Journal of Applied Hematology

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 9  |  Issue : 4  |  Page : 126--130

Evaluation of platelet aggregation in splenectomized beta-thalassemia major and intermedia patients


Mahdi Zahedpanah1, Azita Azarkeivan2, Minoo Ahmadinejad3, Mohamad R Tabatabaiee3, Bashir Hajibeigi2, Mahtab Maghsudlu4,  
1 Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
2 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Thalassemia Clinic, Tehran, Iran
3 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Coagulation Lab, Tehran, Iran
4 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Correspondence Address:
Dr. Mahdi Zahedpanah
Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Qazvin University of Medical Sciences, Bahonar Blv, Qazvin
Iran

Abstract

BACKGROUND: Platelet dysfunction may be one of the pathophysiologic complications in beta-thalassemia patients. However, the results obtained from the platelet aggregation vary in different types of β-thalassemia and among patients. OBJECTIVE: We evaluated the platelet function to determine risk of thrombosis in two groups': beta-thalassemia major (TM) and intermedia (TI). MATERIALS AND METHODS: In a cross-sectional study, platelets of 82 adult patients with beta-thalassemia (46 β-TM and 36 β-TI) who had undergone splenectomy and 85 normal healthy individuals as control were induced by collagen 10 μ g/ml, adenosine diphosphate (ADP) 20 μ M/l, arachidonic acid 500 μ M/l, and ristocetin 1500 μ g/ml. Independent t-test was used to compare the mean values using SPSS 16. P < 0.05 was taken to indicate statistical significance. RESULTS: Although a significant increase was observed in the platelet aggregation by collagen, ADP, and arachidonic acid in β-TM patients as compared with healthy controls, the β-TI patients showed no difference (P < 0.05). There was no significant alteration in response to ristocetin in β-TM but it reduced in β-TI. CONCLUSIONS: The platelet aggregation in β-TM patients is more than β-TI, both of whom splenectomized. The platelet aggregation in beta-thalassemia might be impressed by transfusion. Given these changes, thrombotic risk should be considered in beta-thalassemia patients.



How to cite this article:
Zahedpanah M, Azarkeivan A, Ahmadinejad M, Tabatabaiee MR, Hajibeigi B, Maghsudlu M. Evaluation of platelet aggregation in splenectomized beta-thalassemia major and intermedia patients.J Appl Hematol 2018;9:126-130


How to cite this URL:
Zahedpanah M, Azarkeivan A, Ahmadinejad M, Tabatabaiee MR, Hajibeigi B, Maghsudlu M. Evaluation of platelet aggregation in splenectomized beta-thalassemia major and intermedia patients. J Appl Hematol [serial online] 2018 [cited 2019 Apr 22 ];9:126-130
Available from: http://www.jahjournal.org/text.asp?2018/9/4/126/251493


Full Text

 Introduction



The thalassemia syndromes are a heterogeneous group of inherited anemia characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Beta-thalassemia intermedia (β-TI) differentiation from major (β-TM) (According to International Thalassemia Federation [TIF], major thalassemia if the patient started transfusion protocol before 2 years old and thalassemia intermediate if transfusion started after 2 years old) type is accomplished with assessment of clinical manifestations, severity, laboratory features, and age of presentation.[1],[2] These patients are involved in a lot of complications of the primary disease and of the treatment-related. The hypercoagulable state, meaning that there is more procoagulant or clotting activity (or less anticoagulant activity), has been introduced in beta-thalassemia patients and leads to various thrombotic complications.[1],[3]

Numerous factors have been involved in hypercoagulable state in beta-thalassemia patients that contribute to thrombosis and thromboembolic events (TEEs). The incidence of TEE in β-TI has been reported to be higher than in β-TM.[4] Transfusion and phosphatidylserine (PS) exposure in the outer membrane leaflet of red blood cells are usually two factors that contributed to risk of thrombosis in beta-thalassemia patients. In β-TI with irregular blood transfusion, the clinical complications resulted from red blood cells (RBCs'), PS-exposing might be expected at a higher rate than patients with β-TM who received blood transfusion regularly.[5],[6] Platelet dysfunction may be one of their pathophysiologic complications. There have been several reports of thromboembolic complications in thalassemia due to spontaneous platelet aggregation leading to tribulations such as microthrombi in pulmonary artery and hypoxemia.[7] So in the management of the thalassemia, disorders of hemostasis and coagulation have to be taken into account. However, the results obtained from the platelet aggregation vary in different types of β-thalassemia and among patients.

The aim of this study was to investigate the platelet aggregation in β-TM in comparison with β-TI to identify which patients should be further put under observation for platelet thrombosis.

 Materials and Methods



Our study was approved by the IBTO (Iranian Blood Transfusion Organization) committee. Written informed consent was obtained from patients and their parents or legal guardians in all cases. A cross-sectional examination, for a period of 1 year, was carried out on patients who had complete files with authentic information for a period of 1 year. Basic information such as age, sex, type of thalassemia, and blood groups was extracted from their files from Thalassemia Clinic of Iran Blood Transfusion Organization in Tehran. This assessed 82 participants with beta-thalassemia. Clinically, 46 patients were β-TM and 36 patients TI. For TM patients, we had fixed periodic transfusions or regular transfusion (every 2–4 weeks), but in TI patients, we had transfusion at time of severe anemia, without any fixed period for transfusion. All patients have been splenectomized. No patient had been taking anti-aggregating drugs. Patients with acquired disorders leading to platelet dysfunction were excluded from this survey. As controls, we studied 85 age- and sex-matched normal healthy subjects.

Venous blood was collected by Vacutainer plastic tubes with trisodium citrate. The other specimen was taken from each patient in ethylenediaminetetraacetic acid-containing tube for platelet count. Platelet function of beta-thalassemic patients was measured in vitro by platelet aggregation test. This test can indicate a certain function of the platelets in vivo. If platelets are exposed to a stimulatory agonist they can be aggregated. Adenosine diphosphate (ADP), collagen, arachidonic acid, and ristocetin are usual agonists using for testing.

Platelet-rich plasma (PRP) was obtained by centrifuging at room temperature for 10–15 min at 400 g. Platelet-poor plasma (PPP) was prepared by centrifugation of the remaining blood at 2000 g for 15 min. Testing was completed within 3 h of collecting the blood sample. A platelet count was performed on the PRP. Because of the very high count arising from splenectomy, the patient samples were adjusted by diluting the PRP in the patient's PPP to give a platelet count of 200–400 × 109/1. The control PRP also was adjusted to the same count. The four aggregating agents in a local laboratory concentration references, collagen 10 μg/ml, ADP 20 μM/l, arachidonic acid 500 μM/l, and ristocetin 1500 μg/ml were included in this survey. The agonists obtained from Helena Biosciences. We have performed study on Helena Laboratories PACKS-4® aggregometer (Platelet Aggregation Chromogenic Kinetic System). This procedure is performed on a turbidimetric aggregometer. Platelets were counted in whole blood using Sysmex XT-2000i automated hematology analyzer.

The distribution of results is a normal bell-shaped curve given the assumption Kolmogorov–Smirnov test was greater than the significance level of 0.05. The groups had equal variances regarding to the Levene's test was not significant. Hence, independent t-test was used to compare the mean values for two groups' patients and controls. Data were analyzed with computer software SPSS version 16 (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.

 Results



The mean age of our patients was 29.9 ± 10.5 years. 35 (43%) patients were male and 47 (57%) were female. [Table 1] shows the characteristics of patients. [Figure 1] shows the percentage of the platelet aggregation with agonists. [Table 2] presents the mean percentage of platelet aggregation in the groups.{Table 1}{Figure 1}{Table 2}

In the group with β-TM, the mean percentage of the platelet aggregation was significantly (P < 0.05) increased on induction with collagen, ADP, and arachidonic acid as compared to the controls, but ristocetin had no effect on platelet agglutination. In contrast to β-TM, the TI group showed no increase in platelet aggregation induced by these agonists, but platelet agglutination with ristocetin decreased significantly as compared to the control group. It is apparent from [Table 3] that patients' platelets with TM are aggregated more than intermedia, except when induced with ADP.{Table 3}

 Discussion



In the present study, we found that unlike the patients with the TI, there is an increase in platelet aggregation in the TM in the presence of agonist collagen, ADP, and arachidonic acid. According to reports, in patients with β-thalassemia, especially whom splenectomized, in vitro platelet aggregation may be associated with alterations, whether due to transfusion, or due to disease characteristics, such as PS-exposing RBCs, mutation type, or even due to the existence of racial differences.[8] Hence, the results vary in different studies. Some studies have suggested an increased platelet aggregation; on the other hand, others have reported either decreased or normal reactivity. Impaired platelet aggregation has been reviewed in most of β-TM by Eldor and Rachmilewitz.[8] In a study on children suffering from β-TM and TI, the decreased aggregation in more than 60% of patients and the normal aggregation in others has been shown.[9] In our study, platelets of β-TM patients exhibited an increased aggregation in response to collagen, ADP, and arachidonic acid. Ristocetin had no effect on platelet agglutination. On the whole, there was a platelet hyperaggregation. The presence of a chronic platelet activation in β-TI patients might prevent further stimulation of the platelets by the agonists; actually the activated platelets become refractory to additional stimulation.

Those reported by Shebl et al. who found a platelet hyperaggregation in response to collagen in thalassemic children who had undergone splenectomy.[10] However, Orudzhev et al. suggested that the increased quantity of antibody against blood platelets is the reason of disaggregation in patients with beta-thalassemia, and especially in those who had intact spleen.[11] The investigation on relationship between coagulation and splenectomy has shown that the spontaneous platelet aggregation appears to be more common in splenectomized patients.[6] In this context, a report from Indonesia has demonstrated a significantly increased aggregation in ADP-stimulated platelets in splenectomized β-TM patients compare to nonsplenectomized patients.[12] Atichartakarn et al. have presented that following splenectomy in hemoglobin E/beta-thalassemia, there is a platelet hyperaggregation with ADP and ristocetin using whole-blood analysis.[13] Furthermore, in two other investigations, increase in platelet aggregation in beta-thalassemia after splenectomy has also been described.[14],[15]

It has already been shown that the elevation of PS on the RBCs surface of the thalassemic patients[16],[17] is related to hemostatic anomalies and plays an important role in recognition and removal of erythrocytes by the spleen.[18] Besides, Brown et al. have reported a significant increase in collagen-stimulated platelet aggregation by erythrocytes that they have attributed it to PS exposure on RBC outer membrane.[19] PS allows the assembly of the prothrombinase complex and results in increased thrombin formation,[20] the most potent activator of platelets. Hence, splenectomy might effect on function enhancement of platelets by increasing both PS-exposing RBCs and thrombin in circulation. Furthermore, by noting the role of the red blood cells membrane in hypercoagulable state, the fibrinogen levels, an important agent for platelet aggregation, is reduced by increased thrombin generation in beta-thalassemia patients,[17],[21] while in patients with beta-TI, unlike TM who receive blood regularly cannot be compensated for by transfusion.[5],[8]

We found a slightly more increased but not significant in ADP-induced platelet aggregation in β-TM than β-TI. Although the splenectomized patients had an intravascular hemolysis that it could be associated with hypercoagulable states, it will occur in β-TM after blood transfusion as well.[22] Considering the studies, it suggests that release of intraerythrocytic ADP might increase platelet reactivity through activation of the P2Y12 platelet receptor activated by ADP.[8],[12],[18],[23],[24],[25]

In this study, despite the reduction of the platelet activity in response to ristocetin in β-TI, not in β-TM, the responses to other agonists did not alter. Significant diminished in response to ristocetin in TI group may be attributed to the presence of a plasma inhibitor, or an abnormally high factor VIII antigen/von Willebrand factor activity ratio, as it has reported in patients with sickle cell anemia and beta-thalassemia hemoglobin E.[26],[27],[28] According to the cause, the normoaggregation of the platelets stimulated by ristocetin in β-TM group might be resulted from plasma dilution and relatively compensation of von Willebrand factor by transfusion.[26] Differences in race and the presence of a kind of single nucleotide polymorphisms may also contribute to reactivity of platelets to ristocetin,[29],[30] however, in our patients and controls, there are no known racial differences, whereas they differ obviously in ethnicity, that might affect the interaction between ristocetin and platelet.

 Conclusions



The platelet aggregation in β-TM patients is more than β-TI, both of whom splenectomized. The platelet aggregation in beta-thalassemia might be impressed by transfusion. Given these changes, thrombotic risk should be considered in beta-thalassemia patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Rund D, Rachmilewitz E. Beta-thalassemia. N Engl J Med 2005;353:1135-46.
2Olivieri NF. The beta-thalassemias. N Engl J Med 1999;341:99-109.
3Zahedpanah M, Azarkeyvan A, Hajibeygi B, Ahmadinezhad M, Eshghi P, Tabatabaei MR, et al. Coagulation inhibitors in thalassemia. Sci J Iran Blood Transfus Organ 2010;7:78-8.
4Sirachainan N. Thalassemia and the hypercoagulable state. Thromb Res 2013;132:637-41.
5Cappellini MD, Robbiolo L, Bottasso BM, Coppola R, Fiorelli G, Mannucci AP, et al. Venous thromboembolism and hypercoagulability in splenectomized patients with thalassaemia intermedia. Br J Haematol 2000;111:467-73.
6Cappellini MD, Grespi E, Cassinerio E, Bignamini D, Fiorelli G. Coagulation and splenectomy: An overview. Ann N Y Acad Sci 2005;1054:317-24.
7Sumiyoshi A, Thakerngpol K, Sonakul D. Pulmonary microthromboemboli in thalassemic cases. Southeast Asian J Trop Med Public Health 1992;23 Suppl 2:29-31.
8Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood 2002;99:36-43.
9Chaudhary HT, Ahmad N. Frequency of platelet aggregation defects in children suffering fromg β-thalassemia. Saudi J Health Sci 2012;1:92.
10Shebl SS, el-Sharkawy HM, el-Fadaly NH. Haemostatic disorders in nonsplenectomized and splenectomized thalassaemic children. East Mediterr Health J 1999;5:1171-7.
11Orudzhev AG, Guseĭnova EE, Khalilova IS, Dzhavadov SA. Assessment of endogenous intoxication and thrombocyte functions in beta-thalassemia. Klin Lab Diagn 2003;3:39-41.
12Setiabudy R, Wahidiyat PA, Setiawan L. Platelet aggregation and activation in thalassemia major patients in indonesia. Clin Appl Thromb Hemost 2008;14:346-51.
13Atichartakarn V, Angchaisuksiri P, Aryurachai K, Chuncharunee S, Thakkinstian A.In vivo platelet activation and hyperaggregation in hemoglobin E/beta-thalassemia: A consequence of splenectomy. Int J Hematol 2003;77:299-303.
14Opartkiattikul N, Funahara Y, Fucharoen S, Talalak P. Increase in spontaneous platelet aggregation in beta-thalassemia/hemoglobin E disease: A consequence of splenectomy. Southeast Asian J Trop Med Public Health 1992;23 Suppl 2:36-41.
15Laosombat V, Wongchanchailert M, Kenpitak K, Wisitpongpon C. Spontaneous platelet aggregation in thalassemic children and adolescents. Southeast Asian J Trop Med Public Health 1992;23 Suppl 2:42-6.
16Zahedpanah M, Azarkeivan A, Aghaieepour M, Nikogoftar M, Ahmadinegad M, Hajibeigi B, et al. Erythrocytic phosphatidylserine exposure and hemostatic alterations in β-thalassemia intermediate patients. Hematology 2014;19:472-6.
17Atichartakarn V, Angchaisuksiri P, Aryurachai K, Onpun S, Chuncharunee S, Thakkinstian A, et al. Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/beta-thalassaemic patients. Br J Haematol 2002;118:893-8.
18Kuypers FA, de Jong K. The role of phosphatidylserine in recognition and removal of erythrocytes. Cell Mol Biol (Noisy-le-Grand) 2004;50:147-58.
19Brown GE, Ritter LS, McDonagh PF, Cohen Z. Functional enhancement of platelet activation and aggregation by erythrocytes: Role of red cells in thrombosis. Peer J Preprints 2014;2:e351v.
20Chung SM, Bae ON, Lim KM, Noh JY, Lee MY, Jung YS, et al. Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes. Arterioscler Thromb Vasc Biol 2007;27:414-21.
21Triantafyllou AI, Vyssoulis GP, Karpanou EA, Karkalousos PL, Triantafyllou EA, Aessopos A, et al. Impact of β-thalassemia trait carrier state on cardiovascular risk factors and metabolic profile in patients with newly diagnosed hypertension. J Hum Hypertens 2014;28:328-32.
22Atichartakarn V, Chuncharunee S, Archararit N, Udomsubpayakul U, Aryurachai K. Intravascular hemolysis, vascular endothelial cell activation and thrombophilia in splenectomized patients with hemoglobin E/β-thalassemia disease. Acta Haematol 2014;132:100-7.
23Helms CC, Marvel M, Zhao W, Stahle M, Vest R, Kato GJ, et al. Mechanisms of hemolysis-associated platelet activation. J Thromb Haemost 2013;11:2148-54.
24Andrews DA, Low PS. Role of red blood cells in thrombosis. Curr Opin Hematol 1999;6:76-82.
25Silvain J, Abtan J, Kerneis M, Martin R, Finzi J, Vignalou JB, et al. Impact of red blood cell transfusion on platelet aggregation and inflammatory response in anemic coronary and noncoronary patients: The TRANSFUSION-2 study impact of transfusion of red blood cell on platelet activation and aggregation studied with flow cytometry use and light transmission aggregometry. J Am Coll Cardiol 2014;63:1289-96.
26Leichtman DA, Brewer GJ. A plasma inhibitor of ristocetin-induced platelet aggregation in patients with sickle hemoglobinopathies. Am J Hematol 1977;2:251-8.
27Sarji KE, Eurenius K, Fullwood CO, Schraibman HB, Colwell JA. Abnormalities of platelet aggregation in sickle cell anemia. Presence of a plasma factor inhibiting aggregation by ristocetin. Thromb Res 1979;14:283-97.
28Visudhiphan S, Ketsa-Ard K, Tumliang S, Piankijagum A. Significance of blood coagulation and platelet profiles in relation to pulmonary thrombosis in beta-thalassemia/Hb E. Southeast Asian J Trop Med Public Health 1994;25:449-56.
29Buchanan GR, Holtkamp CA, Levy EN. Racial differences in ristocetin-induced platelet aggregation. Br J Haematol 1981;49:455-64.
30Flood VH, Gill JC, Morateck PA, Christopherson PA, Friedman KD, Haberichter SL, et al. Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor. Blood 2010;116:280-6.