Influence of thrombopoietin on platelet activation in myeloproliferative disorders

Although thrombopoietin itself does not influence platelet aggregation, it enhances platelet activation in response to certain agonists. We evaluated the effects of thrombopoietin on platelet activation using platelet-rich plasma from 16 patients with myeloproliferative disorders (MPD group) and 16 healthy volunteers (control group). Preincubation with thrombopoietin significantly enhanced platelet aggregation stimulated by ADP, collagen, or epinephrine in the MPD group as well as the control group. However, aggregation induced by 3 μ M ADP or 16 μ M epinephrine showed significantly less augmentation by thrombopoietin in the MPD group than in the control group. Thrombopoietin significantly shortened the lag time between the addition of 3 μ M ADP or 16 μ M epinephrine and initiation of secondary aggregation and the lag time between addition of 2 μg/ml collagen and initiation of aggregation in both groups. When platelet-rich plasma was used without adjustment of the platelet count, thrombopoietin itself induced aggregation in two patients. Hypoaggregation after addition of 0.5 μg/ml collagen was observed in seven out of nine patients with normal thrombopoietin levels and only one of six patients with high levels ( P =0.04). Enhancement of 0.5 μg/ml collagen-induced aggregation by thrombopoietin was seen in five out of nine patients with normal thrombopoietin levels and none of the six patients with elevated levels ( P =0.04). These results indicate that platelet activation by certain agonists is enhanced by thrombopoietin in patients with these diseases as well as in normal controls and that the serum thrombopoietin level may regulate the function of circulating platelets in vivo .     

PF 4 versus beta TG as evidence for platelet activation in myeloproliferative disorders

19 patients with MPD have been studied. As described in normals, an age-related increase in beta thromboglobulin (beta TG) release is observed. Such release, however, is greater in patients with myeloproliferative disorders (MPD). MPD seem therefore to cause platelet activation, allowing an earlier and more evident manifestation of physiologic ageing phenomena. PF 4 levels are near zero both in controls and patients, regardless of platelet number. This suggests that increased levels of PF 4 represent only a laboratory artifact, caused by platelet activation in vitro. Mean ability in producing thromboxane B2 (TxB2) is increased, but is perfectly normal in patients with normal platelet count and decreased in 3 thrombocythaemic patients, who seem to present an increased thrombotic risk. TxB2 is reduced almost to zero by the administration of aspirin plus dipyridamole; contrarily, all other parameters were unaffected, either by such drugs or by AD 6, a new coumarin derivative with antiplatelet properties.     

Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders

Chronic myeloproliferative disorders (MPDs) are characterized by a high incidence of thrombohaemorrhagic complications, possibly caused by platelet dysfunction. In an attempt to define platelet functional abnormalities, we assessed the expression of activation-dependent membrane proteins in unstimulated and agonist [ADP and thrombin receptor-activating peptide (TRAP)]-stimulated platelets using quantitative whole blood flow cytometry in samples from 50 MPD patients and 30 controls. The receptor densities of activation markers and glycoproteins (GPs) were quantified using standardized fluorescent beads. Compared with controls, the mean percentage of P-selectin-positive (15.3% vs. 7.2%; P < 0.001) and thrombospondin (TSP)-positive (6.6% vs. 3.7%; P = 0.003) platelets was increased in unstimulated platelets from patients. Patients having experienced a thrombotic event had a higher mean percentage of TSP-positive non-stimulated platelets than patients without a history of thrombosis (9.0% vs. 4.6%; P = 0.02) and a higher GPIV molecules of equivalent fluorochrome (MEF) value (33113 vs. 24471 MEF; P = 0.02). Mean MEF values of monoclonal antibodies (mAbs) against GPIb (34055 vs. 38945 MEF; P < 0.001) and GPIIb/IIIa (1416 vs. 1648 MEF; P < 0. 001) were significantly reduced among patients, whereas surface expression of GPIV was increased in patients (28273 vs. 16258 MEF; P < 0.001). In TRAP (10 micromol/l) stimulated whole blood, the MEF of P-selectin (9611 vs. 13293 MEF; P = 0.004) and CD63 (2385 vs. 5177 MEF; P < 0.001) and the ratio of PAC-1/GPIIb/IIIa MEF (0.98 vs. 2. 00; P < 0.001) was reduced in patients, indicating either a reduced granule GP content or an intrinsic cellular defect in receptor-mediated granule secretion and activation of the GPIIb/IIIa complex. Expressed as the relative change of MEF compared with unstimulated platelets, TRAP induced decrease of GPIb (7.8% vs. 45%; P < 0.001) and increase of GPIIb/IIIa (49.1% vs. 95.7%; P < 0.001) and GPIV expression (17.8% vs. 55.2%; P < 0.001) was attenuated in patients.     

Quantitation of RNA-dependent platelet DNA polymerase in patients with myeloproliferative disorders

Platelets from 28 patients with the myeloproliferative diseases (MPD) polycythaemia vera (9), essential thrombocythaemia (6), myelofibrosis with myeloid metaplasia (5) and chronic myelogenous leukaemia (8) were examined for an RNA-dependent DNA polymerase activity using standardized conditions permitting highly reproducible quantitation. Low levels of activity were detected in platelets of normal individuals, but platelets of nearly all MPD patients (25/28) possessed higher levels. The polymerase activity correlated with diagnosis (P = 0.001) and did not correlate with platelet counts (P greater than 0.2). Quantitation of this RNA-dependent DNA polymerase activity may be a useful parameter in the diagnosis of myeloproliferative disorders.     

Increased circulating platelet-leukocyte aggregates in myeloproliferative disorders is correlated to previous thrombosis, platelet activation and platelet count

Platelet-leukocyte adhesion may occur as a consequence of platelet activation and possibly plays a key role in the deposition of activated platelets and fibrin in the thrombotic plug. The aim of the present study was to assess by whole blood flow cytometry the presence of circulating platelet-leukocyte aggregates (PLA) and the platelet-leukocyte response to platelet agonist stimulation (ADP and TRAP) in 50 patients with chronic myeloproliferative disorders (MPD) and 30 controls. PLA were identified as platelet-granulocyte/monocyte aggregates (PGMA), platelet-monocyte aggregates (PMA) and defined as the percentage of leukocytes coexpressing the platelet-specific marker glycoprotein Ib. Compared to controls the mean percentage of PGMA and PMA was increased in unstimulated whole blood from patients with MPD (7.98 vs. 1.76%; p<0.001 and 12.34 vs. 3.2%; p<0.001, respectively). The percentage of PGMA was correlated to the platelet count (r=0.46; p<0.001), percentage of P-selectin (r=0.69; p<0.001) and thrombospondin (r=0.58; p<0.001) positive platelets and platelet expression of GPIV (r=0.33; p=0.02). The mean percentage of PGMA and PMA was significantly increased in ADP-stimulated whole blood of patients (57.14 vs. 47.92%; p=0.009 and 54.91 vs. 45.89%; p<0.001, respectively). Compared to patients without a history of thrombosis, patients having experienced microvascular disturbances or a thrombotic event had a higher mean percentage of PGMA and PMA in non-stimulated whole blood (10.07 vs. 6.34%; p=0.025 and 14.81 vs. 10.48%; p=0.021, respectively) and a higher percentage of PGMA in ADP stimulated whole blood (64.32 vs. 51.50%; p<0.01). These data document an increased frequency of PLA in non-stimulated whole blood in MPD associated with a previous history of thrombosis or microvascular disturbances.     

Increased leukocyte-platelet adhesion in chronic myeloproliferative disorders with high platelet counts

The heterophilic adhesions between monocytes and platelets may result in the modification of both platelet and monocyte function. This mutual modification includes a greater activation of platelets with increased production of PDGF and other metabolites as well as an enhanced tissue factor expression of monocytes with greater activity in the circulation. The heterophilic aggregation has been well documented during extracorporal circulation, haemodialysis and in diabetic retinopathy. Here we provide evidence that there is significant increase of monocyte-platelet aggregates in disorders associated with high platelet counts, such as chronic myeloproliferative disorders. The presence of these heterophilic aggregates may contribute to the vascular complications observed frequently in polycythaemia vera and essential thrombocythaemia.     

Changes in distribution of platelet membrane glycoproteins in patients with myeloproliferative disorders

Glycoproteins have been discovered to be important to platelet function both in normal and pathological states. We have studied membrane glycoprotein patterns in 16 patients with various myeloproliferative disorders. There was an abnormal ratio of glycoprotein I glycoprotein IV in patients with myeloproliferative disease compared with controls. There was no discernible correlation between glycoprotein pattern and aggregation response or platelet count, but patients with megathrombocytes had higher values for glycoprotein IV than those without megathrombocytes. These experiments suggest that patients with myeloproliferative disorders may have alterations in membrane glycoproteins that could alter platelet function.     

A reversible defect of platelet PDGF content in myeloproliferative disorders

The transport of iron through erythroid cell membrane was studied in a model system, measuring ferrous iron uptake by reticulocytes. It was found that these cells were able to take up ferrous iron and to incorporate it into haem at a rate similar to that observed when diferric transferrin was the iron donor. No comparable iron uptake could be measured when the metal was provided as Fe3+-citrate or when reticulocytes were replaced by mature erythrocytes. The involvement of endogenous transferrin in the Fe2+ uptake by reticulocytes could be excluded, since proteolytic treatment of the cells had no significant effect on the process. Fe2+ uptake by reticulocytes followed saturation kinetics, characteristic to carrier mediated transport processes. Kinetic analysis of the data revealed the following apparent transport parameters: Km = 8.8 +/- 3.8 microM; Vmax = 1.1 +/- 0.2 ng/10(8) reticulocytes/min. These results indicate that a high affinity, carrier mediated iron transport system is present in the reticulocyte membrane, ensuring the efficient translocation of the metal through the membrane barrier between the site of its release from transferrin and the site of its utilization.     

Effect of anagrelide on platelet count and function in patients with thrombocytosis and myeloproliferative disorders.

BACKGROUND. Anagrelide is a quinazolin compound developed initially as an inhibitor of platelet aggregation. Since "in vivo" studies demonstrated that it was responsible for thrombocytopenia in humans, anagrelide has been used recently in a small number of patients with thrombocytosis and myeloproliferative disorders. Platelet count was well controlled in the large majority of patients, and only minimal side effects were observed. PATIENTS. Eight patients (5 with essential thrombocythemia, 2 with chronic granulocytic leukemia, and 1 with idiopathic myelofibrosis) received anagrelide (induction dose 4 mg/die; mean maintenance dose 2 mg/die; mean observation time 26 weeks). Complete blood counts were determined 4 times during the first month, and subsequently every month. "In vivo" and "ex vivo" platelet function was studied before anagrelide and after 4 and 10 days of therapy. RESULTS. Platelet count was reduced and maintained below 500 x 10(9)/L in 5 of 8 patients. Headache, palpitation/tachycardia, gastrointestinal symptoms and a decrease in hemoglobin were the side effects. Anagrelide did not modify the leukocyte count or "in vivo"/"ex vivo" platelet function. CONCLUSIONS. Anagrelide may control thrombocytosis in patients with myeloproliferative disorders, even when traditional drugs have failed. When required, anti-aggregating drugs may be associated with anagrelide, since it has no effect on platelet function.     

PF 4 versus βTG as evidence for platelet activation in myeloproliferative disorders

19 patients with MPD have been studied. As described in normals, an age-related increase in β thromboglobulin (βTG) release is observed. Such release, however, is greater in patients with myeloproliferative disorders (MPD). MPD seem therefore to cause platelet activation, allowing an earlier and more evident manifestation of physiologic ageing phenomena. PF 4 levels are near zero both in controls and patients, regardless of platelet number. This suggests that increased levels of PF 4 represent only a laboratory artifact, caused by platelet activation in vitro. Mean ability in producing thromboxane B₂ (TxB₂) is increased, but is perfectly normal in patients with normal platelet count and decreased in 3 thrombocythaemic patients, who seem to present an increased thrombotic risk. TxB₂ is reduced almost to zero by the administration of aspirine plus dipyridamole; contrarily, all other parameters were unaffected, either by such drugs or by AD 6, a new coumarin derivative with antiplatelet properties.     

Clonality in myeloproliferative disorders.

The myeloproliferative disorders are a group of hematologic diseases that are believed to arise from somatic mutations in an early hematopoietic stem cell. This statement is based on the demonstration of monoclonal involvement of terminally differentiated myeloid and lymphoid elements. The techniques for establishing clonal derivation of cells are discussed and the application of these techniques to myeloproliferative diseases is reviewed. The evidence for limited myeloid involvement, lineage heterogeneity, in some patients with myeloproliferative disorders is summarized.     

Cardiac involvement in patients with myeloproliferative disorders.

INTRODUCTION: To evaluate cardiac involvement in myeloproliferative disorders (MPD), two-dimensional and Doppler echocardiographic studies were performed in 30 patients with MPD. PATIENTS AND METHODS: There were 18 women and 12 men, with an age range from 35 to 76 years. Eighteen patients had polycythemia vera (PV), 8 had essential thrombocythemia (ET), and 4 had agnogenic myeloid metaplasia (AMM). RESULTS: Echocardiography revealed valvular lesions in 19 of 30 patients (63%) compared with only 1 of 22 patients (4.5%) in a control group of patients referred for echocardiography to exclude a cardiac source for idiopathic systemic thromboembolism (chi 2 = 13.39, p < 0.001, by chi 2 test with Yates' correction). Valvular lesions were found in 77% of patients with PV, 50% with ET, and 25% with AMM (p = NS). The aortic and mitral valves were the most commonly involved valves, and the most common echocardiographic lesion was leaflet thickening, which was found in 12 patients (40%), followed by vegetations, which were observed in 5 patients (16%). In their past history, 14 of 30 (47%) MPD patients had arterial or venous thrombosis or embolism. Twelve of 19 (63%) patients with valvular lesions had thromboembolism compared with only 2 of 11 (18%) patients without evidence of valvular lesions (chi 2 = 3.99, p < 0.05, by chi 2 test with Yates' correction). Pulmonary hypertension, unrelated to the severity of valvular disease and probably resulting from pulmonary venous occlusion, was found in four patients (13%). CONCLUSIONS: We conclude that the heart is frequently involved in patients with MPD, particularly when their past history is complicated by a thromboembolic event. Some patients have clinically significant valvular disease. Pulmonary hypertension is another relatively common finding in MPD patients. Echocardiography provides information of clinical significance in MPD patients. A larger number of patients is needed to determine whether the presence of valvular lesions is of prognostic significance and may herald future thromboembolic events.     

Increased platelet CD36 constitutes a common marker in myeloproliferative disorders

Summary. The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb-IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb-IIIa measured using the binding of specific radiolabeled antibodies to platelets. In contrast, 2-3-fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6-152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow-up of MPD patients.
Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2-fold increase of CD36 molecules, resting and thrombin-activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.     

Defective platelet beta-N-acetyl hexosaminidase content and release in chronic myeloproliferative disorders

BACKGROUND AND OBJECTIVES: Abnormalities of platelet function or structure are a hallmark of chronic myeloproliferative disorders (MPD). In vivo platelet activation with the release of alpha- and delta-granules in the circulation is one of the most frequently described alterations in MPD. Platelets contain and release upon activation also lysosomes, and in particular beta-N-acetylhexosaminidase (Hex). We have assessed whether the content and in vivo release of Hex of platelets from MPD patients is altered. DESIGN AND METHODS: Twenty-three MPD patients were compared with 19 age- and sex-matched healthy controls. The activity of platelet beta-N-acetylhexosaminidase was measured in plasma, serum and in the capillary blood emerging from the skin wound inflicted for the measurement of the bleeding time. Lysosome integral membrane protein (LIMP or CD63), lysosome-associated membrane protein (LAMP-2 or CD107b) and P-selectin were evaluated by flow cytometry. Platelet aggregation in vitro and the release of beta-N-acetylhexosaminidase, ATP and beta-thromboglobulin were performed to study platelet reactivity. RESULTS: Hex levels in plasma were significantly higher in MPD than in controls while the release of Hex in the bleeding time blood, i.e. at a localized site of in vivo platelet plug formation, was lower in MPD and the platelet content of Hex was reduced. These changes were accompanied by in vivo platelet activation. Finally, the isoenzymatic pattern of Hex was altered in platelets of MPD patients, with a reduced amount of the Hex A isoform as compared with controls.b INTERPRETATIONS AND CONCLUSIONS: MPD patients present an altered platelet Hex content and release; prospective studies to assess whether altered platelet Hex is related to thrombotic/hemorrhagic complications and/or tissue fibrosis in MPD are warranted.     

Secondary gout associated with chronic myeloproliferative disorders.

The early recognition of acute gouty arthritis, prompt institution of colchicine and/or other antigouty inflammatory drugs, and the use of colchicine prophylactically during the interval periods are necessary measures for the control of secondary gout. The importance of administering allopurinol to prevent the extreme hyperuricemia and excessive hyperuricosuria in blood dyscrasias even before the onset of gouty arthritis should not be overlooked. However, the dosage of allopurinol must be titrated according to the degree of the hyperuricemia and of hyperuricosuria. The control of excessive hyperuricemia in patients receiving chemotherapy is particularly important. The side effects including its impact on the liver and the hemopoietic system should be kept in mind. It must also be remembered that the therapy may change the clinical course of secondary gout, but the course of the underlying blood dyscrasias may not be altered.     

Use of whole blood platelet lumi-aggregometry to optimize anti-platelet therapy in patients with chronic myeloproliferative disorders

Twenty-seven patients with chronic myeloproliferative disorders and in vitro evidence of platelet hyperactivity on whole blood platelet lumi-aggregometry were commenced on anti-platelet therapy comprising aspirin, clopidogrel, and/or odorless garlic and the studies were repeated to assess the efficacy of the therapeutic agent(s). Only 8 patients showed clear evidence of anti-platelet effect while receiving the standard low-dose (100 mg/day) aspirin therapy. Thirteen patients required a higher dosage of aspirin and/or an additional anti-platelet agent to achieve therapeutic adequacy. Lumi-aggregometry also proved useful to optimize therapy in the 6 patients who received clopidogrel or odorless garlic because of aspirin intolerance.     

Effects of in vitro platelet activation on platelet derived nitric oxide production in healthy humans and in chronic myeloproliferative diseases with elevated platelet counts

Intravascular EDRF-NO production is known to be impaired in some diseases, e.g., diabetes. This phenomenon may also contribute to the development of diabetic vascular disease. More recently the presence of NO synthase (ecNOS, iNOS) have been recognized in human platelets. Platelets produce NO only during activation, even though in minute amounts. This platelet derived NO seems to play an important physiological role, as it inhibits further platelet recruitment quite substantially. In the present report washed platelets isolated from healthy persons and patients with chronic myeloproliferative diseases (CMPD) were exposed to common and physiologically relevant activators (i.e., thrombin, collagen, epinephrine etc.). These tests were carried out in 20 healthy volunteers and 15 patients suffering from myeloproliferative disorders associated with thrombocytosis. As a consequence of pathological platelet function observed in CMPD, the in vitro platelet NO response is impaired in the patient group. One may assume, that reduced platelet NO response, at least in part, may contribute to platelet hyperfunction, angiopathy and thrombotic complications in some cases of CMPD.     

Effects of in vitro platelet activation on platelet derived nitric oxide production in healthy humans and in chronic myeloproliferative diseases with elevated platelet counts

Intravascular EDRF-NO production is known to be impaired in some diseases, e.g., diabetes. This phenomenon may also contribute to the development of diabetic vascular disease. More recently the presence of NO synthase (ecNOS, iNOS) have been recognized in human platelets. Platelets produce NO only during activation, even though in minute amounts. This platelet derived NO seems to play an important physiological role, as it inhibits further platelet recruitment quite substantially. In the present report washed platelets isolated from healthy persons and patients with chronic myeloproliferative diseases (CMPD) were exposed to common and physiologically relevant activators (i.e., thrombin, collagen, epinephrine etc.). These tests were carried out in 20 healthy volunteers and 15 patients suffering from myeloproliferative disorders associated with thrombocytosis. As a consequence of pathological platelet function observed in CMPD, the in vitro platelet NO response is impaired in the patient group. One may assume, that reduced platelet NO response, at least in part, may contribute to platelet hyperfunction, angiopathy and thrombotic complications in some cases of CMPD.