Markers of endothelial and in vivo platelet activation in patients with essential thrombocythemia and polycythemia vera

We investigated endothelial and in vivo platelet activation in a cohort of 52 patients with essential thrombocythemia (ET) and polycythemia vera (PV) before and after cytoreductive treatment, 22 healthy controls, and 17 patients with acute cerebrovascular ischemia (ACVI) and normal platelet counts. We measured platelet expression of CD62P and CD63 antigens and levels of soluble vascular cell adhesion molecule 1 (sVCAM-1). We found increased in vivo platelet activation in all patients with ET and PV, both before and after cytoreductive treatment, compared with controls. In patients with arterial thrombosis, platelet expression of CD62P, and in patients with erythromelalgia, expression of both markers was higher compared with expression in patients without thrombotic complications. In patients with ET and PV before and after treatment, sVCAM-1 expression was increased compared with expression in controls but also compared with expression in patients with ACVI and normal platelet counts. In patients with arterial thrombosis and erythromelalgia, in vivo platelet activation correlated with the level of sVCAM-1. Our findings indicated that in vivo platelet activation reflects intrinsic platelet defects in patients with ET and PV, persists after cytoreductive treatment, and results in endothelial damage, probably through release of angiogenic factors and/or activation of white blood cells.     

Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. 2000;96:4261-4266)     

Automated quantitation of peripheral blood neutrophil activation in patients with myocardial ischaemia

BACKGROUND: Coronary ischaemic syndromes are associated with neutrophil activation. The Bayer automated haematology analysers can detect increased light scatter of neutrophil populations, which correlates with neutrophil activation. We aimed to assess the role of an automated analyser in detecting systemic neutrophil activation in peripheral blood samples of patients with coronary ischaemia. METHODS: A prospective cross-sectional study was undertaken in 18 patients with chronic stable angina, 9 with unstable angina and 26 normal control subjects. Whole blood samples were taken to assess neutrophil count and light scatter, and serum samples were taken from some patients for assessment of Troponin T, C-reactive protein (CRP) and myeloperoxidase (MPO). In addition, whole blood was stimulated in vitro with interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine (fMLP) to assess changes in neutrophil light scatter detected by the analyser. RESULTS: Neutrophil light scatter was increased in patients with chronic stable and unstable angina compared to normal control subjects (normal subjects 74.1 (73.3, 75.0) (mean arbitrary units (95% confidence intervals, (CI)) vs. 78.6 (76.9, 80.3) in the chronic stable angina group P<0.001 and 77.1 (75.3, 79.0) in the unstable angina group P<0.007). In vitro stimulation of whole blood produced comparable increases in neutrophil light scatter when morphological changes in neutrophils were demonstrable under electron microscopy. CONCLUSIONS: Automated measurement of neutrophil activation by light scatter is possible using the Advia 120 analyser and is superior to a neutrophil count in discriminating groups with angina. This technique may be useful in monitoring disease activity and progression in coronary artery disease and in guiding the use of anti-inflammatory therapies.     

Indications for lowering platelet numbers in essential thrombocythemia

Essential thrombocythemia (ET), one of the chronic myeloproliferative disorders, exposes individuals to significantly increased risk for thrombohemorrhagic complications. Epidemiologic data indicate that the two most prominent risk factors for thrombosis are age greater than 60 years or a history of or presentation with thrombosis at any age. Age is an important factor in selecting among therapeutic options, as the agents used to treat ET may contribute to acute leukemic transformation and other secondary malignancies. Whether or not hydroxyurea (HU) carries these risks is controversial and unresolved, but the uncertainty is a basis for avoiding it in young patients. Alternatives to HU that have established efficacy in lowering platelet counts in ET are interferon and anagrelide. Both are highly effective in reducing platelet numbers, and are apparently not associated with leukemogenicity or mutagenicity. However, approximately 30% of patients find interferon intolerable for long-term therapy. Anagrelide offers the advantage of oral dosing and long-term effectiveness at managing platelet counts. A recent long-term study of young ET patients treated with anagrelide found that all thrombohemorrhagic events occurred in patients with platelet counts greater than 0.4 × 10⁹/L, adding to the evidence that reduction of platelet counts to normal may be required for optimal control of risk. Semin Hematol 40(suppl 1):22-25. © 2003 Elsevier Inc. All rights reserved.     

Hydroxyurea in old patients with essential thrombocythemia

BACKGROUND AND AIMS: A previous thrombotic event and advanced age are well-known risk factors for thrombosis in essential thrombocythemia (ET). In these patients, therefore, cytotoxic drugs are needed to reduce platelet count. In spite of this convincing idea, in clinical practice, some old patients do not use platelet-reducing drugs, for a variety of causes, and few specific studies in old patients with ET are available. Our retrospective study reports single-center experience in 54 old ET patients with long follow-ups. METHODS: We compared the clinical outcome of 27 ET old patients not taking cytotoxic drugs (group A) with 27 cases treated with hydroxyurea (HU) (group B), evaluating the incidence of thrombosis and thrombosis-free survival. In 16 patients in group A and in 18 in group B, V617FJak2 mutation was sought. About 20% of HU-treated patients developed major side-effects. RESULTS: No significant difference was found in the occurred thrombosis between the 2 groups in either clinical or laboratory features. V617FJak2 was equally common in groups A and B, and in patients with or without thrombosis. CONCLUSIONS: This study is not randomised and includes a small number of patients. However, it shows that it is necessary to identify better patients who really need treatment, as the side-effects of HU are relatively common in old people and their treatment should be discontinued. V617FJak2 does not define the thrombotic risk in old ET patients.     

Aspirin responsive platelet thrombophilia in essential thrombocythemia and polycythemia vera

Essential thrombocythemia (ET) and polycythemia vera (PV) frequently present with erythromelalgia and acrocyanotic complications, migraine-like microvascular cerebral and ocular transient ischemic attacks (MIAs) and/or acute coronary disease. The spectrum of MIAs in ET range from poorly localized symptoms of transient unsteadiness, dysarthria and scintillating scotoma to focal symptoms of transient monocular blindness, transient mono- or hemiparesis or both. The attacks all have a sudden onset, occur sequentially rather than simultaneously, last for a few seconds to several minutes and are usually associated with a dull, pulsatile or migraine-like headache. Increased hematocrit and blood viscosity in PV patients aggravate the microvascular ischemic syndrome of thrombocythemia to major arterial and venous thrombotic complications. Phlebotomy to correct hematocrit to normal in PV significantly reduces major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated erythromelalgia and MIAs. Complete long-term relief of the erythromelalgic microvascular disturbances, MIAs and major thrombosis in ET and PV patients can be obtained with low dose aspirin and platelet reduction to normal, but not with anticoagulation. Skin punch biopsies from the erythromelalgic area show fibromuscular intimal proliferation of arterioles complicated by occlusive platelet-rich thrombi leading to acrocyanotic ischemia. Symptomatic ET patients with erythromelalgic microvascular disturbances have shortened platelet survival, increased platelet activation markers β-thromboglobulin (β-TG), platelet factor 4 (PF4) and thrombomoduline (TM), increased urinary thromboxane B2 (TXB2) excretion, and no activation of the coagulation markers thrombin fragments F1+2 and fibrin degradation products. Inhibition of platelet cyclooxygenase (COX1) by aspirin is followed by the disappearance and no recurrence of microvascular disturbances, increase in platelet number, correction of the shortened platelet survival times to normal, and reduction of increased plasma levels of β-TG, PF4, TM and urinary TXB2 excretion to normal. These results indicate that platelet-mediated fibromuscular intimal proliferation and platelet-rich thrombi in the peripheral, cerebral and coronary end-arterial microvasculature are responsible for the erythromelalgic ischemic complications, MIAs and splanchnic vein thrombosis. Baseline platelet P-selectin levels and arachidonic acid induced COX1 mediated platelet activation showed a highly significant increase of platelet P-selectin expression (not seen in ADP and collagen stimulated platelets), which was significantly higher in JAK2^V617F mutated compared to JAK2 wild type ET.     

Hemostatic complications in young patients with essential thrombocythemia

PURPOSE: The purpose of this study was to determine the incidence of hemostastic complications in young patients with essential thrombocythemia (ET). PATIENTS AND METHODS: The clinical course of 44 patients under the age of 45 with the diagnosis of ET was reviewed in a retrospective manner. Patients were collected from three medical centers in the United States and Italy: the Brigham and Women's Hospital and the Harvard Community Health Plan, Boston, Massachusetts, and the Ospedali Riuniti di Bergamo, Bergamo, Italy. RESULTS: The overall incidence of hemorrhage or thrombosis, or both, in this group of patients was 39% (17 of 44), with serious complications occurring in 23% (10 of 44). Two patients died of thrombotic events. Neither the presence of symptoms at diagnosis nor any single laboratory parameter proved predictive of clinical sequelae. Treatment with antiplatelet drugs or platelet-lowering agents was not protective. CONCLUSION: We conclude that ET in young patients may result in serious and life-threatening hemostatic problems and consequently that young age is not a favorable prognostic factor in this disease.     

Role of platelet counts in the management of essential thrombocythemia: experience with anagrelide

An elevated platelet count is the hallmark of essential thrombocythemia (ET). Therapeutic control of platelet counts in ET has proven effective for minimizing the risk of bleeding complications. By contrast, the relationship between platelet count and thrombotic complications is less well defined. The beneficial effects of lowering the platelet count are greatest when levels are reduced close to or within the normal range, thus supporting the trend within the medical community to reduce platelet treatment targets to below 400-450 × 10(9)/l. Platelet counts have further utility in ET as a marker to indicate intolerance/resistance to hydroxyurea according to recently published guidelines. In the current era, where novel risk factors for ET complications are being discussed, this article takes a back-to-basics approach, considering what platelet counts reveal in practice as a risk factor for complications and a marker of treatment efficacy, with particular focus on data for the platelet-selective agent anagrelide.     

Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.     

Platelet activation and thrombosis: Studies in a patient with essential thrombocythemia

Recent advances permit the detection of activated platelets using specific monoclonal antibodies and flow cytometry. Nevertheless, there are few reports in which activated platelets have been studied over a period of time in patients at risk for thrombosis. Our patient S.D. has essential thrombocythemia and a prothrombotic state manifested in two major thrombotic episodes involving the portal vein and a mesenteric artery. Investigation revealed both spontaneous aggregation and hyperaggregability in response to ADP and the presence of activated platelets in platelet-rich plasma as revealed by flow cytometry. Interestingly, the activated platelets were recognized by an anti-RIBS (“receptor-induced binding site”) monoclonal antibody that recognized bound fibrinogen but not by antibodies reactive with antigens whose presence on the platelet surface was secretion dependent. Treatment with aspirin inhibited spontaneous platelet aggregation but had little effect on the activated platelet profile. A change of therapy to ticlopidine suppressed expression of platelet activation markers. Treatment with ticlopidine has continued for 1 year so far without further thrombotic complications. © 1996 Wiley-Liss, Inc.     

Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia

Background

Most cell types, including blood - and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia.

Design and Methods

In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay.

Results

Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500×10⁶ events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001).

Conclusions

Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.     

羟基脲联合血小板去除术治疗老年原发性血小板增多症的临床疗效分析

目的观察羟基脲联合血小板去除术治疗老年原发性血小板增多症（ET）的临床疗效。方法根据有无临床症状,将28例老年ET患者分为有临床症状组及无临床症状组。所有患者均采用口服羟基脲（Hu）治疗,剂量为O．5～3g／d。对血小板计数〉1000×10^9／L者,确诊后即采用血小板去除术1～3次,并且于血小板去除术后开始接受羟基脲治疗。结果有临床症状组的患者发病时外周血血小板计数（1469．00±521．40）×10^9／L明显高于无临床症状组（892．00±302．10）×10^9／L（P〈0．01）。所有患者治疗前后血小板计数为（1023．00±606．30）VS（468．00±236．20）×10^9／L（P〈0．01）。所有患者接受治疗后CR16例（57．1％）,PR11例（39．3％）,NR1例（3．6％）。结论羟基脲联合血小板去除术治疗老年ET疗效明确,不良反应较少,耐受性良好。