Essential Thrombocythemia Patients with CALR Mutations Have Increased Platelet Activation Markers Compared to JAK2V617F Mutated Patients

Abstract Essential thrombocythemia (ET) is associated with an increased risk for thrombo-hemorrhagic complications. The presence of the JAK2V617F mutation, found in approximately 50% of ET patients, has been associated with increased indices of platelet (PLT) activation suggesting its casual role in thrombus formation. Mutations in CALR were recently described in the majority of JAK2V617F negative ET patients, and are associated with a decreased rate of thrombotic events. This has led us to hypothesize that CALR mutations have a different influence on PLT activation compared to JAK2V617F. To evaluate the PLT activation state, surface expression of two PLT activation markers - p-selectin (CD62P) and PAC1 was studied using specific antibodies. MFI was analyzed by flow cytometry at baseline, as well as following ADP addition to PLT rich plasma. Monocyte-platelet aggregates were studied in whole blood samples by gating CD45+/CD14+ cells and calculating the percentage of CD41+ cells in the monocytes population. The immature PLT fraction (IPF) was analyzed with the XE-5000 hematology analyzer (Sysmex UK Ltd., Milton Keynes, UK), and the absolute number of immature PLT (nIP) was calculated from the total PLT count. Low risk ET patients (N-13, M/F-5/8) and healthy controls (N-10, M/F-4/6) are included in this analysis. JAK2V617F and CALR mutations were present in 8 and 5 patients, respectively; low dose aspirin (range 75-100mg) was taken by 85% of patients and 90% of controls. Median PLT count in CALR mutated, JAK2V617F mutated and healthy subjects was 913, 579 and 247 K/uL, respectively (p=0.0002), and it was higher in CALR compared to JAK2V617F positive patients (p=0.09). Both patient subgroups had a lower baseline MFI of p-selectin and PAC1 compared to healthy controls (p-selectin: 2.8, 3 and 4.5 for JAK2V617F [p=0.01], CALR [p=0.05] and controls; PAC1: 3, 3.3 and 5.2 for JAK2V617F [p=0.01], CALR [p=0.02] and controls, respectively) with no difference between CALR and JAK2V617F mutated patients. CALR compared to JAK2V617F mutated patients had higher median number of immature PLT (30 and 10.6 K/uL, p=0.04), and a higher fraction of monocyte- platelet aggregates (90 and 58%, p=0.05). nIP and monocyte- platelet aggregates were also significantly higher in CALR mutated but not in JAK2V617F mutated patients compared to healthy controls. Interestingly, there was no difference in post ADP PLT activation (post/baseline ratio) between ET patients and healthy controls. Finally, there were correlations between the PLT counts and nIP (R=0.8, p<0.0001), monocyte- platelet aggregates (R=0.5, p=0.02), baseline p-selectin MFI (R=-0.5, p=0.02) and PAC1 MFI (R=-0.5, p=0.01). Our preliminary results suggest a correlation between PLT activation markers and the PLT numbers, which can explain why CALR mutated patients in our cohort had higher nIP and monocyte- platelet aggregates fractions. The absence of an increased ADP induced PLT activation between patients and controls in this cohort compared with previous reports could be explained by the use of aspirin in the majority of patients and the high ADP concentration used for PLT activation. These results will be further studied in a lager cohort of patients. Disclosures No relevant conflicts of interest to declare.

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Neutrophil subpopulations and their activation potential in patients with antiphospholipid syndrome and healthy individuals

Rheumatology ◽

10.1093/rheumatology/keaa532 ◽

2020 ◽

Author(s):

Lisa-Marie Mauracher ◽

Moritz Krall ◽

Johanna Roiß ◽

Lena Hell ◽

Silvia Koder ◽

...

Keyword(s):

Gradient Centrifugation ◽

Absolute Number ◽

Neutrophil Extracellular Trap ◽

Healthy Controls ◽

Activation Markers ◽

Healthy Donors ◽

Increased Risk ◽

High Median ◽

Insight Into ◽

Net Release

Abstract Objectives Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. Methods HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. Results LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58–10.24) vs 0.56% (0.19–1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08–99.13) vs 35.50% (13.50–93.85)], whereas no difference was found in LDNs. NET formation was increased in patients’ HDNs upon stimulation. Conclusion HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.

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Evaluation of Patients with Myelodysplastic Syndromes (MDS) Obtaining Stable Disease with the Use of Decitabine.

Blood ◽

10.1182/blood.v114.22.2790.2790 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2790-2790

Author(s):

Henry G. Kaplan ◽

Michael Milder

Keyword(s):

Stable Disease ◽

Dna Methyltransferase ◽

De Novo ◽

Conflicts Of Interest ◽

Clinical Importance ◽

Hematologic Malignancies ◽

Median Number ◽

Outpatient Setting ◽

Entire Cohort ◽

Increased Risk

Abstract Abstract 2790 Poster Board II-766 BACKGROUND: MDS is a group of hematologic malignancies associated with reduced quality of life related to progressive cytopenias and increased risk of infections and bleeding. Successful treatment in MDS is typically defined in terms of complete remission (CR). Treatment with decitabine, a DNA methyltransferase inhibitor, has led to CR in 9 to 39% of MDS patients. Many patients have responses that do not meet criteria for CR or partial remission, but may be of clinical importance, especially for older MDS patients. Since patients who achieve stable disease may receive benefits from treatment, it was of interest to evaluate patient characteristics and treatment response results of those who achieved stable disease with decitabine. METHODS: 99 patients with de novo (n=88) or secondary (n=11) MDS were treated with decitabine, 20 mg/m2 daily for 5 days every 4 weeks in an outpatient setting (Steensma et al. J Clin Oncol 2009). No dose reductions were allowed but dose delays were permissible. Any FAB, including CMML, were eligible if ECOG 0-2 and normal hepatic and renal function. Supportive care, including blood products, were permitted. G-CSF was permitted for serious infection or sepsis. Twenty-three patients (18 de novo and 5 secondary MDS) achieved stable disease as the best response by IWG 2006 criteria. RESULTS: At baseline, stable disease patients had a median age of 75 years (70% >70 years) and were mainly men (70%). Ten patients had RA, 7 had RAEB, 4 had RAEB-t, and one each had RARS or CMML. The IPSS scores for these patients were Low (n = 1; 4%), INT-1 (n = 8; 35%), INT-2 (n = 5; 22%), and High (n = 9; 39%). Cytogenetics were good 10 (43%), 1 (4%) intermediate, 10 (43%) poor, or unknown 2 (9%). At baseline 19 (83%) were RBC transfusion dependent, 3 (13%) platelet transfusion dependent. 22 patients were ECOG 0-1. Five patients had received prior cytotoxic chemotherapy, none with azacitidine or decitabine. The median number of cycles initiated was 5.0 (range 2 – 19). At the time of the analysis, 12 of the 23 patients had died with a median survival of 19.2 months (95% CI: 9.4, not estimable). This is consistent with the survival response (19.4 months (95% CI: 15, not estimable) for the entire cohort, which included the stable disease patients, 50% who achieved a hematologic improvement or better, and 10% with progressive disease with decitabine (15% not assessable). Median time to AML or death was 16.1 months (95% CI: 7.2, not estimable). Three of 19 RBC-dependent patients at baseline became transfusion independent for at least 8 weeks with treatment. Conversely, 3 of 4 baseline RBC-independent patients became transfusion dependent. One of 3 platelet-dependent patients became transfusion independent. Three of 20 platelet-independent patients at baseline became transfusion dependent. Of ten patients evaluable for cytogenetic responses, 2 patients had partial cytogenetic responses. Eleven out of 23 patients had at least one related SAE. Myelosuppression-related adverse events were common (≥10%) in these 23 patients with grade 3 or higher adverse experiences of anemia (26%), febrile neutropenia (17%), neutropenia (39%), and thrombocytopenia (30%). CONCLUSIONS: In an outpatient setting, approximately one-quarter of MDS patients maintained stable disease with decitabine treatment, with acceptable and manageable toxicity. Overall survival in this subset of patients appeared to be similar to that observed with the entire cohort, which included 50% of patients with an objective clinical benefit. Larger analyses are needed to fully understand the characteristics of and treatment-related benefits for patients who achieve stable disease with decitabine treatment. Disclosures: No relevant conflicts of interest to declare.

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Circulating Microparticles and Risk of Venous Thromboembolism.

Blood ◽

10.1182/blood.v114.22.3987.3987 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3987-3987

Author(s):

Paolo Bucciarelli ◽

Emanuele Previtali ◽

Ida Martinelli ◽

Andrea Artoni ◽

Serena M Passamonti ◽

...

Keyword(s):

Body Mass Index ◽

Venous Thromboembolism ◽

Body Mass ◽

Plasma Levels ◽

Conflicts Of Interest ◽

Factor V Leiden ◽

Control Group ◽

Dependent Manner ◽

Healthy Controls ◽

Increased Risk

Abstract Abstract 3987 Poster Board III-923 Background Microparticles (MPs) are circulating, submicroscopic fragments (<1 μm of diameter) of membrane-bound cytoplasm that shed from the surface of an activated or apoptotic cell and play a role in coagulation, inflammation, cell remodelling and proliferation. There is increasing evidence that MPs are involved in thrombosis, but whether or not they are an independent risk factor for venous thromboembolism (VTE) is not established. Aim of the study To investigate the association between high plasma levels of MPs and risk of VTE Patients and Methods In a case-control study, 186 patients with a first episode of VTE (deep venous thrombosis and/or pulmonary embolism) and 418 healthy controls were included. MPs were analyzed by flow cytometry with a gate defined by a 1 μm beads and using APC-Annexin V together with FITC anti-CD41 or FITC anti-CD142 antibodies in order to identify platelet MPs (MP-Plts) and MPs exposing tissue factor (MP-TF), respectively. MPs levels were expressed as number/μL. Results Patients had significantly higher median plasma levels of both MPs-Plts and MPs-TF than controls [1942 vs 1519 (p<0.0001) and 579 vs 454 (p<0.0001)]. Higher median levels of MP-Plts and MP-TF were found in 41 patients who underwent blood sampling within 6 months from VTE than in those sampled later [2114 vs 1694 (p=0.086) and 652 vs 543 (p=0.120)]. Sex, age, body mass index and factor VIII plasma levels had no influence on MPs levels, as well as the use of oral contraceptives (this latter evaluated only in controls). In the whole study population, carriership of thrombophilia (antithrombin, protein C or protein S deficiency, factor V Leiden, prothrombin G20210A, antiphospholipid antibodies, hyperhomocysteinemia or combined abnormalities) had higher levels of MP-Plts and MP-TF than non-carriers [1907 vs 1565 (p=0.002) and 532 vs 468 (p=0.011)]. The odds ratio (OR) for VTE, adjusted for sex, age, body mass index and thrombophilia was 2.5-fold higher in individuals with MPs plasma levels >95th percentile of the control group (3633/μL for MPs-Plts and 1113/μL for MPs-TF) than in those with MPs levels ≤95th percentile [for MPs-Plts: OR=2.59 (95%CI 1.23 – 5.45); for MPs-TF: OR=2.38 (1.15 – 4.92)]. The risk increased in a dose-dependent manner for both MPs-Plts and MPs-TF, particularly above the 75th percentile of the distribution in controls. The exclusion of patients whose MPs levels were measured within 6 months from VTE (in order to avoid the possible effect of the acute phase on MPs measurements), did not change the results [adjusted OR: 2.63 (1.18 – 5.89) for MPs-Plts and 2.36 (1.10 – 5.19) for MPs-TF]. The Table shows the relative risks of VTE associated with the presence or absence of high MPs levels and thrombophilia. Individuals with MPs >95th percentile or thrombophilia alone had a 2 to 3-fold increased risk of VTE, whereas those with both MPs-Plts >95th percentile and thrombophilia had a 9-fold increased risk of VTE. This synergistic effect was confirmed also for MPs-TF and remained after the exclusion of patients whose blood sample was collected within 6 months from VTE [OR 7.72 (1.68-35.4) for MP-Plts and 8.14 (2.08-31.8) for MP-TF]. Conclusions Plasma levels of MPs are significantly higher in patients with VTE than in healthy controls. MPs levels >95th percentile are associated with a 2.5-fold increased risk of VTE. There is a synergistic interaction between high levels of MPs and thrombophilia on VTE risk. Disclosures: No relevant conflicts of interest to declare.

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Fibrinogen Supports Colitis-Associated Adenoma Progression through a Mechanism Coupled to Engagement of the Leukocyte Integrin αMβ2.

Blood ◽

10.1182/blood.v114.22.334.334 ◽

2009 ◽

Vol 114 (22) ◽

pp. 334-334

Author(s):

Netanel Horowitz ◽

Kris A. Steinbrecher ◽

Maureen A. Shaw ◽

Kelley A. Barney ◽

Matthew Flick ◽

...

Keyword(s):

Cell Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Disease Process ◽

Mutant Form ◽

Short Term ◽

Activation Markers ◽

Vascular Integrity ◽

Tumor Dissemination

Abstract Abstract 334 A growing body of evidence points to a crucial role for fibrinogen in both tumor dissemination and the regulation of inflammation. Given this dual importance, we hypothesized that fibrinogen plays an important role in the progression of inflammation-driven cancers. To test this hypothesis, we induced colitis-associated cancer (CAC) in fibrinogen-deficient mice and controls using a combination of azoxymethane (AOM) and dextran sodium sulfate (DSS). Fibrinogen deficiency resulted in a dramatic diminution in the number of adenomas formed after AOM/DSS challenge in spite of the fact that overt gastrointestinal bleeding appeared more severe in Fib- animals relative to controls. These results suggest that while fibrin deposition is crucial for the maintenance of vascular integrity and control of blood loss in colitis, fibrin(ogen) appears to drive colitis-associated adenoma development/outgrowth. Fibrin(ogen) could promote CAC through multiple mechanisms. One intriguing possibility is that fibrin(ogen) interactions with leukocytes through the integrin αMβ2 are an important determinant of inflammation-induced tumor progression. To test this hypothesis, we explored CAC development in control mice and animals expressing a mutant form of fibrinogen (Fibγ390-396A) that maintains full clotting function and supports thrombus formation normally in vivo, but does not support binding to αMβ2. Consistent with our hypothesis, Fibγ390-396A mice developed significantly fewer adenomas after AOM/DSS challenge. In fact, the majority of Fibγ390-396A mice had no discernable adenomas while the phenotypic penetrance of adenoma development was 100% in control animals. Detailed analyses revealed that one mechanism coupling fibrin(ogen)-mediated αMβ2 engagement to adenoma formation/outgrowth is by supporting inflammatory events during the antecedent colitis. Fibγ390-396A mice manifested a dramatic diminution in inflammatory cell infiltrates, colonic edema, crypt loss and epithelial ulceration relative to control mice after chronic DSS exposure. Analyses of short-term DSS exposure revealed an ∼10-fold diminution in Fibγ390-396A mice relative to controls in the elaboration of key cytokines known to promote CAC progression (i.e., IL-6, TNF-α, IL-1β, IFN-γ). Previous studies suggest that these cytokines promote CAC, at least in part, through changes in epithelial cell function. Consistent with this view, the number of colonic epithelial cells staining positive for established activation markers (i.e., phosphorylated cJun and RelA/p65) were significantly diminished in Fibγ390-396A animals relative to control animals after short-term DSS exposure. Taken together, these data strongly suggest that one mechanism coupling fibrin(ogen) to adenoma progression is through αMβ2-mediated proinflammatory events early in the disease process which lead to epithelial damage/turnover important in adenoma formation. However, the role of fibrin(ogen) in adenoma progression does not appear to be limited to these early inflammatory events. The adenomas harvested from Fibγ390-396A mice were significantly smaller than those from control mice and less proliferative based on mitotic indices, suggesting an additional role for fibrin(ogen) in the growth of established adenomas. These studies demonstrate, for the first time, a unique link between fibrin(ogen) and the development of inflammation-driven malignancy. Given the importance of antecedent inflammation in the progression of numerous cancers, these studies suggest that therapies targeting fibrin(ogen)-αMβ2 interactions may be useful in preventing and/or treating this important subset of malignancies. Disclosures: No relevant conflicts of interest to declare.

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Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

Blood ◽

10.1182/blood.v124.21.1865.1865 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1865-1865

Author(s):

Fiorina Giona ◽

Marica Laurino ◽

Luciana Teofili ◽

Sara Capodimonti ◽

Maurizio Martini ◽

...

Keyword(s):

Children And Adolescents ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Correct Diagnosis ◽

Young Patients ◽

Antiplatelet Agents ◽

Wild Type ◽

Primary Thrombocythemia ◽

Calr Mutations

Abstract The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome. PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months. Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1). JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122). Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies. On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5215-5215

Author(s):

Munazza Rashid ◽

Rifat Zubair Ahmed ◽

Shariq Ahmed ◽

Muhammad Nadeem ◽

Nuzhat Ahmed ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Diagnostic Algorithm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Common Mutation ◽

Hypochondriac Region ◽

Exon 9 ◽

Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

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Predictive Value of Coagulation Markers Concerning Clinical Outcome 90 Days after Anterior Myocardial Infarction

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614557 ◽

1999 ◽

Vol 81 (05) ◽

pp. 701-704 ◽

Cited By ~ 11

Author(s):

Frederic Kontny ◽

Ulrich Abildgaard ◽

Carl-Erik Dempfle

Keyword(s):

Myocardial Infarction ◽

Clinical Outcome ◽

Predictive Value ◽

Thrombus Formation ◽

Left Ventricular ◽

Adverse Clinical Outcome ◽

Coagulation Activation ◽

Activation Markers ◽

Increased Risk ◽

D Dimer

SummaryTo study the predictive value of coagulation markers concerning clinical outcome, prothrombin fragment F1.2 (F1.2), fibrin monomer antigen (FM), D-Dimer (DD), and fibrinogen were measured in plasma samples drawn 2 and 7 days after acute myocardial infarction (AMI) in 314 consecutive patients randomized in a clinical trial of low molecular weight heparin (Dalteparin) (the FRAMI trial). Placebo-treated patients suffering death or new AMI within 90 days had significantly higher levels at day 2 of FM (Enzymun-Test FM), and DD (TINAquant D-dimer) (p = 0.001 and 0.02, respectively), but not F1.2 (Enzygnost F1.2 micro), relative to those without serious clinical events. At day 7 all three coagulation activation markers were significantly higher in patients with subsequent adverse clinical outcome. The Dalteparin group had significantly lower levels of these markers as compared to the placebo group. Left ventricular (LV) thrombus formation was not associated with changes in coagulation activation. However, patients with thrombus had significantly higher fibrinogen levels than those without thrombus (p = 0.004 day 2), independent of treatment group. Thus, markers of coagulation activation may be useful in stratification of patients when estimating risk for adverse clinical outcome after AMI. Furthermore, elevated fibrinogen levels are associated with increased risk of LV thrombus formation.

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Cocaine and Certain of Its Long-Acting Metabolites Stimulate Endothelial Cell Von Willebrand Factor Secretion

Blood ◽

10.1182/blood.v118.21.1148.1148 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1148-1148 ◽

Cited By ~ 2

Author(s):

William E Hobbs ◽

Rebecca P Penkala ◽

Emily E Moore ◽

José A. López

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Intracellular Camp ◽

Thrombotic Risk ◽

Cocaine Use ◽

Cocaine Metabolites ◽

Increased Risk ◽

String Formation

Abstract Abstract 1148 Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures: No relevant conflicts of interest to declare.

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GPIbα As a Selective Bidirectional Modulator Of α-Thrombin Prothrombotic Functions Influencing Fibrin Deposition and PAR-Dependent Platelet Activation

Blood ◽

10.1182/blood.v122.21.32.32 ◽

2013 ◽

Vol 122 (21) ◽

pp. 32-32

Author(s):

Alessandro Zarpellon ◽

Antonella Zampolli ◽

Patrizia Marchese ◽

James R. Roberts ◽

Grazia Loredana Mendolicchio ◽

...

Keyword(s):

Platelet Activation ◽

Defense Mechanism ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Thrombus Formation ◽

Blood Platelets ◽

Human Platelets ◽

Fibrillar Structure ◽

Clotting Time ◽

Platelet Aggregates

Abstract Background Generation of α-thrombin (FIIa) in response to vascular injury is a key host defense mechanism influencing thrombus formation and inflammation. Blood platelets express glycoprotein (GP) Ibα as the most abundant FIIa membrane binding site, as well as different protease activated receptors (PARs) with an effector role in platelet activation after proteolytic cleavage. The functional role of GPIbα, which is not a substrate for FIIa, relative to that of different PARs remains unclear. Aims Goal of these studies was to define with mechanistic understanding whether and how binding to GPIbα can modulate FIIa prothrombotic functions in vivo and ex vivo. Methods Endogenous mouse platelet GPIbα was replaced by the human (hu) counterpart with wild type (WT) sequence; or containing the single substitution of Asp277 (mutated to Asn), which interacts selectively with a site involving FIIa exosite 2; or with the combined substitution of post-translationally sulfated Tyr276, Tyr278 and Tyr279 (each mutated to Phe), which interact with FIIa residues in proximity of exosite 1 as well as exosite 2. These mice were evaluated in intravital models of arterial thrombosis. Moreover, their platelets were tested ex vivo for the response to FIIa-induced activation measuring changes in intracytoplasmic Ca2+ levels; and for effects on fibrinogen clotting and fibrin formation. Comparative ex vivo experiments were conducted with human and huGPIbα-WT mouse platelets in which FIIa binding was similarly blocked by the anti-human GPIbα monoclonal antibody, LJ-Ib10. Ex vivo FIIa effects on platelet activation/aggregation and fibrin clot formation were also evaluated concurrently in a model of thrombus formation in blood perfused over a thrombogenic surface under controlled flow conditions. Results Genetically modified mouse platelets expressed ≈9000 WT or mutant huGPIbα molecules; platelets with huGPIbα-WT bound ≈10,000 FIIa molecules with 1:1 stoichiometry and KD of ≈3 nM. FIIa binding to mutant huGPIbα was essentially abolished. Mice with defective FIIa binding to GPIbα exhibited a pronounced prothrombotic phenotype, with a shorter time to carotid artery occlusion following ferric chloride injury (median 550.5 seconds in 18 mutant huGPIbα, vs. 1980 seconds in 19 huGPIbα-WT mice; P<0.01). Accordingly, the platelet-rich plasma (PRP) of mutant huGPIbα mice exhibited a significantly shorter clotting time in the presence of 4 nM FIIa and significantly enhanced intracytoplasmic Ca2+ transients and platelet aggregation following stimulation by 0.5 nM FIIa. Human platelets, similar to mouse platelets, bound FIIa with a 1:1 stoichiometry relative to GPIbα and KD of ≈3 nM. Remarkably, blocking FIIa binding to GPIbα with antibody LJ-Ib10 essentially abolished activation by 1 nM FIIa in human platelets, in which FIIa effects are mediated predominantly by PAR1; this was in contrast to the enhanced activation seen under the same conditions in hu GPIbα-WT mouse platelets, in which FIIa acts through PAR3 and PAR4. Accordingly, the volume of platelet aggregates and fibrin formed in huGPIbα-WT mouse blood perfused over a thrombogenic surface was enhanced by blocking FIIa binding to platelets; in contrast, the volume of platelet aggregates, but not that of fibrin clots, was decreased under the same conditions in human blood. Antibody LJ-Ib10 shortened the clotting time of both huGPIbα-WT mouse and human PRP; however, in the absence of GPIbα-bound FIIa, fibrin associated with platelet aggregates had a less ordered fibrillar structure. Conclusions Our findings identify GPIbα as a relevant FIIa activity modulator. Through distinct mechanisms influenced by the expression of specific PAR subtypes, GPIbα can modulate FIIa function in hemostasis and thrombosis both enhancing and controlling prothrombotic responses and, thus, size and structure of platelet/fibrin thrombi. The effect of GPIbα on PAR4-mediated platelet activation, as well as fibrinogen clotting, can be explained by competition for FIIa exosites required for substrate binding, but the mechanism supporting the distinct GPIbα-PAR1 functional association remains to be elucidated. Disclosures: No relevant conflicts of interest to declare.

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Activation Status of Integrin αIIbβ3 in Essential Thrombocythemia with Calreticulin Mutation

Blood ◽

10.1182/blood.v124.21.4571.4571 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4571-4571

Author(s):

Jun Yamanouchi ◽

Takaaki Hato ◽

Etsuko Matsubara ◽

Taichi Azuma ◽

Hideyuki Nakanishi ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Healthy Subjects ◽

Conflicts Of Interest ◽

Thrombotic Event ◽

Jak2 V617f ◽

Insertion Mutant ◽

Integrin Αiibβ3 ◽

Calr Mutations ◽

Calr Mutation ◽

Activation Status

Abstract Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations. It has been reported that CALR is a potential regulatory protein of integrin activation based on the interaction between CALR and a conserved sequence of GFFKR in the integrin α cytoplasmic tails. Recent studies suggest that calreticulin activates β1 integrin and modulates integrin-associated signaling. In this study, we examined the effect of CALR mutations on integrin αIIbβ3 activation. We first identified mutations of JAK2, MPL, and CALR genes in 37 patients with WHO defined ET and explored clinical characteristics of patients with CALR mutation. The patients with JAK2 V617F were 22 (59%), MPL W515L was 1 (3%), and CALR mutations were 10 (27%). The two types of CALR mutations were found; deletion (52-bp deletion; c.1092_1143del) and insertion (5-bp insertion; c.1154_1155insTTGTC) mutations. The patients with CALR mutations had lower hemoglobin and leukocyte count compared with JAK2 V617F patients, but platelet count did not have a difference between the CALR and JAK2 mutation groups. Nine (41%) of 22 patients with JAK2V617F had a thrombotic event while 1 (10%) of 10 patients with CALR mutation did (p<0.05), suggesting that patients with CALR mutation had a lower risk of thrombosis than JAK2 V617F patients. Two patients with CALR mutations developed myelofibrosis while no patient with JAK2V617F did. One patient with CALR mutation developed acute myeloid leukemia, with persistence of the CALR mutation in his leukemic cells.To see if the CALR mutation affects activation status of αIIbβ3, we examined the binding of PAC1, a monoclonal antibody recognizing the active conformation of αIIbβ3, to platelets from 5 patients with CALR mutation and 12 patients with JAK2V617F in the presence or absence of ADP. Platelets from all the 5 patients with CALR mutation showed the same level of PAC1 binding as platelets from healthy subjects. Overexpression of recombinant CALR proteins in CHO cells expressing αIIbβ3 by transfection of a protein-expression vector containing wild-type, deletion, or insertion mutant CALR had no effect on PAC1 binding. On the other hand, platelets from 4 of 12 patients with JAK2V617F had an increase in PAC1 binding in the presence and absence of ADP compared with platelets from healthy subjects. All 4 (100%) of 4 patients with increased PAC1 binding had a thrombotic event while 4 (30%) of 13 patients with normal PAC1 binding did. Our study suggests no activation of integrin αIIbβ3 by CALR mutation, which may explain a clinical finding of a low risk for thrombosis in patients with CALR mutation. Disclosures No relevant conflicts of interest to declare.

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