was no significant difference in the incidence of other presenting symptoms between the case and the comparison groups.There were no significant differences in the standard hemostatic workup between the two groups, including platelet count, PT, PTT, platelet function assay (PFA-100), platelet aggregation, and von Willebrand panel (Table 1). In both case and comparison groups, 20% of patients had abnormal PFA-100 and 50% had abnormal platelet aggregation with at least one agonist. The only significant difference in the laboratory findings was platelet EM results used to categorize cases (Figure 1 and Table 1).Conclusions:This study showed that in adult patients referred for hemostatic workup, abnormal platelet EM does not correlate with bleeding symptoms or the results of other laboratory tests such as PFA-100 or platelet aggregation. This calls into question the relevance of abnormal platelet EM alone and the appropriate “reference range” in the adult population. Patients with delta granules below the reference range were commonly diagnosed with PSPD, and interventions such as desmopressin and/or antifibrinolytic agents were subsequently recommended. Over-diagnosis of PSPD is a concern, but under-diagnosis in patients with normal EM but persistent bleeding symptoms is also problematic. Future research is needed to characterize correlation of platelet EM values with severity of clinical presentation and to establish appropriate reference range. Furthermore, better diagnostic tools are needed to evaluate patients with bleeding symptoms. [Display omitted] DisclosuresWang:Daiichi Sankyo: Consultancy, Other: Travel.

Novel Hermanksky-Pudlak Syndrome Type 6 Missense Variant Associated with Subclinical Oculocutaneous Albinism and Mild Bleeding

▪Qualitative disorders of platelets are often missed at clinical evaluation. Hermansky-Pudlak (HPS) syndrome is a rare genetic metabolic disorder with subtype specific clinical associations most prevalent in Puerto Rico with strong link to consanguinity. HPS is usually associated with albinism, visual impairment and a qualitative platelet dysfunction due to absence of dense granules. Ceroid accumulation can be associated with inflammatory bowel disease, pulmonary fibrosis and kidney disease. Ten variants have been described with types 1, 2 and 4 associated with severe disease whilst type 3, 5 and 6 is associated with mild disease. Little is known about types 7, 8, 9 and 10. A Caucasian adult female presented with a history of intermittent episodes of severe bleeding. She carried the diagnosis of probable von Willebrand's disease at presentation. This particular patient did not respond to cryoprecipitate infusion but bleeding stopped secondary to infusion of normal platelets, a clue to a platelet storage disorder. Hence she was re-investigated for a bleeding disorder and initial coagulation testing identified abnormal platelet aggregation and amplitude pattern to epinephrine. She was subsequently evaluated with platelet transmission electron microscopy and platelet flow cytometry at the Mayo Clinic Hematopathology. Platelet TEM showed complete absence of dense granules and a normal flow cytometry. HPS was suspected and initial genetic studies identified a variant genetic abnormality. Further studies were done at the NIH. Classical clinical features of HPS like nystagmus, and ocular albinism was not identified at initial neurological and ophthalmologic examination. But more detailed evaluation revealed subclinical oculocutaneous albinism. Genome wide SNP analysis showed regions of homozygosity including HPS 1 and HPS 6; deletions were not identified in these genes. Full length HPS1 transcript was amplified by PCR of genomic DNA. Targeted next-generation sequencing identified a novel homozygous missense variant [c.383T>C (p.V128A)] in HPS6. Reduced HPS6 mRNA levels were found in the patient's skin fibroblasts compared to cells from patients with HPS-1 and normal control cells. HPS6 protein expression in the patient's cells was also low and approximately 60% lower than that of normal cells. HPS is a rare platelet storage disorder that can often be missed. Platelet aggregation tests need to be followed up by platelet TEM and genetic testing to definitively diagnose this condition. Further work up has defined a new missense variant by SNP analysis, next generation sequencing and fibroblast culture. It is important to identify the subtype of HPS, because certain subtypes of HPS (HPS 1, 2 and 4) have clinical manifestations like progressive pulmonary fibrosis. Identification of the platelet storage disorder also helps in management of the bleeding diathesis. DisclosuresNo relevant conflicts of interest to declare.

Histone-Complexed DNA Fragments Levels are Associated with Coagulopathy, Endothelial Cell Damage, and Increased Mortality after Severe Pediatric Trauma.

The release of damage-associated molecular pattern molecules in the extracellular space secondary to injury has been shown to cause systemic activation of the coagulation system and endothelial cell damage. We hypothesized that pediatric trauma patients with increased levels of histone-complexed DNA fragments (hcDNA) would have evidence of coagulopathy and endothelial damage that would be associated with poor outcomes. We conducted a prospective observational study of 149 pediatric trauma patients and 62 control patients at two level 1 pediatric trauma centers from 2013 to 2016. Blood samples were collected upon arrival and at 24 h, analyzed for hcDNA, coagulation abnormalities, endothelial damage, and clinical outcome. Platelet aggregation was assessed with impedance aggregometry (Multiplate) and coagulation parameters were assessed by measuring prothrombin time ratio in plasma and the use of viscoelastic techniques (Rotational Thromboelastometry) in whole blood. The median age was 8.3 years, the median injury severity score (ISS) was 20, and overall mortality was 10%. Significantly higher levels of hcDNA were found on admission in patients with severe injury (ISS > 25), coagulopathy, and/or abnormal platelet aggregation. Patients with high hcDNA levels also had significant elevations in plasma levels of syndecan-1, suggesting damage to the endothelial glycocalyx. Finally, significantly higher hcDNA levels were found in non-survivors. hcDNA is released following injury and correlates with coagulopathy, endothelial glycocalyx damage, and poor clinical outcome early after severe pediatric trauma. These results indicate that hcDNA may play an important role in development of coagulation abnormalities and endothelial glycocalyx damage in children following trauma.

20 The polyphenol delphinidin induces antioxidant effects in human umbilical vein endothelial cells through activation of endogenous glutathione: importance of using relevant concentration in in vitro systems

Polyphenols are regarded to have a wide range of health-promoting effects. Increased consumption of polyphenol-rich food is known to be associated with numerous cardioprotective effects. Polyphenols have been shown to improve endothelial function, inhibit abnormal platelet aggregation, reduce inflammation and improve plasma lipid profiles. Moreover, polyphenols have been widely recognised as powerful antioxidants. Given that oxidative stress plays a key role in initiation and progression of atherosclerosis, antioxidant therapy with polyphenols has potential. However, the concentrations required to mediate sufficient antioxidant effect appear to be unattainable under in vivo conditions. Polyphenols are characterised by poor absorption, rapid degradation and extensive metabolism, culminating in poor bioavailability (~1 µM). In addition, they can also exhibit paradoxical pro-oxidant activities. Spectrophotometric and mass spectrometry (LC-MS/MS) analysis of the phenolic compound, delphinidin, confirmed its low stability and rapid degradation (t 1 / 2 ~30 min) under physiologically relevant conditions. Delphinidin degraded to smaller phenolics: gallic acid and phloroglucinol aldehyde. Moreover, both the parent compound and its main metabolite, gallic acid, generated oxygen-centred free radicals at concentrations≥10 µM, as determined by electron paramagnetic resonance spectrometry (EPR). Interestingly, the tested phenolics offered significant protection to human umbilical endothelial cells (HUVECs) against chemically induced oxidative stress. The protective effect of both phenolics were hormesic in profile; supraphysiological concentrations (100 µM) were cytotoxic, whereas physiologically relevant concentrations (100 nM – 1 µM) were protective against oxidative stress. The observed protection was associated with increased intracellular glutathione. The results confirm that physiologically relevant concentrations of delphinidin and its major metabolite, gallic acid, are sufficient to induce antioxidant benefits, but via an indirect, xenobiotic mechanism that induces upregulation of endogenous antioxidant capacity. The findings emphasis that stability, rate of absorption, distribution and metabolism of phenolic compound needs to be taken into consideration when designing in vitro studies to test their mechanism of action.

Novel labdane diterpenoids from the aerial parts of Leonurus japonicus

Three new labdane diterpenoids, leojapones A–C (1–3), along with a new artifactual analogue (4) and four known terpenoids, were isolated from the EtOAc extract of the aerial parts of Leonurus japonicus Houtt. Their structures were elucidated by spectroscopic analyses and physicochemical evidences. The effects of new compounds against the abnormal increase in platelet aggregation induced by adenosine diphosphate were examined. But instead of inhibiting the abnormal platelet aggregation, compounds 1–3 boosted it significantly.

Molecular phenotype and bleeding risks of an inherited platelet disorder in a family with a RUNX1 frameshift mutation.

Inherited defects in RUNX1 are important causes of platelet function disorders. Our goals were to evaluate RUNX1-related platelet disorders among individuals evaluated for uncharacterized, inherited platelet function disorders and test a proof of concept that bleeding risks could be quantitatively estimated for typical families with an inherited platelet function disorder. Index cases with an uncharacterized inherited platelet function disorder were subjected to exome sequencing with confirmation of RUNX1 mutations by Sanger sequencing. Laboratory findings were obtained from medical records and persistence of platelet non-muscle myosin heavy chain IIB (MYH10), a biomarker of RUNX1 defects, was assessed by Western blotting. Bleeding histories were assessed using standardized assessment tools. Bleeding risks were estimated as odds ratios (OR) using questionnaire data for affected individuals compared to controls. Among 12 index cases who had their exomes sequenced, one individual from a family with eight study participants had a c.583dup in RUNX1 that segregated with the disease and was predicted to cause a frameshift and RUNX1 haploinsufficiency. Unlike unaffected family members (n = 2), affected family members (n = 6) had increased bleeding scores and abnormal platelet aggregation and dense granule release responses to agonists but only some had thrombocytopenia and/or dense granule deficiency. This family's mutation was associated with persistence of MYH10 in platelets and increased risks (OR 11-440) for wound healing problems and mild bleeding symptoms, including bleeding interfering with lifestyle in women. Inherited platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.

The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy) neuron survival in the mouse anorexia (anx) mutation.

ABSTRACTSevere appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia (anx) mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3−/− mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo. Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting detrimental weight loss.

PCTP (Phosphatidylcholine Transfer Protein) is Regulated By RUNX1 in Platelets/Megakaryocytes and is Associated with Adverse Cardiovascular Events

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5’ upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors.Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Landscape of APL Identifies Aberrant Podoplanin Expression As a Defining Feature and Missing Link for the Bleeding Disorder of This Disease

Background:Acute promyelocytic leukemia (APL) is a favorable-risk subgroup of AML characterized by the t(15;17) translocation. The leading cause of early death in APL is uncontrolled bleeding mostly attributed to aberrant expression of tissue factor (F3) and annexin A2 (ANXA2) on leukemic promyelocytes leading to disseminated intravascular coagulation and hyperfibrinolysis, respectively. To prevent or treat such complications, early suspicion of APL and rapid initiation of therapy and supportive measures are critical.Podoplaninor PDPN is a surface glycoprotein expressed in most cell types (http://www.gtexportal.org), but not in blood cells. CLEC-2, the PDPN receptor, is expressed on normal platelets and was found to be necessary for the separation of blood and lymphatic vessels during embryogenesis. PDPN expression (whether endogenous or ectopic) in cell lines induces platelet aggregation, which can be inhibited by chemical tool compounds or by monoclonal antibodies (Chang et al, Oncotarget, 2015, Kato et al, Biochem. Biophys. Res. Commun., 2006).Aims and Methods:We analyzed the transcriptome of 30 APL comprised in the Leucegene 430 AML cohort, aiming to identify clinically useful markers and to better understand the hemostasis-related transcriptomic landscape of this subgroup. Patient cohorts and sorted normal hematopoietic cell populations (n=63) were previously reported (Lavallée et al, Nature Genetics, 2015 and Lavallée et al, Blood, 2016). Comparative analysis of gene expression and mutations were performed as previously described.Results:Our analytical pipeline identified several mutated genes in this cohort, most of which are non-specific and previously identified. CEBPE mutations were the only exception and were specific to APL specimens in this cohort (2/30 vs 0/400, p= 0.005). Most interestingly, we identified PDPN as the single most differentially overexpressed gene in APL (median 2.6 vs 0 RPKM, q = 7 x 10-29, Fig A-B). We also found that PDPN is not expressed in whole blood, bone marrow and in any sorted cell subpopulations from these normal tissues, including promyelocytes (median PDPN expression = 0 RPKM, range 0-0.018). This indicates that platelets are never exposed to PDPN in the adult vasculature and reveals that this gene is ectopically expressed in APL promyelocytes. Accordingly, our hypothesis is that aberrant PDPN expression on leukemic promyelocytes contributes to abnormal platelet aggregation in APL patients. We found that high PDPN expression is associated with lower platelet counts at presentation (18 vs 34 x 1012/L, median PDPN expression ≥ 10 vs < 10 RPKM, p = 0.016, Fig C). Furthermore, a strong inverse correlation was observed between the number of estimated circulating PDPN+ promyelocytes and platelet counts (leukocyte count x [% PDPN+ cells] > 1 x 109/L, Fig C, p=0.007). By incorporating anti-PDPN antibody (clone NC-08, Biolegend) in the EuroFlow protocol, we observed that PDPN expression test was 90% sensitive and 100% specific for APL (n= 48 and 50 APL and non-APL primary AML, respectively). Of note, 5 APL cases considered positive expressed low levels of PDPN.Interestingly, by comparing expression of all coagulation and fibrinolysis genes in APL (n=30) to that of non-APL specimens (n=400), we found that PDPN was the most discriminatory transcript (Fig. A). This result stands in sharp contrast with that found with F3 and ANXA2 which largely overlap in these APL versus non-APL human AML (Fig. A-B). This reinforces the hypothesis that aberrant PDPN expression may be a strong and unappreciated contributor to platelet consumption in APL.Conclusion:I) CEPBE is recurrently mutated in a small subset of APL patients. II) PDPN is the most specific aberrant transcript in this disease and is a new biomarker for APL. Systematic assessment of podoplanin by flow cytometry in newly diagnosed AML could lead to an earlier detection of unsuspected APL cases. III) PDPN expression by leukemic promyelocytes likely contributes to defective primary hemostasis, representing a new mechanism of APL-related bleeding. This provides a strong rationale for evaluating PDPN-CLEC-2 axis inhibitors in this setting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of Primary Hemostasis Alterations Induced By Ibrutinib Assessed By Shutter Speed and Aggregometry Impedance

Introduction:Ibrutinib is a covalent inhibitor of Bruton Tyrosine Kinase (BTK), approved for the treatment of Mantle Cell Lymphoma (MCL), Chronic Lymphocytic Leukemia (CLL) and Waldenstrom's Macroglobulinemia (WM). BTK is expressed in platelets and is believed to have a central role in platelet activation through GPIb and GPIV pathway. However, the clinical significance of inhibition of BTK is unclear in terms of bleeding and it is known that patients with congenitally deficient BTK do not exhibit increased risk of bleeding.Objectives:The main objectives were: (1) identify possible alterations in hemostasis induced by Ibrutinib in patients who start taking the drug, and (2) to monitor the development of hemorrhagic adverse effects in patients who start taking Ibrutinib.Methods:From August 31, 2015 and January 31, 2016 we conducted a prospective study including all consecutive patients with the diagnosis of MCL, CLL or WM treated in 2nd line or more, with Ibrutinib as single-drug therapy. The following parameters were analyzed on day 0 (baseline), 10 and 28 after starting treatment with Ibrutinib: APTT, PT, platelet count, PFA-100 Collagen/Epinephrine and Collagen/ADP, aggregometry impedance in whole blood by multiplate assay with AA, ADP, Ristocetin and TRAP as agonists. Von Willebrand factor antigen (vWF:Ag), Ristocetin cofactor (vWF:RCo) and coagulant factor VIII were also analyzed. We collected prospectively bleeding adverse events.Results:In the period of study, we enrolled 11 patients, 7 were males and 4 females, with a median age of 63 years old (range, 53-88). Five patients had the diagnosis of CLL, 5 patients had MCL and 1 patient had WM. After a median follow-up of 6 months, 3 out of 11 (27.3%) developed grade 1 bleeding adverse events. Of these, 1 patient was under anticoagulant treatment with intermediate dose of enoxaparin, and another patient had low levels of vWF:Ag and vWF:RCo, suggesting a possible acquired von Willebrand disease. Five out of 11 (45.5%) of patients had a prolonged shutter speed Collagen/Epinephrine at baseline, with a median of 175 sec (range, 94-270). After Ibrutinib exposure, median shutter speed Collagen/epinephrine at 10 days was 231 sec (range, 108-287), p=0.02; and at 30 days was 142 sec (range, 79-300), p=n.s. Four out of 11 patients (36.4%) had a prolonged shutter speed Collagen/ADP at base line, with a median of 105 sec (range, 63-190). After Ibrutinib exposure, median shutter speed Collagen/ADP at 10 days was 107 sec (range, 66-192), p=n.s; and at 30 days was 78 sec (range, 75-149), p=n.s. Regarding platelet aggregometry impedance, 2 patients had abnormal platelet aggregation baseline at TRAP assay, 6 at ADP assay, 3 at AA assay and 4 at Ristocetin assay. After Ibrutinib exposure, there was a worsening of platelet aggregation in ADP analysis at 10 days of treatment but became normal after 30 days of treatment. There was an improvement with TRAP at both 10 and 30 days of treatment. Of notice, all 3 patients with minor bleeding had abnormal hemostasis studies baseline before starting with Ibrutinib.Conclusions:Almost half of patients showed an altered haemostasis baseline studies before starting with Ibrutinib. The biology of the disease appears to cause in subjects primary hemostasis changes that result in a basal bleeding risk regardless of the treatment used. Only minor bleeding complications were observed under Ibrutinib treatment, all with baseline altered platelet function; 2 of the three patients who developed bleeding complications had other bleeding risk factors associated. PFA-100 Collagen/Epinephrine is prolonged in almost 50% of patients after Ibrutinib is started compared with baseline study. Aggregometry impedance only worsened with ADP at 10 days, but went into normal level at 30 days. There is an interindividual variability in alterations of haemostasis and hemorrhagic clinical signs. DisclosuresCordoba:Janssen: Research Funding, Speakers Bureau.

All too common: bleeding and genetic variation

Inherited bleeding disorders are both common and challenging to diagnose, particularly when the disorder is mild (reviewed in references [1, 2]). Most mild bleeding disorder patients are not routinely bleeding in their daily lives, and it can be difficult to differentiate bleeding events common in healthy populations from events which rise to the level of pathology. Fundamental to the diagnosis of bleeding disorders is a detailed assessment of the history of the patient and the family. This article is protected by copyright. All rights reserved.

Light Transmission Aggregometry Does Not Correlate With the Severity of δ-Granule Platelet Storage Pool Deficiency.

Delta-granule platelet storage pool deficiency (δ-PSPD) is a poorly studied bleeding diathesis resulting from either decreased granule content or decreased average number of platelet δ-granules. Light transmission aggregometry (LTA) is commonly used to evaluate for δ-PSPD and platelet electron microscopy (EM) is used to confirm the diagnosis. Currently, little data exist examining the relationship between the likelihood of abnormal platelet aggregation findings, severity of δ-granule deficiency on platelet EM, and severity of bleeding symptoms in patients with δ-PSPD. Patients diagnosed with δ-PSPD by platelet EM who also underwent LTA testing were identified at a single institution for correlation between severity of bleeding, average number of platelet δ-granules, and number of agonist abnormalities on LTA. No statistically significant association was identified between the average number of δ-granules per platelet and likelihood of an abnormal LTA. LTA abnormalities were quite varied and only 50% diagnosed with δ-PSPD on EM had abnormal aggregation testing. Also, no correlation was seen between the number of clinical bleeding symptoms, number of average δ-granules per platelet, and the number of LTA agonist abnormalities. Our findings highlight the difficulties inherent in the laboratory evaluation of platelet function.

Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It?

Objective:The aim of this study was to investigate the effects of imatinib mesylate on platelet aggregation and adenosine triphosphate (ATP) release in chronic myeloid leukemia patients.Materials and Methods:Platelet aggregation and ATP release induced by 5.0 mM adenosine diphosphate, 0.5 mM arachidonic acid, 1.0 mg/mL ristocetin, and 2 µg/mL collagen were studied by whole blood platelet lumi-aggregometer in 20 newly diagnosed chronic myeloid leukemia patients before and after imatinib mesylate treatment.Results:At the time of diagnosis, 17/20 patients had abnormal platelet aggregation results; 8 (40%) had hypoactivity, 6 (30%) had hyperactivity, and 3 (15%) had mixed hypo- and hyperactivity. Repeat platelet aggregation studies were performed after a mean of 19 months (min: 5 months-max: 35 months) in all patients who received imatinib mesylate during this period. After therapy, 18/20 (90%) patients had abnormal laboratory results; 12 (60%) had hypoactive platelets, 4 (20%) had mixed hypo- and hyperactive platelets, and 2 (10%) had hyperactive platelets. Three of the 8 patients with initial hypoactivity remained hypoactive, while 2 developed a mixed picture, 2 became hyperactive, and 1 normalized. Of the 6 patients with initial hyperactivity, 4 became hypoactive and 2 developed a mixed pattern. All of the 3 patients with initial hypo- and hyperactivity became hypoactive. Finally, 2 of the 3 patients with initial normal platelets became hypoactive while 1 remained normal. There was a significant decrease in ristocetin-induced platelet aggregation after therapy (p<0.001), while platelet aggregation and secretion induced by other agonists showed no difference after treatment (p>0.05).Conclusion:These findings indicate that a significant proportion of chronic myeloid leukemia patients have different patterns of platelet function abnormalities and imatinib mesylate has no effect on these abnormalities, with a significant impairment in ristocetin-induced platelet aggregation.

Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

The Class Effect of Tyrosine Kinase Inhibiotrs on Impaired Platelet Aggregation on Stimulation with Collagen and Arachidonic Acid

▪INTRODUCITONTyrosine kinase inhibitors (TKIs), the effective therapy for chronic myeloid leukemia (CML), has been reported to induce bleeding in patients, independent of thrombocytopenia. Some TKIs especially dasatinib and ponatinib can cause platelet function defect. We tested whether platelet dysfunction is associated with other TKIs, including imatinib and nilotinib. The clinical correlation of bleeding and platelet function has been explored.METHODSPatients with CML either newly diagnosed or receiving TKIs including imatinib, nilotinib an dasatinib were enrolled into the study. Those taking antiplatelet medications or having thrombocytopenia less than 100 x 109/μL were excluded. We tested platelet aggregation upon stimulation by 4 agonists, including 2.5 and 20-mmol/L ADP, 10-mmol/L epinephrine, 2-mg/mL collagen and 1-mmol/L arachidonic acid (AA). Abnormal platelet aggregation was defined as less than 60% of platelet aggregation upon stimulation by platelet agonists except single abnormality of platelet aggregation on stimulation with low-dose ADP or epinephrine. Clinical significance of bleeding was evaluated by bleeding scores.ResultsThere were 111 patients with CML, including 14 newly diagnosed patients and those receiving imatinib (n=75), nilotinib (N=18) and dasatinib (n=4). Majority of treatment naïve patients were in chronic phase (n = 11, 79%) whereas 2 and 1 patients were in blast phase and accelerated phase, respectively. Most of the patients on TKIs were in chronic phase (n = 94, 97%) while there were few patients in accelerated (n = 2) and blast phase (n =1). Dose intensity of imatinib was 300 mg (n=15), 400 mg (n=54), 600 mg (n=5) and 800 mg (n=1), while nilotinib dose was 600 mg (n=4) and 800 mg (n=14). Three and 1 patients received 100 mg and 50 mg of dasatinib, respectively. Of those taking TKIs, complete cytogenetic response (CCyR) and major molecular response (MMR) were achieved in 59% and 27% of patients while the minority of them achieved partial cytogenetic response (PCyR) or less (8%). Impaired platelet aggregation on stimulation with 20-mmol/L ADP was observed in 14%, 7%, 17% and 50% of patients who were treatment naïve, on imatinib, nilotinib and dasatinib, respectively (P =0.036). Patients on TKIs had higher proportion of patients with abnormal platelet aggregation on stimulation with collagen compared to treatment naïve patients (72% for imatinib, 56% for nilotinib, 75% for dasatinib and 36% for treatment naïve, P = 0.05). The corresponding figures for proportion of patients with abnormal platelet aggregation on stimulation with AA were 78%, 39%, 50% and 29%, P <0.001). Two out of 3 patients with normal platelet aggregation at diagnosis of CML had abnormal platelet aggregation on stimulation with collagen and AA after imatinib treatment. Combination of abnormal platelet aggregation upon stimulation with collagen and AA was the most common pattern of impaired platelet aggregation for TKIs (63% for imatinib, 33% for nilotinib and 50% for dasatinib). Assessed by bleeding score, none of patients with impaired platelet aggregation had significant clinical bleeding.ConclusionThe effect of TKIS on abnormal platelet aggregation appeared to be class effect, though, the prevalence was more common among patients treated with imatinib and dasatinib. Although, there was no apparent clinical correlation of TKIs induced abnormal platelet aggregation and clinical bleeding, CML patients on TKIS should be closed monitoring when undergoing any hemostatic challenges DisclosuresNo relevant conflicts of interest to declare.

Platelet Function Disorders in Bleeding Patients without Other Coagulopathies

▪Introduction:We present a series of 105 patients referred for hematologic consultation to evaluate a clinical bleeding disorder. Little is known regarding the prevalence of non-genetic qualitative platelet disorders and which of these might respond to desmopressin.Methods:Patients were assessed over a two-year period, from December 2011 through December 2013. Patients, who were found to have neither von Willebrand's disease nor coagulation factor deficiencies, nor quantitative platelet defects, were tested for platelet function abnormalities by PFA-100 (Dade Behring, Marburg, Germany) and platelet aggregation and secretion studies using lumi aggregation (Chrono-Log Corp., Havertown, PA, USA). Selected patients with abnormal platelet function were given a trial of intravenous desmopressin to determine efficacy, and platelet function studies were repeated 2 hours later.Results:Of the 105 referred patients (26 males, age range 15-83 years; 79 females, age range 21-86 years), 67 with either normal (n=43) or abnormal (n=24) PFA-100 results were not evaluated further based on their clinical history and presentation. 18 patients with abnormal PFA-100 results, consisting of Collagen/ADP, Collagen/Epinephrine, or both, had platelet aggregation and secretion studies performed. 16 of these patients had abnormal platelet aggregation and secretion studies, while 2 patients had normal studies. In addition, 20 patients with normal PFA-100 results underwent platelet aggregation and secretion studies. Of these, 17 had abnormal platelet aggregation and secretion predominantly in response to two or more weak agonists, including ADP, epinephrine and/or arachidonic acid. Only 3 of these 20 patients had normal platelet aggregation and secretion. Of the 16 patients with both abnormal PFA-100 and abnormal platelet aggregation and secretion tests, 11 underwent subsequent pre- and post- desmopressin platelet function testing. 7 of the 11 patients showed improvement or complete normalization of at least one PFA-100 parameter, i.e., either Collagen/ADP, Collagen/Epinephrine, or both. However, results of platelet aggregation and secretion tests remained unchanged, irrespective of PFA-100 results.Conclusions: 1.In the present study, platelet aggregation and secretion studies in patients with no other coagulopathies revealed platelet function defects in approximately 30% of the study cohort. Platelet function defects were in response to weak agonists, including ADP, epinephrine, and/or arachidonic acid.2.Our data support the recent SCC ISTH recommendations against the use of PFA-100 for platelet function screening.3.We recommend that the hematologist carefully assess the bleeding history, and, if significant, pursue platelet aggregation and secretion studies to identify potential platelet function defects. DisclosuresAledort:Baxter Healthcare: Membership on an entity's Board of Directors or advisory committees, Other: DSMB Participation; Kedrion BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Evaluation of a Standardized Approach to the Diagnosis of Mild Platelet Function Defects: Recommendation for a Targeted Approach to the Diagnosis of Mild Platelet Function Defects and Identification of a Subset of Children with a "Developmental" Form

Mild platelet function defects (MPFD) are a variable group of common inherited disorders characterized by mucocutaneous bleeding and excessive bleeding with trauma, surgery and procedures. The diagnosis is often based on subjective interpretations of expensive platelet function tests that require a substantial volume of blood. Furthermore, the diagnosis of MPFD can significantly limit participation in contact sports and physically demanding careers. To explore these issues, a retrospective chart review of patients from the Comprehensive Center for Bleeding Disorders of the BloodCenter of Wisconsin with a diagnosis of MPFD, and who had at least one follow-up evaluation was undertaken. The majority of patients had been evaluated by a two-step process; initially screened with CBC, PT, PTT, TT and a von Willebrand factor (VWF) panel. If these tests were normal, platelet function was assessed with light transmission aggregometry and chemiluminesence detection of ATP release in platelet rich plasma by the agonists TRAP, arachidonic acid (0.5 nM), ADP (10 uM), collagen (2.5 ug/mL) and high and low dose ristocetin.One hundred forty-eight patients diagnosed with a primary MPFD between 1982-2014 and with at least one recorded follow-up were identified. Bleeding scores were assigned by a system incorporating principles developed for the ISTH bleeding assessment tool. Patients ranged in age from 2 months to 57 years. For assessment of age on platelet function, patients were grouped as follows: Group A <2 years (N=23), Group B 2-4 years (N=33), Group C 5-8 years (N=36), Group D 9-16 years (N=30), and group E >16 years (N=26).At diagnosis, median bleeding score was 3 (range 0-12). Bleeding scores increased with age (p<.0001); the median (range) score was 2 (0-8) for A, 2 (0-5) for B, 3 (0-9) for C, 3 (0-7) for D and 4 (0-12) for E. There was no difference in gender by age group except group E with 23 females and 1 male; twenty of the 23 females reported menorrhagia. One hundred twenty eight (86%) of the patients had abnormal ATP release (122) or platelet aggregation (6) in the presence of ADP, the most common defect identified. Of those with abnormal release of ATP, 50% had another platelet function abnormality; there was no difference in bleeding scores between those with either single or multiple abnormalities. Of those who did not have abnormal aggregation or release with ADP, 8 had an abnormal response(s) with collagen alone and 1 had abnormal ATP release with arachidonic acid alone. At the most recent follow-up visit, (median 97 months, range 1-368 ) after the initial evaluation, the interim median bleeding scores were lower; for each age group the bleeding score (range) was 2 (0-8) for A, 1 (0-6) for B, 3 (0-7) for C, 2 (0-8) for D and 3.5 (0-10) for E. When compared to the oldest group (E), groups B and D showed a statistically significant difference (p <0.05).Twenty-nine of the patients had follow-up platelet function studies; of these, 13 (all were children) had a significant decrease in bleeding score and normalization of platelet function studies. There were no differences in age, gender, or family history of bleeding between those with persistent MPFD and those with normal follow-up platelet function tests. The persistent group had higher initial (p<0.03) and follow-up (p<0.02) bleeding scores. Those with normalization had a significant decrease in bleeding scores from diagnosis to follow-up (p<0.01) while the persistent MPFD group had no difference.These results suggest that a more targeted approach utilizing the analysis of ATP release by ADP, with TRAP release to confirm the presence of granules, will identify the majority of patients with mild platelet function defects. For those with suspected MPFD and normal ATP release with ADP, a full panel of agonists followed by additional step-wise evaluation as recommended by the ISTH should be undertaken. Limiting initial platelet function testing will reduce the volume of blood required and the cost. The reassessment of platelet function studies in patients with MPFD whose bleeding symptoms subside will identify a subset of these patients with normalization of platelet function. This group will no longer require treatment for procedures and restrictions of career choices or athletic pursuits. Thus, by utilizing a standard approach, patients with MPFD can be diagnosed and managed more efficiently and effectively with less cost to both the patient and healthcare system. DisclosuresFriedman:Alexion: Speakers Bureau; Novo Nordisk: Consultancy; CSL-Behring: Consultancy; Instrumentation Laboratories: Consultancy. Flood:Baxter: Consultancy; CSL Behring: Consultancy. Jobe:Bayer: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Membership on an entity's Board of Directors or advisory committees. Gill:Bayer: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Membership on an entity's Board of Directors or advisory committees.

A combination of pharmacophore modeling, molecular docking, and virtual screening for P2Y12 receptor antagonists from Chinese herbs

P2Y12, a member of the G-protein-coupled receptors, is associated with abnormal platelet aggregation, a condition that contributes to thrombus formation. As receptor antagonists are effective solutions for anti-thrombus, the P2Y12 receptor is a popular drug target. After the recent resolution of the P2Y12 receptor’s crystal structure, pharmacophore modeling and docking were combined to discover potential natural antagonists. Various approaches were used for the validation of the pharmacophore models and the optimization of docking algorithms. Hypo18, which was generated by 24 known antagonists, was determined to be the best hypothesis and is comprised of one ring aromatic, one hydrogen bond acceptor, one exclude volume, and three hydrophobic features. Hypo18 was thus utilized to screen TCMD (version 2009) to identify any potential active compounds, which then resulted in a hit list of 121 compounds with drug-likeness analysis. In addition, docking was used to refine the pharmacophore-based screening results as a cross-linking method. Then, the top 20 compounds with high docking scores were reserved. This paper provides a reliable source for discovering natural P2Y12 receptor antagonists from traditional Chinese herbs.

Isolated Factor V Deficiency in Myeloproliferative Neoplasms: A Mayo Clinic Series of 33 Patients

Background: Although the association of acquired isolated factor V (FV) deficiency with myeloproliferative neoplasms (MPN) has been described, it has not been well characterized. Objective: To characterize the clinical and laboratory features of patients with MPN and isolated FV deficiency. Methods: After IRB approval, patients with MPN who underwent coagulation profile diagnostic testing from January 1980 to December 2012 were identified via the Mayo Clinic Special Coagulation Laboratory Registry. Medical records of those with MPN and concurrent isolated FV deficiency or those with mild coagulopathy who had at least 20% discordantly lower FV activity were reviewed. Excluded were patients with known inherited bleeding diathesis, clinically significant liver disease, or on concurrent warfarin treatment. Data on patient demographics, clinical features and laboratory data were abstracted. Results: Thirty three patients met our study criteria (median age 67 years; 55% males; median BMI 25 kg/m2). Twenty one (64%) patients had either primary or post-polycythemia vera (PV) or post-essential thrombocythemia (ET) myelofibrosis; five (15%) had PV; three (9%) had unclassified MPN; two (6%) had ET; and one (3%) each had chronic myeloid leukemia and chronic myelomonocytic leukemia. Majority of these patients had splenomegaly (85%), with a median palpable spleen size of 14.3 cm below the left costal margin. Nearly 40% had hepatomegaly (median 4 cm below the right costal margin). Twelve (36%) patients were being treated with hydroxyurea; nine (27%) with aspirin and seven (21%) with an investigational agent. Nearly two-thirds (n=21; 64%) of these individuals had bleeding episodes, nine (43%) of which were provoked either by a surgical intervention or trauma. Majority of the bleeding episodes (n=15; 71%) were clinically significant/major bleed, as defined by the standard International Society of Thrombosis Haemostasis criteria, of whom eight required red cell transfusion support. Most of the bleeding were from muco-cutaneous surfaces (45%), followed by excessive bleeding from surgical site (24%). Median time from MPN diagnosis to bleeding was 5.4 years (range: 0 – 25). Of note, 42% (n=14) of all patients had thrombotic events (5 ischemic stroke/ TIA; 4 peripheral artery disease; 1 myocardial infarction; 1 splenic artery thrombosis; 3 leg DVT; and 3 splanchnic vein thrombosis) prior to their bleeding episode. Median time from MPN diagnosis to thrombosis was 1.4 years (range: 0.1 – 19.7). The median (range) leukocyte and platelet count were 17.8 (3.6 – 212.7) x 1000/ mm3 and 315 (16 – 1284) x 1000/ mm3, respectively. The median (range) PT [reference range: 10.3 – 12.8 sec] and aPTT [reference range: 26 – 36 sec.] were 13.3 sec (10.1 – 18.7) and 38 sec (29 – 66), respectively. The median FV activity [reference range: 70 – 165%] was 44% (range: 18% - 60%). Seven of the 18 individuals (39%) tested for von Willebrand syndrome had ristocetin cofactor activity (RCoA)/von Willebrand antigen (vWF Ag) ratio of &lt;0.7, although their vWF Ag and RCoA were within the normal reference range. Seventeen (52%) patients had platelet aggregation studies; all had at least one aggregation abnormality. Abnormal or absent response to epinephrine was the commonest noted abnormality (n=15), followed by an abnormal response to collagen (n=6). On univariate analysis, there was no significant correlation of FV activity with age, sex, BMI, myelofibrosis, hepatomegaly, splenomegaly, WBC count, platelet count, or use of hydroxyurea or investigational drugs. Further, on univariate analysis, neither the aforementioned covariates, nor aspirin use, WBC count, platelet count, FV activity, abnormal platelet aggregation, RCoA/vWF Ag ratio &lt; 0.7 were predictive of either overall bleeding, provoked bleeding or clinically significant/major bleed. Conclusions: Isolated FV deficiency associated with MPN often co-exists with platelet dysfunction and/or an acquired vWF abnormality, which together may contribute to a clinically significant bleeding diathesis. Our experience did not lead to identification of specific risk factors for MPN-associated isolated FV deficiency. Disclosures No relevant conflicts of interest to declare.