abnormalities of platelet aggregation in chronic myeloproliferative disorders
TRANSCRIPT
J.Cell.Mol.Med. Vol 5, No 1, 2001 pp. 79-87
Abnormalities of platelet aggregation in chronic myeloproliferative disorders
Simona Avram, Anca Lupu, Silvana Angelescu, Nicoleta Olteanu, Delia Mut-Popescu*
Department of Hematology, "Coltea" Clinical Hospital, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania
Received: December 18, 2000; Accepted: March 14, 2001
In chronic myeloproliferative disorders (MPD), thedefects of leukocytes and red cells are associatedwith various qualitative and quantitative platelet
abnormalities. Therefore, bleeding and thromboticcomplications may occur in these patients and oftenare major causes of morbidity and mortality. Thequalitative defects of platelets may contribute tohemostatic abnormalities in these disorders, besidesother factors like increased whole-blood viscosityor elevated platelet counts.
A variety of qualitative platelet abnormalities havebeen described in these patients [1-5]. Platelets are
*Correspondence to: Prof. Dr. Delia MUT-POPESCU, "Carol Davila" University of Medicine and Pharmacy,Department of Hematology, "Coltea" Clinical Hospital, 1, I. C. Bratianu Blvd, 70453 Bucharest, Romania. Phone: 0040-1-3142744, Fax: 0040-1-3110153.E-mail: [email protected]
Introduction
Keywords: platelets • polycythemia vera • chronic myelogenous leukemia • essential thrombocythemia •myelogenous metaplasia and myelofibrosis • aggregation • bleeding • thrombosis
Abstract
A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnor-malities may be due to deficiency of platelet granules, arahidonic acid metabolism defects or platelet membraneglycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro bymeasuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. Theseresults were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We foundcomplex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent,decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almostabsent platelet response to all agonists while in some patients a normal platelet aggregation was noted. Thecorrelation between these results and template bleeding time, thrombotic or hemorrhagic events and the type ofdiseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigatingplatelet aggregation may be useful not only for the assesment of the hemostatic balance in chronic myeloproli-ferative disorders but also for a better insight into cell abnormalities occuring in these pathologic conditions.
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often hypogranular, heterogenous in size, with giant,bizarre-shaped forms and, occasionally, megakary-ocyte fragments circulating in peripheral blood maybe found [5,6]. The degree of platelet function impair-ment can be evaluated by platelet aggregation abnor-malities which usually are not characteristic for thisgroup of diseases. Abnormal platelet aggregationespecially to epinephrine but also to collagen andADP were however described [6-8].
The causes of these abnormalities may include:1. defects in arachidonic acid metabolism (decreasein agonist-induced release of arachidonic acid frommembrane phospholipids; reduced conversion ofarachidonic acid to prostaglandine endoperoxidesor lipoxygenase products; reduced platelet respon-siveness to tromboxane A2) [9-11]; 2. abnormalitiesof platelet granules (increased platelet alpha-gran-ule secretion and an aquired storage pool defect ofdense granules) [3,12,13]; 3. decreased number ofá2-adrenergic receptors, abnormalities of specificplatelet membrane glycoproteins (GP) such asGPIIb/IIIa, GP Ib/IX or increased number of GPIVmolecules and receptors for the Fc component ofIgG [3,14-16]. An aquired von Willebrand disease[17,18], a reduction of platelet procoagulant activi-ty and an aquired form of Bernard-Soulier syn-drome were also reported in these disorders [19].
The bleeding and thrombosis occur in variedpatterns. The template bleeding time is usually nor-mal; when prolonged, it does not corellate with anincreased risk of hemorrhage or with aggregationabnormalities [20, 21].
The aim of this study was to detect plateletaggregation abnormalities in 76 patients withchronic myeloproliferative disorders, to correlatethese platelet aggregation defects with template
bleeding time and with clinical aspects, and todetermine the utility of platelet aggregation study inthe MPD diagnosis.
Materials and methods
We selected 76 patients with various entities of chronicmyeloproliferative disorders at which were carried outaggregometry studies. These include : 19 patients withchronic myelogenous leukemia (CML), 13 patients withmyeloid metaplasia and myelofibrosis (MMM), 22patients with polycythemia vera (PV) and 22 patientswith essential thrombocythemia (ET).
Platelet aggregation was analized in vitro on aChrono-Log aggregometer (source : Chrono-Log Corp.,Havertown, Pa., USA) through the optical aggregationmethod [22]. Platelets from citrated platelet rich plasma(PRP) were incubated at 37°C , subjected to stirring andstimulated with various agonists (ADP, epinephrine,collagen, arachidonic acid and ristocetin). The changesin light transmission from PRP versus platelet poorplasma (PPP) were recorded over time by the recordingdevice, as a wave. The amplitude of waves representsthe percent of platelet aggregation in PRP (the sample)arbitrarily considered to be 0% light transmission or0% aggregation before stimulation, in comparison withPPP (the reference) that is considered to be 100% lighttransmission or 100% aggregation. Normal aggregationcurves that have an amplitude above 60% for each ago-nist were used as controls (Fig. 1). We should mentionthat in cases with very high platelet counts, it wasneccesary to work with diluted PRP (diluted with PPPfrom the same patient) in order to exclude a false nor-mal-like response due to excesively increased numberof platelets.
The results were then correlated with template bleed-ing time (Duke’s method) [22a] and with bleeding orthrombotic complication of these diseases.
Response (Cases %)
Epinephrine (10ì M)
ADP (10ì M)
Collagen (10ì g/ml)
Arachidonic Acid (0,5mM)
Ristocetin (1mg/ml)
normal 33% 45% 42% 68% 75% absent 21% 7% 4% 17% 2% decreased 46% 47% 42% 14% 22% "lag"phase prolongation - - 12% - -
Table 1. Platelet aggregation response to various agonists in 76 patients with chronic myeloproliferative disorders
µ µµ
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Fig. 1 The normal platelet aggregation in response tovarious agonists. Arrowheads mark the moment of agonistaddition. A. 10 µM epinephrine; first aggregation wavecorresponding to platelet aggregation in response to addedagonist (arrow 1); secondary aggregation wave correspon-ding to enhanced aggregation in response to platelet secre-tion (arrow 2); B. 10 µM ADP; C. 10 µg/ml collagen.Note in B and C a "lag" phase (arrow 3) that occurs inADP and collagen-induced aggregation; the normal dura-tion of the lag phase is less than 60 seconds and reflectsthe shape changes of platelets in response to these ago-nists. D. 0,5 mM arachidonic acid; E. 1 mg/ml ristocetin.
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Results
The patterns of platelet aggregation response in thepresently investigated group are presented in Table 1.
When analyzing aspects of aggregation tracing,it was observed that the most common defect inplatelet function was an abnormal aggregationresponse to epinephrine (Fig. 2). The most frequentabnormal response of platelets to epinephrine wasthe decrease of platelet reactivity to this agonist.The complete loss of both first and secondaryaggregation waves to epinephrine was the mostcommon finding among the cases with absence ofplatelet response to the investigated stimuli (Fig. 3).In 9 from 35 cases with decreased epinephrine-induced aggregation, it was observed a peculiaraspect of the curve: a prolonged flat tracing withblunted slope that may be interpreted as an absenceof secondary wave of aggregation and a very retard-ed response of the platelets (Fig. 4A).
The responses to the other agonists werealtered in various proportions. The pattern ofresponse to ADP and collagen was more as adiminution than as a loss of platelet response. Themost common ADP-induced aggregation responsewas the loss of the secondary wave aggregationsuggesting impaired platelet release. In 22 cases, atendency to aggregation reversal was noted (Fig.4B). Another peculiar aspect was a prolongation ofthe “lag” phase of collagen-induced responseabove the normal range of 60 seconds, sometimeswith normal amplitude of the platelet response(Fig. 4C). The abnormal response of platelet toarachidonic acid is rather characterized by absencethan by decrease of platelet reactivity. The leastchanges of platelet aggregability were found toristocetin (Fig.2).
In two cases (one polycythemia vera and oneessential thrombocythemia) the response wasmarkedly decreased ( almost absent ) to all agonists.
Fig. 2. Incidence ofplatelet aggregationimpairment in res-ponse to various ag-gregating agents.
Fig. 3. Number ofcases with absentplatelet aggregationin response to vari-ous agonists.
Normal aggregation response to all agonists wasobserved in a not negligible percent - 15 cases from76 (~20%) - and this fact demonstrates that plateletsmay be normal or not yet altered in these diseases.
Analysis of obtained platelet aggregation curvesemphasized that in a certain number of cases a cer-tain combination of platelet aggregation abnormali-ties with various agonists may be detected. Thiscombination include (fig. 5):
- complete loss or a peculiar very flat wave toepinephrine
- absence of aggregation secondary wave toADP with consecutive unstable aggregates anddeaggregation tendency
- decrease of platelet response to collagen
- lost or markedly decreased platelet aggregatingresponse to arachidonic acid
- normal or decreased ristocetin-induced aggre-gation.
This panel of aggregation abnormalities wasobserved in 19 from 76 cases (25%).
The bleeding syndrome was found in 26 cases(34%) and includes the most common mucocuta-neous hemorrhages. The thrombotic complicationsoccurred in 21 cases (28%) and include mostly peri-pheral venous thrombosis. In 5 cases (7 %) we ob-served both bleeding and thrombotic complicationsand this fact seems to be more common in essentialthrombocythemia. The analysis of clinical aspects de-monstrated that bleeding syndrome is more frequent
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Fig. 4. Various abnormal aspects of platelet aggregatingresponse in the presently investigated patients: A. Abnormalplatelet aggregation response to 10 µM epinephrine -peculiar aspect - a very prolonged wave with bluntedslope. B. Abnormal platelets aggregation response with10µM ADP - the loss of the secondary wave of aggrega-tion and deaggregation tendency. C. Abnormal plateletaggregation response to 10 µg/ml collagen - prolonged"lag"phase: more than 60 seconds. Arrowheads mark themoment of agonist addition.
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Fig. 5 A frequent pattern of platelet response in the inves-tigated chronic myeloproliferative disorders: A. markedlydecreased of platelet response to 10 µM epinephrine.B. decrease of platelet aggregation to 10 µM ADP withdeaggregation tendency. C. decreased platelet response to10 µg/ml collagen. D. absent platelet aggregation to 0,5mM arachidonic acid. E. normal platelet response to1mg/ml ristocetin. Arrowheads mark in each diagram themoment of agonist addition.
A B
C D
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than thrombosis; the former was most frequent in my-eloid metaplasia with myelofibrosis and the latter wasmost common in essential thrombocythemia (Fig. 6).
The template bleeding time was normal in 62cases (82%) and prolonged in 14 cases (18 %).
We tried to correlate platelet aggregationabnormalities with thrombohemorrhagic manifes-tation and with bleeding time but the results werenot significant.
It was difficult to establish a correlation betweenthe type of platelet aggregating alterations and thesubtype of MPD, suggesting that aggregometry isnot very useful in differential diagnosis betweenthese entities.
Discussion
The thrombohaemorrhagic complications occurringin MPD incited many authors to investigate thefunction of platelets and to correlate the detectedabnormalities with these clinical aspects and withchanges of bleeding time [3,4,23-27].
In the present study we tried to detect variouspatterns of platelets aggregation response in MPDand to determine the possible relevance of theseabnormalities for a closer diagnostic of these dis-eases. It could be emphasized that platelet aggre-gating responses to various inducers are quite fre-quent and display several patterns in MPD. Theincidence of impaired platelet aggregation to vary-ing agonists is in agreement with those generallyreported in the literature [ 24,28,29].
Also, in agreement with most previous studies,we found that the most frequently detected abnor-mality was the impaired epinephrine-induced plateletaggregation [7,8,21,28-30]. This result consolidatesthe hypothesis that an important biochemical defectof platelet in MPD is the loss of α2-adrenergic recep-tors [31]. Also, this impaired response seems to behighly suggestive for a MPD and can be useful indiscriminating ET from secondary thrombocytosis[21,28]. Referring to this agonist, we observed apeculiar aspect of aggregation that could be consid-ered rather specific for MPD.
Abnormalities of ADP-induced platelet aggrega-tion response occurred less frequently and weremainly represented by an absence of the secondarywave of aggregation, a fact mentioned by otherauthors [6,7,24,28]. A question raised by our exper-iments is why not all decreased responses to ADPshow reversibility of aggregation.
To our knowledge, the peculiar aspect of colla-gen-induced aggregation (prolongation of “lag”phase) observed in MPD was not commented byother authors. We consider that this aspect shouldbe approached in further studies of MPD.
Despite the fact that any combination of plateletaggregation defects may be seen in this pathology[24,32], we observed in a not negligible percent ofcases a certain pattern of abnormal responses.
As previously noted [20,21,28] the low percentageof template bleeding time alteration demonstrate thatthis parameter is not correlated with the degree offunctional platelet defects in MPD and cannot be con-sidered as a reliable marker for these diseases.
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Fig. 6. Incidence ofbleeding, thrombosis orboth in various types ofchronic myeloprolifera-tive disorders. (chronicmyelogenous leukemia- CML, myeloid meta-plasia and myelofibro-sis - MMM, polycythe-mia vera - PV, essentialthrombocythemia - ET)
The occurrence of thrombohemorrhagic com-plications in our study group was in agreementwith other studies [25-27, 30, 33]. There is also aninsufficient correlation of these manifestationswith platelet aggregation defects [12, 23-27].
We intend to continue this study and extend it tothe determination of the relationship betweenplatelet aggregation abnormalities and the develop-ment stage of the disease.
Conclusions
According to these observations, we conclude thatplatelet aggregation abnormalities in chronicmyeloproliferative disorders are frequent, com-plex and varied.
Although these abnormalities are not specificfor MPD we consider that their detection may beuseful for a complete diagnosis of disease. A nor-mal platelet aggregation cannot exclude a chronicmyeloproliferative disorder but we may suspect achronic myeloproliferative disorder if plateletsaggregation is abnormal in a certain pathologicalcontext. Impaired epinephrine-induced plateletaggregation is the most common defect found atthese patients. Other aggregation abnormalitieswere found in varied degree.
We observed some peculiar abnormal respons-es to diverse agonists, for example a prolonged flatcurve (blunted slope) to epinephrine, deaggrega-tion to ADP and prolonged “lag” phase when col-lagen was the agonist.
We tried to achieve a characteristic pattern ofabnormal platelet aggregation in MPD but furtherinvestigations are required in order to demonstratethat the presently detected pattern is specific forthese diseases, so that it might be used as an addi-tional diagnostic tool.
The bleeding complication is more frequentthan thrombosis and this fact demonstrate thatfunctional platelet defects are common in thesedisorders.
The type of disease couldn’t be correlated witha certain type of platelet aggregation response.Also, a correlation between clinical aspects, tem-plate bleeding time and platelet aggregation is dif-ficult to achieve.
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