Qualitative Platelet Abnormalities Research Articles

Heavy Menstrual Bleeding in Adolescent: Normal or a Sign of an Underlying Disease?

Heavy, and often irregular, menstrual bleeding (HMB) is a common gynecologic complaint among adolescents. During the first few post-menarcheal years, anovulatory cycles related to immaturity of the hypothalamic-pituitary-ovarian axis are the most common etiology for abnormal uterine bleeding and should be considered as a part of normal pubertal development rather than a disease. If an already regular menstrual cycle becomes irregular, secondary causes of anovulation should be ruled out. Inherited and acquired bleeding disorders, such as von Willebrand disease, and quantitative and qualitative abnormalities of platelets are relatively common findings in adolescents with HMB from menarche. History of excessive bleeding or a diagnosed bleeding disorder in the family supports this etiology, warranting specialized laboratory testing. First-line treatment of HMB among adolescents is medical management with hormonal therapy or nonhormonal options. Levonorgestrel-releasing intrauterine device is an effective tool also for all adolescents with menstrual needs.

Platelets, extracellular vesicles and coagulation in pulmonary arterial hypertension.

Pulmonary arterial hypertension is a rare disease of the pulmonary vasculature, characterised pathologically by proliferation, remodelling and thrombosis in situ. Unfortunately, existing therapeutic interventions do not reverse these findings and the disease continues to result in significant morbidity and premature mortality. A number of haematological derangements have been described in pulmonary arterial hypertension which may provide insights into the pathobiology of the disease and opportunities to explore new therapeutic pathways. These include quantitative and qualitative platelet abnormalities, such as thrombocytopaenia, increased mean platelet volume and altered platelet bioenergetics. Furthermore, a hypercoagulable state and aberrant negative regulatory pathways can be observed, which could contribute to thrombosis in situ in distal pulmonary arteries and arterioles. Finally, there is increasing interest in the role of extracellular vesicle autocrine and paracrine signalling in pulmonary arterial hypertension, and their potential utility as biomarkers and novel therapeutic targets. This review focuses on the potential role of platelets, extracellular vesicles and coagulation pathways in the pathobiology of pulmonary arterial hypertension. We highlight important unanswered clinical questions and the implications of these observations for future research and pulmonary arterial hypertension-directed therapies.

Enfermedades de la hemostasia primaria. Púrpuras vasculares. Enfermedades de las plaquetas

Diseases of primary hemostasis. Vascular purpura. Platelet disorders: This update addresses the group of disorders that affects blood vessel walls and platelets, which are a fundamental part of primary hemostasis. They are divided into purpura (vascular involvement) or quantitative or qualitative platelet abnormalities and can be congenital or acquired. The incidence of many of these entities is low; some are considered rare diseases. The typical hemorrhagic manifestations include the appearance of cutaneous and mucosal bleeding with trauma. Some congenital coagulopathies form part of groups of syndromes in which involvement goes beyond bleeding symptoms, requiring a multidisciplinary approach. In all coagulopathies, it is important to take a thorough personal and family medical history. In this regard, platelet function and aggregation tests are useful. More and more, the genetic alterations behind these cases are becoming known, with molecular diagnostic techniques gaining greater importance. In congenital forms, treatment is symptomatic, targeted according to the localization, and accompanied by multidisciplinary teams. For cases with platelet function involvement, a platelet transfusion can also be opted for.

Clonal hematopoietic mutations linked to platelet traits and the risk of thrombosis or bleeding.

Platelets are key elements in thrombosis, particularly in atherosclerosis-associated arterial thrombosis (atherothrombosis), and hemostasis. Megakaryocytes in the bone marrow, differentiated from hematopoietic stem cells are generally considered as a uniform source of platelets. However, recent insights into the causes of malignancies, including essential thrombocytosis, indicate that not only inherited but also somatic mutations in hematopoietic cells are linked to quantitative or qualitative platelet abnormalities. In particular cases, these form the basis of thrombo-hemorrhagic complications regularly observed in patient groups. This has led to the concept of clonal hematopoiesis of indeterminate potential (CHIP), defined as somatic mutations caused by clonal expansion of mutant hematopoietic cells without evident disease. This concept also provides clues regarding the importance of platelet function in relation to cardiovascular disease. In this summative review, we present an overview of genes associated with clonal hematopoiesis and altered platelet production and/or functionality, like mutations in JAK2. We consider how reported CHIP genes can influence the risk of cardiovascular disease, by exploring the consequences for platelet function related to (athero)thrombosis, or the risk of bleeding. More insight into the functional consequences of the CHIP mutations may favor personalized risk assessment, not only with regard to malignancies but also in relation to thrombotic vascular disease.

Platelets as Mediators of Thromboinflammation in Chronic Myeloproliferative Neoplasms

Chronic myeloproliferative neoplasms (MPN) are stem cell disorders driven by mutations in JAK2, CALR, or MPL genes and characterized by myeloid proliferation and increased blood cell counts. They encompass three closely related conditions, including essential thrombocythemia, polycythemia vera, and primary myelofibrosis. Elevated levels of cytokines released by clonal and non-clonal cells generate a chronic proinflammatory state that contributes to disease pathogenesis. Thrombosis represents the most common cause of morbidity and mortality in MPN, although paradoxically, patients may also present with a bleeding diathesis. The mechanisms leading to thrombosis are complex and multiple and include increased blood cells together with qualitative abnormalities of red cells, leukocytes, and platelets that favor a prothrombotic activated phenotype. The functional interplay between blood cells, the clotting cascade, and dysfunctional endothelium contributes to hypercoagulability and this process is perpetuated by the effect of inflammatory cytokines. In addition to their well-known function in hemostasis, platelets contribute to innate immunity and inflammation and play a key role in MPN thromboinflammatory state. In vivo platelet activation leads to platelet aggregate formation and exposure of adhesion molecules which favor their interaction with activated neutrophils and monocytes leading to circulating platelet-leukocyte heterotypic aggregates. Platelets are recruited to the activated endothelium further enhancing the reciprocal activation of both cell types. Crosstalk between activated cells drives cytokine production, further fuelling the self-reinforcing thromboinflammatory loop. In addition, MPN platelets provide a procoagulant scaffold which triggers the coagulation cascade and platelet-derived microparticles amplify this response. Markers of platelet, leukocyte, endothelial and coagulation activation are increased in MPN patients although prospective studies are required to determine the potential value of these parameters for identifying patients at increased thrombotic risk. Thrombosis remains the main complication of MPN patients, with a high risk of recurrence despite adequate cytoreductive and antithrombotic treatment. Deeper insight into the mechanism favoring thrombosis development in this setting may lead to novel therapeutic approaches for MPN thrombosis. Considering the critical role of inflammation in the vascular risk, concomitant targeting of inflammatory pathways could potentially impact on primary or secondary prevention strategies.

Platelet count and indices in acute uncomplicated Malaria in Kano, Nigeria

Background: Quantitative and qualitative abnormalities of platelets are common among patients with malaria infection, and these correlate with severity. Few studies documented platelet changes in uncomplicated malaria in the endemic regions. This study aimed to describe the changes in platelet counts (PLTs) and automated platelet indices in uncomplicated malaria infection in a Nigerian population. Materials and Methods: This is a comparative cross-sectional study of 152 patients presenting with symptoms of uncomplicated malaria to the general outpatient Department of Aminu Kano Teaching Hospital, Kano, Nigeria. Malaria diagnosis was made using gold-standard microscopy of Giemsa-stained thick blood smear, while PLTs and indices (mean platelet volume [MPV], plateletcrit [PCT], platelet distribution width [PDW] and platelet-large cell ratio [P-LCR]) were determined using automated haematology analyser. Data were analysed using STATA v13 and results presented in descriptive terms. P =0.05 was considered statistically significant. Results: There were more female 8 (51.3%) than male 74 (48.7%) patients. A total of 84 patients (55.3%) were malaria positive. The prevalence of malaria-positive smears was higher in males 48 (31.6%) and among the age groups of 5–14 and 15–24 years (13.8% each). Thrombocytopenia was seen in 15 (19.7%) and 2 (2.6%) of malaria-positive and malaria-negative individuals, respectively, and the difference was statistically significant (P = 0.001). Platelet indices reveal significant differences in the MPV (P = 0.0016) and P-LCR (P = 0.0265) between malaria-positive and malaria-negative patients. Although both lower mean PLT and PCT, and higher PDW were found in malaria-positive than in malaria-negative individuals, this difference was not statistically significant (P > 0.05). Conclusion: This study showed that abnormalities in PLT and indices are more common among patients with an acute febrile illness who are malaria positive than those who are malaria negative.

A guideline for the management of specific situations in polycythaemia vera and secondary erythrocytosis: A British Society for Haematology Guideline.

The previous BSH guideline for the management of erythrocytosis was published in 2005 (McMullin et al, 2005) and amended in 2007 (McMullin et al, 2007). Here, we re‐evaluate the literature formulate guidance on the management of specific situations encountered in polycythaemia vera (PV) and the management of the other types of secondary erythrocytosis. Recommendations for the diagnostic pathway of investigation of an erythrocytosis, risk stratification and management of PV are in the accompanying guideline (McMullin et al, 2018). We review evidence and outline guidance on management of acute thrombotic events and secondary prevention of thrombosis in PV. The unusual thrombotic events, splanchnic vein and cerebral vein thromboses are discussed and haemorrhage. The specific situations of surgery and pregnancy and guidance on management of pruritus are included. The evidence for the management of other causes of erythrocytosis, including idiopathic erythrocytosis, congenital erythrocytosis, hypoxic pulmonary disease and post‐transplant erythrocytosis, is reviewed and recommendations made.

Observational retrospective study of vascular modulator changes during treatment in essential thrombocythemia

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU+ASA, 15 on ANA+ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA+ASA (P<0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P<0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

Platelet Disorders

A bleeding disorder may be suspected when a patient reports spontaneous or excessive bleeding or bruising, often secondary to trauma. Possible causes can vary between abnormal platelet number or function, abnormal vascular integrity, coagulation defects, fibrinolysis, or a combination thereof. This review addresses hemorrhagic disorders associated with quantitative or qualitative platelet abnormalities, such as thrombocytopenia, platelet function disorders, thrombocytosis and thrombocythemia, and vascular purpuras. Hemorrhagic dis­orders associated with abnormalities in coagulation (e.g., von Willebrand disease and hemophilia) are not covered. An algorithm shows evidence-based practice guidelines for the management of immune thrombocytopenic purpura. Tables list questions regarding bleeding and bruising to ask patients, clinical manifestations of hemorrhagic disorders, typical results of tests for hemostatic function in bleeding disorders, causes of thrombocytopenia, other forms of drug-induced thrombocytopenia, classification of platelet function disorders, and selected platelet-modifying agents. This review contains ­1 highly rendered figure, 7 tables, and 82 references.

Retroperitoneal hemorrhage associated with bone marrow trephine biopsy

Patient: Male, 19Final Diagnosis: Hyperleukocytosis • thrombocytosisSymptoms: Hyperleukocytosis • retroperitoneal hemorrhage • thrombocytosisMedication: —Clinical Procedure: Bone marrow trephine biopsySpecialty: Hematology • RadiologyObjective:Diagnostic/therapeutic accidentsBackground:Bone marrow (BM) trephine biopsy is generally a safe procedure, but adverse events such as retroperitoneal hemorrhage (RPH) may occur. We report 3 cases of this complication.Case Report:A 19-year-old male with thrombocytopenia and coagulopathy underwent BM trephine biopsy to confirm relapse of acute lymphoblastic leukemia. Two hours later, he developed severe hypotension and a CT scan revealed a massive RPH, and was treated conservatively. The RPH recurred 2 weeks after chemotherapy and was successfully treated with gel foam embolization. A 55-year-old male with coagulopathy underwent BM trephine biopsy for hyperleukocytosis and thrombocytosis. He developed a large RPH preceded by left lumbar dermatome sensory neuropathy. He was treated conservatively. A 56-year-old overweight woman on aspirin underwent BM trephine biopsy for polycythemia. Twelve hours later she developed severe abdominal pain with hypotension. A CT scan showed a massive RPH and secondary hemothorax. She was treated conservatively and the RPH resolved after several months.Conclusions:We and others showed that myeloproliferative neoplasm, quantitative or qualitative platelet abnormalities, aspirin, coagulopathy, and obesity are associated with development of RPH following BM trephine biopsy. Early diagnosis and intervention are crucial. Correction of coagulopathy and cessation of anti-platelet treatment prior to biopsy can prevent this serious complication.

Blood Cells Derived Microparticles and Vascular Markers Study in Essential Thrombocythaemia Patients

AbstractAbstract 5193Essenthial thrombocythemia (ET) patients are at risk of developing an arterious or venous thrombotic event. Recent studies have shown that there is no clear association between platelet (PLT) count and thrombotic events, but that white cell count plays a role. Qualitative PLT abnormalities, activation of endothelial cells (EC) and PLT may be involved in these processes. Following activation of blood cells, plasma membrane vescicles are released, these are known as microparticles (MP) (<1. 5I′¼m of size). They may originate from PLT (PMP), EC (EMP) or red cells (RMP). MP may affect endothelial modulators such as vasorelaxant nitric oxide (NO), adrenomedullin (ADM) and endothelin-1 (ET-1), a potent vasocostrictor agent. Previous studies [Trappenburg MC et al 2009] demonstrated that MP are present in elevated numbers in ET. However, the relationship between MP with treatment and MP with endothelial modulators is unknown. We investigated the relationship between MP, NO, ADM and ET-1 in ET patients on treatment with hydroxyurea (HU), anagrelide (AN) and aspirin(ASA) only. 52 patients with ET diagnosis were studied: 18 on HU, 15 on AN, and 19 on ASA. Platelet-poor-plasma aliquotes were stored at −80A′°C and subsequently processed. Samples were analysed for MP numbers (absolute total values) and functional markers (percentage values) by flow cytometry, as previously described [Jy W et al JTH 2004]. PMP was identified using CD62P, CD36, and CD63, EMP using CD105 and RMP using CD235a. Endothelial modulator markers NO, ADM and ET-1 were measured by ELISA. Statistical analysis included Kruskal-Wallis test, Bonferroni test, correlation matrix, principal component (PCA) and partial least square regression analysis (PLSR).Total-MP were increased in all patients groups compared to controls (p<0. 01). Strong correlations were seen for tot-MP and %MP CD62P (R=0. 89, p<0. 05), tot-MP and %MPCD36 (R=0. 97, p<0. 05), tot-MP CD62P+ and CD36+ (R=0. 85, p<0. 05) and for tot -MP TF+ and %MP CD105+ (R=0. 66, p<0. 05). Bonferroni test, showed higher MP total number in the AN group compared to the HU and the ASA group (p <0. 05). The percentage of CD63 MP was higher than control both in the HU and in the AN group (p <0. 05), while CD36 MP% was higher in the AN group compared to the HU group (p <0. 05). NO and ADM values were higher in the HU group, while ET-1 values were lower in the AN group (p <0. 05). The principle components (PC) responsible for value variations were PC1 [positive loading for WCC, PMP and EMP] reflecteing PLT and EC activation, and PC4 [positive loadings for PLT, PMP; negative loadings for RMP, NO]. Both PC1 and PC4 were statistically significant for treatment.This study confirms previous findings that MP are present in ET. We also showed that MP are affected by treatment and that MP are mainly PMP and express CD62P and CD36, a marker of immature PLT, which suggests that reticulated PLT partecipate in these processes. We also showed that HU increase NO and ADM levels, while AN reduces ET-1. As NO and ET-1 have antagonistic actions on endothelial cells, we suggest that HU and AN may have overlapping effects on endothelium, although using different biochemichal pathways. [Display omitted] The finding that treatment influences PC1 (PMP and EMP) supports the idea that in ET, treatment impacts directly on MP. The finding that NO and RMP have a negative loading within PC4, suggests that they have an antagonist-action to PMP and EMP in ET. Disclosures:Pizzolo:Hoffmann-La Roche: Consultancy, Honoraria.

Thrombocytosis and thrombocytopenia in the NICU: incidence, mechanisms and treatments

Objective: Quantitative and qualitative platelet abnormalities of neonates must be defined using evidence-based reference ranges, constructed according to gestational and postnatal age. Methods: Platelet counts, and demographic and outcome data, were obtained from neonates in the Intermountain Healthcare system in the western USA and template bleeding times were determined from neonates in Italy. Results: Reference ranges were constructed by excluding values from neonates with diagnoses associated with abnormal platelet counts (small for gestational age (SGA), pregnancy-induced hypertension (PIH), infection and necrotizing enterocolitis (NEC)). Values remaining in the database after excluding these diagnoses were organized into 5th to 95th percentile ranges. At 23–25 weeks gestation, thrombocytopenia (<5th percentile) was defined by a platelet count <100,000/µl. Severe thrombocytopenia (platelet count <50,000/µl) occurred in 2.4% of neonatal intensive care unit (NICU) admissions and was largely due to acquired consumptive causes (bacterial and fungal sepsis, NEC and extracorporeal membrane oxygenation). No correlation was found between platelet count and subsequent central nervous system (CNS), pulmonary or gastrointestinal (GI) bleeding. The mortality rate did not correlate with the lowest platelet count but was proportionate to the number of platelet transfusions received. Platelet transfusions, administered according to guidelines, were given to 7% of NICU admissions, but a change in the guidelines from “count-based” to “mass-based” was associated with a reduction to 4%, with no increase in CNS, pulmonary, GI or cutaneous haemorrhage. Bleeding times were twice as long in neonates <33 weeks gestation as in term neonates, and shortened to term values by day of life ten. Conclusions: When reference ranges for platelets, appropriate to gestational and postnatal ages, are used, more uniformity occurs in definitions. This uniformity will foster consistency in diagnosis, treatment and outcomes-reporting.

33 A STUDY ON COAGULATION PROFILE IN PATIENTS OF CHRONIC LIVER DISEASE

Background: The liver plays not only a central role in coagulation but also closely correlates with qualitative and quantitative deficiency of platelets. Chronic liver disease (CLD), particularly in the end stage, is characterized by disorders of bleeding and coagulation abnormalities. Aim: (i) To determine the coagulation profile and platelet count in the patients of CLD and (ii) to determine the relationship between coagulation profile and severity of CLD in these patients. Method: Sixty-four adult patients of CLD admitted in the Department of Medicine, Gauhati Medical College over a period of 6 months were studied, and prothrombine time (PT), activated partial thromboplastin time (APTT), international normalized ratio (INR), and platelet counts were determined in these patients. Liver disease severity was graded using Child’s score. Result: Alcohol (73.43%) was found to be the most common etiology for CLD in the study. Males were affected more than female (M:F = 8:1). Forty-four (68.75%) patients had increased PT, and 40 (62.5%) patients had increased APTT. Forty-one (64.06%) had increased INR, and 36 (56.25%) patients had decreased platelet count. Mean PT, mean APTT, mean INR, and mean platelet count in the total patients were found to be 18.6, 41.80, 1.67, and 127,905, respectively, while in Child C grade, mean PT, mean APTT, mean INR, and mean platelet count were found to be 24.4, 47.62, 2.15, and 110,538, respectively. Conclusion: Abnormalities in hemostasis is fairly common in CLD. The PT, APTT, and INR abnormalities have a positive correlation with severity of disease. However, qualitative platelet abnormality was also detected in CLD. Therefore, qualitative tests for platelet functions are necessary to arrive at a definite conclusion regarding role of platelets. 34

Microparticle-Associated Thrombin Generation and Procoagulant Activity Is Increased In Patients with Essential Thrombocythemia

AbstractAbstract 1985Essential Thrombocythemia (ET) is a chronic myeloproliferative neoplasm characterized by an increased thrombotic risk. Numerous quantitative and qualitative abnormalities of platelets and leukocytes, arising from the clonal proliferation of hematopoietic progenitor cells, have been described as responsible of the thrombophilic state in this disease. Recently, a high number of plasma microparticles (MP) of different cellular origin has been described in ET patients. MP are a key component of the hemostatic response and have been found increased in diseases at high thrombotic risk. To explore the contribution of plasma MP to the hypercoagulable state of patients with ET, in this study we aimed to characterize the MP functional procoagulant features. We used two different methods: the calibrated automated thrombogram (CAT), to determine the MP-associated thrombin generation (TG), and the P-PPL/1 assay (Stago R&D) to measure the MP-associated procoagulant activity (PCA). Both assays were performed in platelet free plasma (P-FP) obtained from 69 ET patients (24M/45F; 33 carriers of JAK2V617F mutation) and 67 control subjects (32M/35F). In a subgroup of 23 ET patients and 23 controls MP-associated TG and PCA were also determined in MP-free plasma (MP-FP). PFP was obtained by two serial centrifugations (4,000 rpm for 15 min, then 11,000 rpm for 10 min) and MP-FP by re-centrifuging P-FP at 14,000 rpm for 30 min. MP were isolated from the pellet. For the TG assay, 80 ul of P-FP or MP-FP and 20 ul of buffer were mixed, and TG started by adding CaCl2. The results were expressed as lag-time, peak, area under the curve (ETP), and time-to-peak (ttPeak). For P-PPL/1 assay, 100 ul of P-FP or MP-FP were mixed with 50 ul of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds.The results show that P-FP from ET patients generated significantly higher quantity of thrombin compared to controls, as demonstrated by the shorter lag-time (22.1±8.7 vs 29.5±14.7 min; p=0.004) and time to peak (26.6±8.4 vs 33.3±13.2 min; p=0.004) and the significantly greater peak (52.9±24.9 vs 38.24±20.9 nM; p=0.002) and ETP (765.6±206.4 vs 537.5±295 nM*min; p=0.001). Similarly, the MP-associated PCA was significantly increased in ET patients (79±11 sec) compared to controls (89±11 sec; p<0.05). This increase was due to the presence of MP, as no TG and little PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples to the original values of P-FP for both assays. TG was significantly (p<0.05) increased in the JAK2V617F mutation carriers (lag-time: 19.5±7.4 min; peak: 59.8±26.9 nM; ttpeak: 23.9± 4.9 min) compared to wild-type subjects (lag-time: 24.5±9.1 min, peak: 45.4±20.6 nM, ttpeak: 29.3±10.3 min), while no significant differences were found for PCA. Significant correlations were found between the PCA by PPL-assay and the different parameters of TG assay [lag-time (R2= 0.414), peak (R2= -0.542), ETP (R2= -0.514)].In conclusion, our results show that MP-associated TG capacity, as well as PCA, are increased in plasma from ET patients. The highest MP-associated TG was found in JAK-2 mutation carriers, who also are at higher risk for thrombosis compared to wild-type subjects. Our data provide evidence for a contribution of MP to the thrombophilic state of these patients and suggest to test MP associated TG and PCA in prospective studies to predict thrombosis in ET patients. Disclosures:No relevant conflicts of interest to declare.

High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder

Familial platelet disorder (FPD), a rare autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities, is considered as a model of genetic predisposition to acute myeloid leukemia (AML). So far, monoallelic RUNX1 germline mutations have been found in 19 of 20 families with reported FPD, and the analysis of blast cells from only 5 patients at acute leukemia (AL) stage has shown no additional RUNX1 abnormality. Here, we performed RUNX1 analysis at constitutional and somatic levels in 8 persons with FPD who developed AL from 4 independent families. In addition to the germline RUNX1 mutation, we identified a second RUNX1 alteration in 6 AML cases (acquired point mutations in 4 cases and duplication of the altered RUNX1 allele associated with acquired trisomy 21 in 2 other cases). Although haploinsufficiency of RUNX1 causes FPD, our findings suggest that a second genetic event involving RUNX1 is often associated with progression to AML.

Multiple congenital coagulopathies co‐expressed with Von Willebrand’s disease: the experience of Hemophilia Region III Treatment Centers over 25 years and review of the literature

The haematologist medical directors of the Hemophilia Region III Treatment Centers in the mid-Atlantic Region III of the United States identified individuals in their databases diagnosed with additional congenital coagulopathies co-expressing with Von Willebrand's disease (VWD) and its variants. Sixteen individuals from 14 unrelated families, originating from five institutions, had been evaluated comprehensively. They represented approximately 1.5% of the calculated VWD population (n=986) registered in region III at the end of year 2002. Eleven of the 16 (68.75%) had type 1 VWD; two (12.5%) had type 2A; two (12.5%) had type 2 Normandy (2N) and one (6.25%) had coexisting type 2B and type 2N VWD. The accompanying coagulopathies consisted of severe or moderate factor VIII (FVIII) deficiency (haemophilia A) in six (37.5%); severe or moderate factor IX deficiency (haemophilia B) in three (18.75%); severe or moderate factor XI deficiency in two (12.5%); severe factor VII deficiency in two (12.5%); combined factor XI and XII deficiencies in one (6.25%); qualitative platelet abnormality in one (6.25%); and one had combined type 2B and type 2N VWD (6.25%). The reversal of overt bleeding often required multiple therapeutic modalities, mandated by the specific combination of coagulation disorders. This included administration of specific purified clotting factor concentrates, cryoprecipitate, fresh frozen plasma, 1,8 deamino-d-arginine vasopressin, antifibrinolytic agents, platelet transfusions, etc. In summary, combined inherited coagulopathies coexisting with VWD are uncommon and frequently are phenotypically divergent from VWD. Successful therapy required an individualized approach, employing multiple replacement strategies, based on the laboratory definition of the specific additional coagulation defect.

Molecular Pathogenesis and Therapy of Polycythemia Induced in Mice by JAK2 V617F

BackgroundA somatic activating mutation (V617F) in the JAK2 tyrosine kinase was recently discovered in the majority of patients with polycythemia vera (PV), and some with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis. However, the role of mutant JAK2 in disease pathogenesis is unclear.Methods and FindingsWe expressed murine JAK2 WT or V617F via retroviral bone marrow transduction/transplantation in the hematopoietic system of two different inbred mouse strains, Balb/c and C57Bl/6 (B6). In both strains, JAK2 V617F, but not JAK2 WT, induced non-fatal polycythemia characterized by increased hematocrit and hemoglobin, reticulocytosis, splenomegaly, low plasma erythropoietin (Epo), and Epo-independent erythroid colonies. JAK2 V617F also induced leukocytosis and neutrophilia that was much more prominent in Balb/c mice than in B6. Platelet counts were not affected in either strain despite expression of JAK2 V617F in megakaryocytes and markedly prolonged tail bleeding times. The polycythemia tended to resolve after several months, coincident with increased spleen and marrow fibrosis, but was resurrected by transplantation to secondary recipients. Using donor mice with mutations in Lyn, Hck, and Fgr, we demonstrated that the polycythemia was independent of Src kinases. Polycythemia and reticulocytosis responded to treatment with imatinib or a JAK2 inhibitor, but were unresponsive to the Src inhibitor dasatinib.ConclusionsThese findings demonstrate that JAK2 V617F induces Epo-independent expansion of the erythroid lineage in vivo. The fact that the central erythroid features of PV are recapitulated by expression of JAK2 V617F argues that it is the primary and direct cause of human PV. The lack of thrombocytosis suggests that additional events may be required for JAK2 V617F to cause ET, but qualitative platelet abnormalities induced by JAK2 V617F may contribute to the hemostatic complications of PV. Despite the role of Src kinases in Epo signaling, our studies predict that Src inhibitors will be ineffective for therapy of PV. However, we provide proof-of-principle that a JAK2 inhibitor should have therapeutic effects on the polycythemia, and perhaps myelofibrosis and hemostatic abnormalities, suffered by MPD patients carrying the JAK2 V617F mutation.

Molecular basis of the diagnosis and treatment of polycythemia vera and essential thrombocythemia

AbstractRecent insights into the molecular mechanisms of polycythemia vera (PV) and essential thrombocythemia (ET) are challenging the traditional diagnostic classification of these myeloproliferative disorders (MPDs). Clonality analysis using X-chromosome inactivation patterns has revealed apparent heterogeneity among the MPDs. The recently discovered single somatic activating point mutation in the JAK2 gene (JAK2-V617F) is found in the great majority of patients with PV, but also in many patients with phenotypically classified ET and other MPDs. In contrast to the acquired MPDs, mutations of the erythropoietin receptor and thrombopoietin receptor have been identified in familial forms of nonclonal erythrocytosis and thrombocytosis, respectively. The mechanisms of major clinical complications of PV and ET remain poorly understood. Quantitative or qualitative abnormalities of red cells and platelets do not provide clear explanations for the thrombotic and bleeding tendency in these MPDs, suggesting the need for entirely new lines of research in this area. Recently reported randomized clinical trials have demonstrated the efficacy and safety of low-dose aspirin in PV, and an excess rate of arterial thrombosis, major bleeding, and myelofibrotic transformation, but decreased venous thrombosis, in patients with ET treated with anagrelide plus aspirin compared to hydroxyurea plus aspirin.

Investigation of Platelet Functions in Chronic Myeloproliferative Disorders by Optical and Luminesance Method.

Background: Bleeding and thrombosis are common causes of morbidity and mortality in patients with myeloproliferative disorders (MPD). Qualitative platelet abnormalities are frequently found in these patients and range from platelet hypofunction as demonstrated by defective invitro platelet aggregation, acquired storage pool disease and/or platelet membrane defects, in addition to enhanced platelet aggregation, increased plasma beta thromboglobulin levels or shortened platelet survival. In this study we aimed to perform platelet aggregation studies by optical method (on platelet rich plasma=PRP) and luminesance method (on whole blood) in chronic myeloproliferative disorders.Methods:A total of twenty-five patients with MPD (17 chronic myeloid leukemia, 6 polycythemia vera, 2 essential thrombocytosis) were enrolled. Median age was 54,4 (29–76). Platelet aggregation was measured using the optic and luminesance method. The agonists used were adenosine diphosphate (ADP), arachidonic acid (AA) ristocetin and collagen. Platelets were considered to be hyperactive if at least one result (i.e. aggregation or ATP release with one agonist) was above the reference range, and hypoactive if at least one result (i.e. aggregation or ATP release) was below the reference range. Mixed hypo- and hyperactive platelets were considered present when at least one result (i.e. aggregation or ATP release) was below and above the reference range respectively.Results:Platelet aggregation test results by two methods in myeloproliferative disorders were shown in Table 1. The percent for detection of platelet function abnormality by luminesance method was found to be higher than the optic method and a significant difference was shown between two methods (p<0,05).Conclusion Our findings suggest that;1. Luminesance platelet aggregation study is more valuable than optic platelet aggregation study for invitro assessment of platelet function in patients with MPD. 2. The use of luminesance platelet aggregation study appears useful to select patients for antiplatelet therapy.Platelet aggregation test results in myeloproliferative disorders (n=25)NormalHypofunctionHyperfunctionMixTotal abnormalityLuminesance method1(%4)4(%16)8(%32)11(%44)24(%96)Optic method9(%36)11(%44)2(%8)3(%12)16(%64)

Qualitative and quantitative platelet abnormalities in hepatitis C virus associated chronic liver disease

Qualitative and quantitative platelet abnormalities in hepatitis C virus associated chronic liver disease