Platelet Function Defects Research Articles

PO-54 - Clinical and laboratory characterization of platelet dysfunction caused by ibrutinib treatment in patients with chronic lymphocytic leukemia

Ibrutinib treatment in patients with chronic lymphatic leukemia (LLC) is associated with bleeding-related adverse events. About 50% of treated patients display minor bleedings and about 5% major bleedings. A defect of platelet function has been hypothesized and inhibition of signaling by glycoprotein (GP) VI, which is the receptor for collagen, has been previously described. Ibrutinib-associated bleedings and platelet dysfunction may be relevant in the context of aged patients, who are often under antithrombotic treatments. Aim of this study is to investigate and characterize the effect of ibrutinib on platelet function in vitro in patients with CLL. Nine patients recruited in ongoing ibrutinib clinical trials were studied. Assessment of spontaneous bleeding was performed using the WHO bleeding scale. The following tests were performed before and after initiation of treatment with ibrutinib: 1) Light transmission aggregometry (LTA) using platelet-rich-plasma (ADP 2-4μM, PAR1-AP 25 μM, Collagen 10 ug /mL, arachidonic acid 1 mM, ristocetin 0.6-1.2mg/mL) 2) Measurement of vWF antigen and ristocetin cofactor activities by chemiluminescent immunoassay. I) Five patients displayed grade 1 bleeding (cutaneous bleeding) and one patient grade 2 bleeding (rectal bleeding) (66.7%) after initiation of ibrutinib treatment. II) Eight patients displayed abnormalities of the aggregation by 10 ug/ml collagen after initiation of ibrutinib treatment. At high collagen concentration, only significant prolongation of the lag phase was measured (60.4±10.6sec vs basal 38.4±17sec), whereas the maximal aggregation was not impaired (67.9±21.4% vs basal 85.5±5.8%). Compared to previous reports, these results confirmed an impairment of collagen induced aggregation, but at these concentrations only the lag phase was affected. III) Five patients displayed a significant improvement of the aggregation by 2 uM (91.25±5.26% vs basal 39.3±24.62%) and 4 uM (91±2.83% vs basal 65.42±19.43%) ADP after initiation of ibrutinib treatment. IV) vWF antigen and ristocetin cofactor activity were measured in 3 patients. In all patients vWF levels were higher at the onset of the disease (169±38%) and reduced up to normal values under Ibrutinib treatment (111.4±47%). Ibrutinib treatment in LLC patients causes a mild bleeding phenotype most probably due to platelet dysfunction. In this study, collagen induced aggregation resulted impaired, whereas the aggregation by PAR1-AP, ristocetin and arachidonic acid was not affected. On the contrary, the aggregation by ADP was improved upon ibrutinib treatment. The levels of vonWillebrand factor are significantly higher in LLC patients before treatment and were normalized by ibrutinib.

Modulating platelet reactivity through control of RGS18 availability

AbstractMost platelet agonists activate platelets by binding to G-protein–coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL−/− mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

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.

Mechanisms and Therapeutic Modulation of the Bleeding Tendency in Genetically-Engineered Von Willebrand Disease Type 2B Mice

Von Willebrand disease type 2B (VWD2B) is characterized by an abnormal increased affinity of von Willebrand factor (VWF) for platelets and related to mutations in the A1 domain of VWF. In patients, the VWD2B-mutant p.V1316M is specifically associated with a severe phenotype including hemorrhagic tendency, loss of high molecular weight VWF multimers, thrombocytopenia and platelet dysfunction related to defective αIIbβ3 integrin activation (Casari et al. JCI 2013, 123:5071). Genetically-engineered mice carrying the p.V1316M mutation (V1316M+/+ mice) perfectly mimic the human phenotype. We used this murine model to investigate the relative contribution of platelet count and platelet dysfunction to the occurrence of bleeding tendency and explore new therapeutic options.Bleeding tendency was evaluated using a novel tail-vein transection bleeding model involving the specific transection of the lateral vein at standardized depth and diameter (0.7 and 2.3mm respectively). Time (TVT) and blood loss were measured in wild-type (WT) and V1316M+/+ mice at baseline and after platelet count alteration.Compared to WT mice (platelet count: 1019±154G/L, n=58), V1316M+/+ mice (platelet count: 455±139G/L, n=52) have a significant increase (p<0.01) in TVT (25±9min, n=12 vs 2±1min, n=14) and blood loss (448±273µl vs 34±19µl). Induced thrombocytopenia (after infusion of anti-GPIb antibody) in WT mice (platelet count: 347±93G/L, n=6), to a similar level of V1316M+/+ mice, did not induce a hemorrhagic tendency (TVT: 2±1min, blood loss: 14±13µl). An increased bleeding phenotype in WT mice was exclusively observed in case of severe thrombocytopenia (platelet count: 70±25G/L, TVT>30min, blood loss: 532±168ml, n=6). Conversely, romiplostim-treated V1316M+/+ mice with normalized platelet count (platelet count: 866±100G/L, n=8) did not improve their bleeding tendency (TVT: 21±8min, blood loss: 448±282ml). These results suggest the bleeding phenotype in V1316M+/+ mice is independent of their thrombocytopenia, and could be related to the p.V1316M-associated defect in platelet function in general, and αIIbβ3 integrin activation in particular.Based on the hemostatic efficacy of recombinant activated factor VII (rFVIIa) in Glanzmann thrombasthenia, we tested rFVIIa in thrombocytopenic V1316M+/+ mice.A single infusion of rFVIIa (3 mg/kg) induced a complete and immediate correction of the bleeding phenotype (TVT: 4±2min, blood loss: 69±25ml, n=6). Thrombin generation measured in platelet-rich plasma before and after treatment showed a significant reduction of the lag time (1.13min, n=12 vs 1.8min, n=8 respectively, p<0.01).In conclusion, genetically-engineered mice carrying the VWD2B p.V1316M mutation display a bleeding phenotype that is not explained by their reduced platelet count, but is possibly related to the impaired platelet function induced by this VWF mutant. The bleeding tendency was rapidly and completely controlled by using rFVIIa. Based on these promising data, it would be of interest to consider rFVIIa as a therapeutic option in VWD-2B patients characterized by a severe hemorrhagic phenotype. DisclosuresNo relevant conflicts of interest to declare.

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.

Telehematology: Expanding Comprehensive Care for Pediatric Patients with Blood Disorders through Telemedicine at Several Delivery Sites (Medical Homes, Hospital Clinics, Home Setting and Hemophilia Treatment Centers (HTC)) and with Interdisciplinary Providers

Background: Telemedicine (TM) is the exchange of medical information from one site to another via electronic communications to improve patients' health status and access to care. We used telemedicine to provide comprehensive hematology services to patients with blood disorders at a variety of sites that included medical homes, hospital specialty clinics, HTC, patients home and teleconsultation with out of state HTC, all located at a distance from the Michigan State University Center for Bleeding and Clotting Disorders (MSU CBCD). The goals were to increase access to family centered and culturally competent specialty care and increase the numbers of patients with blood/bleeding/clotting disorder that were timely and accurately diagnosed, managed and referred for specialty care.Objectives: 1) To understand the feasibility of telemedicine between specialists and a variety of remote sites for children with bleeding/blood disorders 2) To assess the acceptability by patients, families, primary care physicians, remote site staff, specialist physicians, and specialist staff and 3) To assess the cost of telemedicine visits versus traditional visits from the societal perspective 4) To provide education and resources to distant providers.Methods: Telemedicine sites were 1) two medical homes-one at a pediatrician's office, Upper Great Lakes Family Health Center (UGLFHC)/Portage Health in Houghton MI and the other at a family medicine office, Iron Mountain/Iron River Upper Peninsula, MI 2) specialty clinics at Marquette General Hospital, Marquette MI 3) Hemophilia Treatment Centers (HTC) at Traverse City MI and Rush University IL 4) and a patient's home in MI. PolycomTM and/or VidyoTM systems were used to deliver HIPAA (Health Insurance Portability and Accountability Act) regulated bidirectional videoconferencing technology. Records of patient visits seen by local providers and via TM by MSU CBCD pediatric hematologists, nurse and social worker were obtained. Data included patient diagnosis, distance traveled, estimated time and travel costs saved, and education of distance provider. Measurable objectives included feasibility, acceptability. Technology issues were addressed. Site visits with Portage and Iron River Health Department were conducted to discuss local barriers and needs.Results: Beginning 1998, a total of 68 patients, ages 2 weeks to 17 years and their families were seen by TM at various sites. There were 97 TM visits and frequency of clinics varied monthly to as needed. Average distance travelled by patients to local clinics ranged from 12 - 70 miles (range 2-170 miles). In addition to coagulation disorders (hemophilia, von Willebrands disease, factor deficiencies, patients with bleeding symptoms such as epistaxis, menorrhagia etc.), other diagnosis such as cytopenias, spherocytosis, hemoglobinopathies, lymphadenopathy, Hereditary Hemorrhagic Telangiectasia, Ehlers Danlos Syndrome, and pediatric oncology patients in follow-up phase were also seen and in some cases family members tested. Physician and staff were educated regarding disease management; guidelines and educational materials shared. Estimated costs per patients to fly or drive to MSUCBCD were $999 ($579 -$1275) and $1653 ($1405-1887) respectively compared to obtaining care locally $87.6 ($73-117) via TM. This resulted in cost savings per patient of $911.4 and $1565.7 for driving and flying respectively. The MSU CBCD team also incurred significant cost savings by avoiding travel/lodging costs. Interviews with staff and physicians resulted in improvement in TM acceptance and delivery. A majority of laboratory testing were send-outs and local testing for platelet function defects were not available. Technology issues addressed included adjustments in bandwidth, firewall and network settings. It was feasible to do consultations with out-of-state HTCs as well as follow patients at home.Conclusion: Besides improving access to specialty care, TM with remote areas is feasible and acceptable in a variety of settings with considerable time and cost savings for both patients as well as the specialty care treatment team. Our study is in accordance with the American Academy of Pediatrics recent policy statement (Pediatrics 2015;136:202-209) recommending that TM services be coordinated through the medical homes thereby involving local primary care providers. DisclosuresKulkarni:Biogen: Research Funding, Speakers Bureau; Baxter: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; BPL: Membership on an entity's Board of Directors or advisory committees. Witkop:Pfizer: Other: Advisory Board, Research Funding; Baxter Bioscience: Other: Advisory Board; Novo Nordisk: Other: Advisory Board, Speakers Bureau.

Novel RUNX1 Mutation in a Family with an Uncharacterized Secretion Defect

Background: Platelet function disorders are a common cause of a bleeding problem. While many rare and severe forms of platelet function disorders are well studied, many common platelet function defects are uncharacterized. In a family with uncharacterized platelet secretion defects, we identified a single base pair insertion in the gene RUNX1 by exome sequencing. We report on the clinical and laboratory phenotype of the defect in this family.Methods: Bleeding histories of affected and unaffected family members, obtained using a standardized questionnaire, were scored using the International Society on Thrombosis and Haemostasis Bleeding Assessment Tool (ISTH BAT). Laboratory data evaluated included blood counts, platelet aggregation responses, dense granule ATP release findings (lumiaggregometry) and platelet dense granule counts, evauated by whole mount preparations and electron microscopy. Affection status was based on the recorded opinion of the subject's hematologist, which concurred with diagnostic laboratory findings. Exome sequencing was performed on the index case, followed by evaluation of all family members for the candidate mutation by polymerase chain reaction (PCR) amplification and Sanger sequencing.Results: Family members investigated (median age: 25.5, range 1-69] included 5 males (affected: n=4, unaffected: n=1) and 3 females (affected: n=2, unaffected: n=1). ISTH BAT bleeding scores for affected members were elevated (median: 10.5, range 4-20), unlike unaffected family members (n=2) and healthy controls (n=40) (ranges: 0-1 and 0-2). Platelet counts were low in 2/6 affected individuals (109 platelets/L : affecteds: median: 164, range: 125-169). While unaffected family members had unremarkable platelet function findings, the affected individuals had absent or reduced dense granule secretion with all agonists tested (including thrombin, collagen, epinephrine, U46619 and arachidonic acid), and in aggregation tests, they had absent secondary aggregation with epinephrine, reduced maximal aggregation with collagen (1.25 and 5.0 μg/mL) and thromboxane analogue U46619, variable responses to arachidonic acid (reduced in 4/5 affecteds), and normal responses to ADP and ristocetin. Only some (3/6) affected family members had dense granule deficiency (lower reference interval limit: 4.9/platelet; affecteds: median: 5.2, range: 4-6). A single base pair insertion (A) in exon 6 of the RUNX1 gene (NM_001754.4:c.583dup) was identified in the index case using exome sequencing. The mutation results in a reading frame shift at codon Ile195, that is expected to end in a premature termination codon 17 positions downstream. Sanger sequencing further confirmed this mutation was present in 6/6 affected family members and it was absent in both unaffected family members. Further evaluation of the family history indicated that only one family member (deceased aunt of the index case) had developed leukemia or myelodysplasia although her mutation status is unknown.Conclusion: We identified a novel, frameshift RUNX1 mutation in a family with an inherited platelet function disorder associated with increased bleeding symptoms. This variant has not been previously reported in exome sequencing of over 60 000 individuals as documented by the ExAC consortium. The variable platelet count and dense granule numbers among affected individuals in this family emphasize the importance of phenotyping multiple family members. RUNX1 defects should be considered as a potential cause of inherited abnormalities in dense granule secretion, even in individuals without dense granule deficiency or a striking family history of leukemia/myelodysplasia. DisclosuresNo relevant conflicts of interest to declare.

Identification of Molecular Defects in ITGA2B and ITGB3 Genes and Phenotypic Correlation in Pakistani Patients with Glanzmann Thrombasthenia

Background:Glanzmann thrombasthenia (GT) is most common inherited platelet functional defect. It is an autosomal recessive disorder, characterized by a bleeding diathesis. Incidence is increased in locations where consanguineous marriages are common. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3. This results in qualitative or quantitative abnormalities of the platelet receptor, αIIb-β3 integrin.Objectives:The aim of this study was to identify and correlate the mutations in GT patients with phenotype of the patient.Subjects and methods:20 patients with GT were enrolled in the study to identify the molecular defects and to correlate their phenotype with their genotype. CBC with peripheral film, PT, APTT and Fibrinogen levels were done initially. Platelet aggregation studies, flow cytometry, and mutation analysis was done by Sanger sequencing. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for GT gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). For any variation wild type was matched on HGMD (Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/index.php) and wild type color fasta sequence (http://pga.gs.washington.edu/). Pathogenecity score was evaluated by using software tools including :Polyphen-2(http://genetics.bwh.harvard.edu/pph2/) ,SNP&GO(http://snps.biofold.org/snps-and-go/index.html), MUpro (http://mupro.proteomics.ics.uci.edu/) ,SIFT (http://sift.jcvi.org), Provean (http://provean.jcvi.org/about.php) .All samples were sequenced at the Gene Sequencing Lab of NIBD (National Institute of Blood diseases and Bone Marrow Transplantation) Karachi.Results:Mutations were identified in all patients. Missense mutations were seen in most of the GT patients. The remaining mutations were heterogeneous and were distributed throughout the length of the gene.Conclusions:The severe type I GT was the most common subtype found in this study.Carrier detection and genetic counseling in these families is a potentially effective alternative for decreasing the burden of severe type of GT. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor RUNX1 Regulates Pctp (Phosphatidylcholine Transfer Protein) in Platelets: A Potential Role in Regulating Platelet Function

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a critical hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, numerous platelet function defects, and increased leukemia risk. We have previously described a patient with a heterozygous RUNX1 nonsense mutation in the conserved runt domain necessary for DNA binding (Sun et al Blood 2004;103;948-54). The patient's platelet abnormalities included impaired aggregation and secretion in response to multiple agonists, granule deficiency, and decreased phosphorylation of myosin light chain and pleckstrin, activation of GPIIb-IIIa, 12-HETE production, and platelet protein kinase C-θ. Transcript expression profiling of patient platelets (Sun et al, J Thromb Haemost 2007;5:146-54) showed numerous 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 is a member of the START (Steroidogenic Acute Regulatory Protein-Related Transfer) domain superfamily and is responsible for the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Several findings indicate that PCTP is important for platelet function. PC is the main fraction of platelet phospholipids and source of arachidonic acid (AA) upon activation. Release of free AA from PC is the rate-limiting step in thromboxane production. PC is also a substrate for phospholipase D, which yields phosphatidic acid that can generate the second messenger diacylglycerol. Importantly, platelet PCTP has recently been demonstrated to have a major role in the racial differences in platelet responses - increased PCTP expression has been linked to the higher platelet aggregation and calcium mobilization induced by thrombin receptor protease-activated receptor 4 (PAR4) in blacks as compared to whites (Edelstein et al Nat Med 2013;19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1. 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 995 bp 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 24 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding sites 1 (-232/-227) and 2 (-345/-340), at site 3 (-519/-514), and encompassing sites 4 and 5 (-861/-856, -884/-879). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-232/-227) and both sites 4 and 5 (-861/-856, -884/-879). To assess the effect of modulating RUNX1 expression on PCTP expression, PMA-treated HEL cells were transfected with RUNX1 overexpression plasmid or siRNA. PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown, suggesting that PCTP is regulated by RUNX1.Conclusions: Our results provide evidence that PCTP is a direct transcriptional target of and regulated by RUNX1, and a cogent molecular mechanism for downregulation of platelet PCTP in our patient with RUNX1 haplodeficiency. Regulation of PCTP by RUNX1 may be relevant to the platelet dysfunction in RUNX1 haplodeficiency as well as to racial differences in platelet responses linked to the differential platelet expression of PCTP. DisclosuresNo relevant conflicts of interest to declare.

Platelet Function Testing Is Commonly Performed in Patients with Known Confounding Factors

Introduction: Commonly used platelet function tests include Platelet Function Analyzer -100 Closure Time (PFA-100), used as a screening test, and platelet aggregation testing by Light Transmission Aggregometry (LTA), which can help differentiate between various types of platelet function defects and guide further testing. In this study, we assessed the indications for platelet aggregation testing at our center, and the effect of factors that may interfere with the outcomes of testing.Methods: This was a retrospective electronic medical records based study in which we included all patients in our healthcare network who had platelet aggregation testing done using LTA between August 2008 and August 2013. Routine descriptive measures were used for frequencies and proportions. McNemar test was used for comparison of paired nominal variables and Wilcoxon signed-rank test was used for comparison of paired continuous variables. P value < 0.05 was considered significant.Results: Four hundred ninety seven patients had platelet aggregation testing performed during the 5-yr study period. Among these, 315 (63%) patients had antecedent screening with PFA-100 with 157 (31%) showing at least one abnormality. Two hundred forty nine (50%) patients underwent some form of further testing after LTA, including VWF analysis (42%), platelet flow cytometry (19%) and/or electron microscopy (1%). Two hundred fifty six (51%) patients had at least one factor previously known or suspected to interfere with platelet aggregation testing (platelet count <100,000, hematocrit <25% or medications), and 205 (80%) of these patients had abnormal results in response to one or more agonists. Eighty three of these patients had repeat aggregation testing performed - 26 were re-tested after correction of 1 or more suspected factor (s), while 57 were re-tested without correction. The former group showed a significant improvement with subsequent testing, both in the average number of abnormalities in the LTA panel (P = 0.01) and in the number of patients with abnormal aggregation to ≥2 agonists (P = 0.02), while the outcomes of platelet function testing in the latter group did not significantly change on subsequent testing (P = 0.15 and 0.21 respectively for the above two parameters). Three hundred forty five (69%) patients had a current or past history of abnormal bleeding. Of these, 81% had history of spontaneous bleeding while 29% had surgical or procedure-related bleeding. The most common sites of spontaneous bleeding were skin (47%) and mucosa (45%). Eight percent of patients had a history of spontaneous bleeding in deeper sites. Fourteen percent had a family history of abnormal bleeding. The most common indication for testing was documented abnormal bleeding (62%) - recent or past. Twenty five percent underwent testing for peri-operative prediction of bleeding risk and 13% to monitor efficacy of anti-platelet therapy.Conclusions: A majority of the patients in our study did not have abnormalities in PFA-100 testing despite abnormal results on subsequent platelet aggregometry. Fifty one percent of patients who underwent aggregometry were on medications that may affect platelet function or had other factors known or suspected to interfere with aggregation testing. Eighty percent of these patients had abnormal results. Repeat testing after correction of the known factor(s) led to a significant improvements in the results of LTA. The most common indication for platelet aggregation testing was a current or past history of abnormal spontaneous bleeding and the most common sites of bleeding in these patients were the skin and mucosal surfaces. DisclosuresMcCrae:Momenta: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Halozyme: 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.

Expression and Release of Platelet Protein Disulfide (PDI) Isomerase Is Increased in Patients with Hemophilia a

Hemophilia A is an X-linked, recessive bleeding disorder caused by congenital factor VIII (FVIII) deficiency. Although the bleeding tendency largely depends on residual FVIII activity (FVIII:C), there is tremendous heterogeneity in bleeding frequency and severity among individuals with similar FVIII:C plasma levels. It is therefore likely that additional factors modulate thrombin generation and fibrin deposition in patients with hemophilia A. PDI is an abundant oxidoreductase with chaperone activity that is also present in human platelets and released upon activation. Preclinical studies indicate that extracellular PDI is critical to hemostasis, thrombosis and vascular inflammation. In particular, PDI has been implicated in monocyte/macrophage tissue factor activation, integrin regulation and platelet-associated thrombin generation. Furthermore, impaired PDI release has most recently been shown to contribute to the bleeding tendency of Hermansky-Pudlak syndrome, an inherited platelet function defect. To explore the role of platelet PDI in hemophilia A, we studied 24 patients (15 severely, 5 moderately and 4 mildly affected) in comparison to 12 age- and sex-matched controls. Expression of PDI antigen on resting platelets and platelets stimulated with either 20 µM ADP or 50 µM thrombin receptor activator peptide 6 (TRAP-6) was assessed by flow cytometry using a fluorescently labeled monoclonal antibody. Analysis of CD41 and CD62P (P-selectin) served as positive controls for constitutive platelet antigen expression and α-granule secretion, respectively. In addition, release of soluble PDI antigen into platelet supernatants was measured by ELISA. There was no significant difference in baseline CD41, CD62P and PDI antigen expression between patients and controls. Furthermore, ADP- and TRAP-6-induced CD62P expression was similar between the two groups (percent positive platelets in patients vs. controls: 28±14 vs. 32±15% and 80±12 vs. 83±9% for ADP- and TRAP-6-treated platelets, respectively). However, expression of PDI antigen on platelets stimulated with either ADP (3.3±2.1 vs. 1.5±1.2%, P<0.01) or TRAP-6 (3.4±1.7 vs. 2.1±1.3%, P<0.05) was significantly increased in patients compared to controls. While ADP-induced release of PDI antigen into platelet supernatants was similar between the two groups and not significantly different from baseline, stimulation with TRAP-6 resulted in significantly increased PDI antigen levels in platelet releasates from patients vs. controls (median, range): 1.5, 0.2-23.2 ng/mL vs. 0.4, 0.2-1.9 ng/mL (P<0.01). Importantly, in two patients with exceedingly high TRAP-6-induced PDI release over baseline (4.8 vs. 0.3 ng/mL and 23.2 vs. 2.8 ng/mL), findings were consistent when platelets were isolated and stimulated on a separate occasion (5.5 vs. 1.3 ng/mL and 10.2 vs. 0.2 ng/mL). Taken together, agonist-induced platelet PDI expression was significantly increased in patients with congenital hemophilia A. Furthermore, release of PDI antigen into supernatants of TRAP-6-activated platelets was significantly increased in patients compared to healthy controls. Up-regulation of platelet PDI may thus represent a compensatory mechanism under conditions of defective thrombin generation and fibrin deposition, and variations in platelet PDI expression and release could at least partially explain the heterogeneity in bleeding severity among patients with congenital hemophilia A and similar FVIII:C plasma levels. DisclosuresLanger:Baxalta: Consultancy, Other: Travel support; Pfizer: Research Funding; CSL Behring: Consultancy, Other: Travel support, Research Funding. Voigtländer:CSL Behring: Other: Travel support. Holstein:CSL Behring: Consultancy, Other: Travel support, Research Funding.

Evaluation of platelet function in horses undergoing colic surgery using the PFA-100 platelet function analyser

Acute colic in horses, especially presentations requiring surgical correction, such as large colon volvulus, very often involves defective or excess platelet activation. The PFA-100 is a new point-of-care analyser that evaluates platelet function by measuring closure time (CT) in the whole blood of healthy horses in a stan - dardised manner. However, there are no reports on platelet function in horses with colic measured by the PFA-100. The aim of the present study was to investigate platelet function in a group of horses suffering ascending colon displacement (ACD) and who underwent surgery compared to a group of control (C) horses. Thirty ACD cases and twenty untreated clinically healthy control horses were included. Blood samples were collected from the ACD horses prior to and 24, 48 and 72 h after laparotomy. Red blood cell count (RBC), haematocrit (HCT), hae - moglobin (Hb) concentration, white blood cell count (WBC), total plasma protein (TP) concentration, platelet count (PLT), platelet volume (MPV) and ADP closure time (CT-ADP) were measured. CT was determined on a PFA-100® device using collagen/ADP cartridges. There were no significant differences in RBC, PLT and MPV between C and ACD horses pre-operatively. Reduced PLT was observed in the post-operative period. The mean CT-ADP of C horses was 96.43 ± 12.53 and 91.43 ± 2.51 seconds in the ACD group prior to surgery. CT-ADP was changed after surgery whereby CT-ADP time was more than doubled. We conclude that the PFA-100 ® can be used to detect platelet function defects in horses.

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function-Associated Antigen-1 and Kindlin-3.

Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA β2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA β2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

SummaryPlatelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF ≥ 1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Remote platelet function testing – Significant progress towards widespread testing in clinical practice

Platelets play a significant role in the physiological process of haemostasis by ensuring the integrity of the vessel wall and initiating primary haemostatic mechanisms when the vessel wall is damaged. They also contribute to the formation of the blood clots that give rise to heart attack and stroke. It is well known that platelet function can be impaired – either elevated or decreased – due to inherited or acquired factors. Reduced platelet function is most often the result of inherited defects in platelet morphology/ function or a consequence of the use of a variety of pharmacological agents that either specifically inhibits platelets to reduce the risk of heart attacks or stroke or those with alternative therapeutic targets that can also affect platelet function. Several dietary components and herbs are also known to affect platelet responsiveness [1]. Accordingly, there are several clinical scenarios when platelet function needs to be assessed. A range of techniques are available to test platelet reactivity, however, one of the major limitations of all available methodologies is that the testing needs to be performed on a fresh blood sample within 2–4 hours following venipuncture [2]. Even if the technique is not too complex and time consuming, it still limits platelet testing to a specialized laboratory or at least a laboratory where the appropriate equipment is installed and experienced personnel are available to perform the tests. Such a limitation is a significant obstacle. For example, in the case of diagnosis of platelet function defects, there may be an underestimation of their prevalence with cases tending to be clustered near specialised diagnostic centres [3]. One of the most commonly used methodologies for measuring platelet function is light transmission aggregometry (LTA), which is still considered the ‘‘gold standard’’ method despite the fact that it was developed long ago in the early 1960s [4]. This method can assess platelet aggregation together with its potentiation via amplification mechanisms, such as TxA2 synthesis and granule release, and register a two-phase aggregation response. This dynamic information is perhaps the most useful characteristic of the LTA and is not available with any other aggregation technique. Although informative, the whole procedure is timeand labourintensive, and involves multiple steps from preparation of plateletrich plasma (PRP) to the actual aggregation measurements. There is also significant variability in performance of LTA between different laboratories [5, 6], and this reduces the diagnostic value of the test as the results become incomparable. Recently, recommendations on standardisation of the LTA procedure were issued [2] with the aim to improve reproducibility and comparability of the LTA; however, the technique is still too cumbersome for routine clinical use. Another limitation of LTA is that the method requires large volumes of blood for PRP preparation, which presents a significant challenge when platelet function needs to be assessed in individuals in whom the volume of blood sample is limited. To overcome some of these limitations, modern technologies have been developed, such as the VerifyNow system (Accumetrics, San Diego, CA) and Multiple Electrode Aggregometry (MEA, Multiplate , Roche Diagnostics International Ltd, Rotkreuz, Switzerland). Currently, their application is mostly restricted to assessing the effectiveness of antiplatelet therapy, although MEA has been used in two studies to diagnose one of the most prominent inherited platelet defects – Glanzmann thrombasthenia [7, 8]. These tests are certainly simpler, and hence less time consuming and labour intensive, they are performed on whole blood and are much better standardised than the traditional LTA. However, their downside is that they utilise a dedicated piece of sophisticated equipment and use of specific consumables, which together make these methodologies quite expensive to run. Another important application of platelet function testing is within the drug discovery area, when novel drugs designed to affect platelet function are being developed or when the effect on platelets needs to be excluded as an unwanted effect for a drug with the therapeutic target other than platelets. In this scenario, complex or expensive methodology is not ideal, as pharmaceutical industry studies often require multiple samples to be processed in a limited time period, in multiple centres, and require that methodology is standardized across all participating centres. Keywords

Current status and future prospects for platelet function testing in the diagnosis of inherited bleeding disorders.

Platelets play a crucial role in haemostasis by preventing bleeding at the site of vascular injury. Several defects in platelet morphology and function have been identified and described over the years. Although a range of methodologies is available to assess platelet function, a significant proportion of subjects with bleeding symptoms and normal coagulation parameters still appear to have normal results on platelet function testing. This might suggest that the reason for bleeding is multifactorial and is due to a combination of several minor defects in platelet function and/or other parts of the haemostatic system or might indicate that the currently available platelet function tests do not provide optimal diagnostic power. This review will summarize the established platelet function tests used for diagnosing inherited platelet abnormalities in adults and children, and discuss the newly developed methodologies as well as unmet challenges and potential areas for further improvement in this field.

Point-of-Care Testing in Critically Ill Patients

Point-of-care (POC) testing in hemostasis has experienced a significant increase in the spectrum of available tests and the number of tests performed. Short turn-around time and observation of rapid changes in test results are facilitated. The quality control process in POC testing must encompass a preanalytic (collection), analytic (measurement), and postanalytic (clinical response) phase. Erroneous interpretation of findings and difficult quality controls can outweigh the advantages of POC testing.Only a limited number of hemostatic POC tests have proven useful so far: prothrombin time POC-monitoring of oral vitamin K antagonists; activated clotting time POC-monitoring of high-dose heparin therapy; platelet function analyzer (PFA; Siemens, Marburg, Germany) closure time (CT)-detection of von Willebrand disease and severe platelet function defects; whole blood aggregometry (WBA) Multiplate (Roche Diagnostics, Rotkreuz, Switzerland), and the VerifyNow system (Accumetrics, San Diego, CA)-detection of platelet dysfunction due to antiplatelet drugs; thromboelastography-continuous observation of clot formation and fibrinolysis. The use of various agonists in WBA and thromboelastography (TEG) requires some expertise. In experienced hands the PFA CT and WBA and TEG are recommended combinations.Application of POC testing depends strictly on whether it improves medical care and patient outcome. More POC test systems are in the research pipeline, but only a few will resist the ravages of time.

Analysis of platelet function and dysfunction.

Although platelets act as central players of haemostasis only their cross-talk with other blood cells, plasma factors and the vascular compartment enables the formation of a stable thrombus. Multiple activation processes and complex signalling networks are responsible for appropriate platelet function. Thus, a variety of platelet function tests are available for platelet research and diagnosis of platelet dysfunction. However, universal platelet function tests that are sensitive to all platelet function defects do not exist and therefore diagnostic algorithms for suspected platelet function disorders are still recommended in clinical practice. Based on the current knowledge of human platelet activation this review evaluates point-of-care related screening tests in comparison with specific platelet function assays and focuses on their diagnostic utility in relation to severity of platelet dysfunction. Further, systems biology-based platelet function methods that integrate global and specific analysis of platelet vessel wall interaction (advanced flow chamber devices) and post-translational modifications (platelet proteomics) are presented and their diagnostic potential is addressed.

Referral Practices in Management of Heavy Menstrual Bleeding in Adolescents

▪Background: Due to the prevalence of mild bleeding disorders (MBDs) among adolescent females with heavy menstrual bleeding (HMB), the American College of Obstetricians and Gynecologists (ACOG) and the American Academy of Pediatrics (AAP) recommend screening select adolescent patients with HMB for mild bleeding disorders (MBDs) under the guidance of a hematologist, as coagulation labs can be difficult to interpret. The goal of this study was to describe referral patterns for adolescents with HMB and compare evaluation and management practices among different pediatric specialties using Medicaid claims data.Methods: Our patient population comprised 10-17 year old females enrolled in Medicaid in central and southeastern Ohio with at least 2 separate diagnoses of HMB and at least 12 months of continuous enrollment prior to their first HMB diagnosis. While there is no standard definition of “severe” HMB, for this study, patients with iron deficiency anemia, an inpatient hospital stay for HMB, or an outpatient blood transfusion were defined as having severe HMB. Medicaid claims data and electronic medical records from Nationwide Children’s Hospital, the region’s only children’s hospital, were linked to collect study data.Results: We identified 509 patients with HMB. Referral to a pediatric specialist occurred in 17.9% of patients; 39.5% of those with severe HMB and 13.8% of those with non-severe HMB were referred. 10.8% of patients were referred to adolescent med, 6.7% were referred to hematology, and 0.4% were referred to endocrinology. Of the patients seen by a specialist at a children’s hospital, 69% were screened for a MBD. When stratified by specialty, 100.0% of patients seen by hematology were screened for a MBD while 51.0% of patients seen by adolescent medicine were screened for a MBD. Approximately one-third (35.7%) of referred patients were diagnosed with a MBD, most commonly platelet function defects and von Willebrand disease (VWD). 48.3% of patients with severe HMB were diagnosed with a MBD, while 29.1% of patients with non-severe HMB were diagnosed with a MBD. Both hematologists (100.0%) and adolescent medicine doctors (97.0%) almost uniformly prescribed treatment for HMB, most commonly hormonal contraception (95.2%). Patients had significantly higher odds of referral to a pediatric specialist if they had severe HMB [2.3, 95% CI (1.42-3.71)]. Patients had significantly lower odds of referral if they lived farther away from the region’s children’s hospital [0.16, 95% CI: (0.09-0.29)]. There was no significant difference in frequency of referral between patients initially diagnosed by family practitioners, pediatricians, or gynecologists.Conclusions: Despite ACOG and AAP recommendations, we found that only a small percentage of adolescents with HMB are referred to a pediatric specialist. Efforts should be made to increase education regarding which patients are at higher risk of an underlying MBD, and increasing the number of these adolescents who are referred to hematology so that coagulation lab studies can be ordered and interpreted appropriately. Such efforts should be targeted at patients in rural areas, since this population was particularly unlikely to receive care from a hematologist. DisclosuresNo relevant conflicts of interest to declare.