Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients

Hanan M Abdel Aziz; Esam A S El-Beih; Douaa M Sayed; Ola A Afifi; Ahmad F Thabet; Sahar Elgammal; Shima G Mansor; Sawsan M Moeen

doi:10.4103/ejh.ejh_14_20

ORIGINAL ARTICLE

Year : 2022 | Volume : 47 | Issue : 3 | Page : 187-193

Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients

Hanan M Abdel Aziz¹, Esam A S El-Beih¹, Douaa M Sayed², Ola A Afifi³, Ahmad F Thabet¹, Sahar Elgammal³, Shima G Mansor², Sawsan M Moeen¹
¹ Department of Internal Medicine, Clinical Hematology Unit, Faculty of Medicine, Assiut, Egypt
² Flow Cytometry Lab., Department of Clinical Pathology, South Egypt Cancer Institute, Assiut, Egypt
³ Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt

Date of Submission	09-Mar-2020
Date of Acceptance	01-Sep-2022
Date of Web Publication	03-Jan-2023

Correspondence Address:
Hanan M Abdel Aziz
Faculty of Medicine, Assiut University, Assiut 71515
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_14_20

Abstract

Introduction Thromboembolism is a frequent complication in thalassemia. Platelet microparticles (PMPs) may have a role in the hypercoagulability in thalassemia. Objective To determine the levels of PMPs in β-thalassemia intermedia (β-TI) patients and whether increased levels of PMPs in these patients are correlated with coagulation parameters or not. Patients and methods Sixty-three β-TI patients, classified into 46 nonsplenectomized (NS) patients, 17 splenectomized (S) patients, and 20 age-matched and sex-matched volunteers as controls. For all, full medical history, through clinical examination, and laboratory investigations: complete blood count, prothrombin time, prothrombin concentration, activated partial thromboplastin time, FVIII, FXI, fibrinogen, D-dimer, and PMPs were detected by using flow-cytometry approach. Results PMPs were significantly higher in S and NS β-TI patients than controls (P<0.001, P=0.002), respectively. FVIII and D-dimer were significantly higher in S patients than controls (P=0.004, 0.048), respectively. Fibrinogen level was significantly lower in both NS and S β-TI patients than in controls (P<0.001). A significant positive correlation between PMPs and platelet count in S patients (r=0.549, P=0.023). Conclusion Thrombotic risk in β-TI patients is related to increased circulating PMPs and platelet count. Splenectomy was considered a risk factor of thrombosis in our study.

Keywords: hypercoagulability, platelet microparticles, β-thalassemia intermedia

How to cite this article:
Abdel Aziz HM, El-Beih EA, Sayed DM, Afifi OA, Thabet AF, Elgammal S, Mansor SG, Moeen SM. Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients. Egypt J Haematol 2022;47:187-93

How to cite this URL:
Abdel Aziz HM, El-Beih EA, Sayed DM, Afifi OA, Thabet AF, Elgammal S, Mansor SG, Moeen SM. Increased levels of circulating platelet microparticles as a risk of hypercoagulable state in β-thalassemia intermedia patients. Egypt J Haematol [serial online] 2022 [cited 2023 Mar 30];47:187-93. Available from: http://www.ehj.eg.net/text.asp?2022/47/3/187/366859

Introduction

Thalassemia is a common genetic blood disease developed as a result of the defect in hemoglobin-chain formation [1]. The disease distribution occurs among populations from the Mediterranean sea, Middle East, Southeast Asia, and Melanesia [2]. The most frequent form of heterozygotes is βeta-thalassemia and represents from 5.3 to 9% of the Egyptian population [3,4].

Thromboembolism (TE) is a common complication in thalassemia due to hemostatic changes in thalassemic patients [5,6]. The incidence of TE in β-thalassemia intermedia (β-TI) is 3.9–29%, the age at which patients develop thrombosis is 24 years [7].

Thrombotic events in β-TI patients are higher than β-thalassemia major patients [8]. TE in thalassemia includes venous and arterial manifestation that involve various organs due to red-blood cell (RBCs), platelets, and endothelial activation [5,9]. Thrombosis varies from subclinical disorders such as silent cerebral infarction up to manifest TE such as pulmonary embolism or deep venous thrombosis [1]. There are numerous mechanisms for TE, including platelet activation and thrombocytosis [7].

Microparticles (MPs) are subcellular membrane structures that shed from different cells, including platelets and endothelial cells [10]. MPs are involved in endothelial-cell dysfunction and hence thrombotic risk [11]. In thalassemia, increased oxidative stress in platelets and RBCs is associated with MP release from activated platelets and RBCs [12].

MPs in thalassemia are involved in hypercoagulability and thrombosis [13]; splenectomy is associated with RBC damage and chronic platelet activation and increased MP shedding [14]. Increased MP levels in splenectomized β-thalassemia patients increase their susceptibility to develop TE manifestations [11]. The etiologies for the thrombotic risk in thalassemia are numerous, but with unclear mechanisms, increased levels of platelet microparticles (PMPs) may be considered a risk factor for hypercoagulability in thalassemia [12].

Patients and methods

This case–control study was conducted at Clinical Hematology Unit, Internal Medicine Department, Assiut University.

This study included 63 patients diagnosed as β-TI. Their ages ranged from 15 to 48 years with a mean±SD of 26.87 ± 8.07 and a median of 25 (20–32) years, classified as nonsplenectomized (NS) (46/63) patients, splenectomized (S) (17/63) patients, as well as 20 age-matched and sex-matched healthy controls, demographic data of different studied groups are demonstrated in [Table 1].

Table 1: Demographic data of the studied groups

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Exclusion criteria

Any participant with decompensated liver disease, receiving aspirin or drugs that could alter the hemostatic function 2 weeks before sample collection, previous history of thrombosis was excluded from the present study. The clinical data of our patients are demonstrated in [Table 2].

Table 2: Clinical data of the patients

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In NS patients, age of onset of the disease ranges from 2 to 20 years. Thirty two (69.6%) were transfusion independent, duration of the last transfusion ranged between 2 and 120 months, and 28 (60.9%) patients presented with pallor, 31 (67.4%) jaundiced, 22 (47.8%) presented with hepatomegaly, 40 (87%) were having splenomegaly, eight (17.4%) were on iron-chelation therapy, and no patient developed thrombosis during the study. In S patients, age of onset ranged from 2 to 21 years, 14 (82.4%) were transfusion dependent, duration of the last transfusion ranged between 3 and 96 months, and eight (47.1%) patients presented with pallor, 14 (82.4%) jaundiced, and three (17.6) presented with hepatomegaly, mean duration of splenectomy was 9.06 years, nine (52.9%) were on iron chelation.

Our patients did not have a previous history of manifest thrombosis before the study, and on follow-up, one (5.9%) patient in S group developed deep venous thrombosis followed by pulmonary embolism. Full medical reports about this thromboembolic event could not be obtained as he was not admitted to our hospital during that event, but this history was taken from his relatives.

Specimen collection and analysis

About 2 ml of venous blood was collected by venipuncture for flow cytometry, 2 ml obtained for hemostatic assessment aliquoted into tubes containing sodium citrate, and another 5 ml for chemistry aliquoted into a plain tube. The following routine investigations were performed for all studied groups, including: complete blood count, liver-function tests and serum ferritin, prothrombin time (PT), prothrombin concentration (PC), activated partial thromboplastin time (aPTT), fibrinogen, FVIII, and FIX performed on Sysmex CA-1500 (Siemens, Marburg, Germany). All reagents used were supplied by Siemens, Marburg, Germany. D-dimer: Assay of D-dimer, using ELISA kit (WKEA MED, China), was also performed on STAT FAX 2100 (Awareness Technology Inc., Germany).

Flow-cytometric detection of MPs by direct immunofluorescence technique using fluorescein isothiocyanate-conjugated Annexin-V (IQ produces) and anti-CD41 phycoerythrin (PE) Ab. FACS Calibur flow cytometric analysis using Cell-Quest software (Becton Dickinson Biosciences, USA) was used to measure the results. It was used to identify PMPs. PE- and fluorescein isothiocyanate-conjugated isotype IgG controls were used to define the background noise of labeling isolation of MPs, flow-cytometric analysis used to quantify and characterize MP methods was conducted from Sewify et al. [15] on FACS Caliber (Becton Dickinson Biosciences, USA) in the flow-cytometry lab, South Egypt Cancer Institute, Assiut University.

Isolation of microparticles

(1) Blood sample was collected into a 5-ml tube containing 3.2% sodium citrate.

(2) In order to isolate platelet-rich plasma, the cells were removed by centrifugation for 20 min at 1550 g at 20°C within 15 min after collection.

(3) Then 250 μl of plasma was centrifuged for 30 min at 18 800 g at 20°C. To isolate the MPs after centrifugation, the supernatant was removed, and the pellet was resuspended in phosphate-buffered saline and centrifuged for 30 min at 18 800 g at 20°C.

(4) The supernatant was removed again and the MP pellet was resuspended in phosphate-buffered saline.

[Figure 1]

Figure 1: Flow cytometry of PMPs in β-TI patients. PMP, platelet microparticle.

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Statistical analysis

Continuous variables were reported as mean±SD. The comparison between more than two independent groups with quantitative data was done by using one-way analysis of variance followed by post-hoc analysis using the LSD test. Spearman correlation coefficients were used to assess the correlation. The P value was considered significant as the following: P value more than 0.05: nonsignificant, P value less than 0.05: significant, and P value less than 0.01: highly significant. Data were collected, revised, coded, and entered to the Statistical Package for Social Science (IBM SPSS), version 23.

Ethical consideration

The study was approved by the Institutional Review Board (IRB), Faculty of Medicine, Assiut University (IRB 17200266). Informed written consent was taken from all enrolled cases and controls.

Results

As regards demographic data of our study, there were no statistically significant differences between age and sex of the studied groups and controls ([Table 1]).

In our study, platelet count was higher in both NS and S patients than controls (P=0.009, P<0.001), respectively.

Hemostatic parameters, including PT, PC, aPTT, fibrinogen and D-dimer FVIII, and FIX demonstrated in [Table 3], show higher PT in both NS and S groups than controls (P<0.001 and 0.002), respectively. PC was significantly lower in NS patients than controls (P<0.001) and in (S) β-TI patients than controls (P<0.001).

Table 3: Hemostatic parameters of the studied groups

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FVIII levels were higher in β-TI patients when compared with controls, and it was only observed in S group (P<0.002). Fibrinogen levels were significantly lower in both NS and S patients than controls (P<0.001).

D-dimer levels were significantly higher in S group in comparison with controls (P=0.048).

No statistically significant difference in aPTT and FIX levels in NS and S β-TI patients in comparison with controls.

The percentage of PMPs in blood samples from both NS and S β-TI patients was higher when compared with blood samples from healthy controls (P<0.002 and P=0.001), respectively. There was no statistically significant difference in PMP percentage between NS and S β-TI (mean±SD: 46.67 ± 18.45, 56.39 ± 18.7), respectively (P=0.090) ([Figure 2]).

Figure 2: Platelet microparticle level in different studied groups, higher levels of PMPs were noticed in patients than controls, particularly in S patients. PMP, platelet microparticle.

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There was a statistically significant positive correlation between PMPs and platelet count in S patients (r=0.549, P=0.023), but in NS patients, there was no correlation (r=0.089, P=0.567), no other statistically significant correlation was noticed between PMP levels and other hemostatic parameters ([Table 4], [Figure 3]).

Table 4: Correlation between platelet microparticles and other hemostatic parameters

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Figure 3: Correlation between PMPs and platelet count in splenectomized patients. A significant positive correlation between PMPs and PLT count is noticed in S patients. PLT, platelet; PMP, platelet microparticle.

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Discussion

Ineffective erythropoiesis in splenectomized β-TI patients is associated with abnormal erythroid precursors that act as activated platelets and enhance the conversion of prothrombin to thrombin and hence thrombosis, chronic platelet activation occurs in splenectomized patients due to the associated post-splenectomy thrombocytosis, which involved in the thrombotic tendency in thalassemia patients [16].

Our study shows higher platelet count in β-TI patients than controls, particularly in S group. These results are similar to the previous results [5,6].

As our result, Faraj [17] reported prolonged PT in thalassemia patients than controls and he found a positive correlation between PT prolongation and serum ferritin. Prolonged PT may be attributed to the hepatic dysfunction resulting from hepatic iron overload. Tripatara et al. [18] reported no statistically significant difference between thalassemic patients and controls as regards PT, they suggested that although there was liver-cell damage in thalassemic patients, hepatic protein synthesis was not affected.

D-dimer is a marker for fibrin formation and fibrinolysis and hence thrombotic risk in the patients. The high plasma levels of D-dimer, can be taken as another indirect index of thrombin activity. Cappellini et al. [16] study and Tripatara et al. [18] also reported higher D-dimer level in β-thalassemia/HbE patients compared with controls, this was markedly noticed in S group, similar results were observed in Hassan et al. [19] study and more recently Hashemieh et al. [20] study. In contrast, Naithani et al. [21] reported no significant difference in D-dimer levels in β-thalassemia patients and controls, but his study was done on thalassemia major patients.

We and Hashemieh et al. [20] reported a higher level of FVIII in S patients than controls and they added that the level of proteins C and S, antithrombin, and fibrinogen showed a significant reduction in TI patients compared with controls. In contrast, Tripatara et al. [18] reported lower FVIII level in TI patients than controls, but this result was noticed in patients with severe form of the disease and on regular blood transfusion every 4 weeks, and they suggested that in spite of receiving normal RBCs in regular blood transfusions, thalassemic red cells still present after splenectomy and have procoagulant character due to the presence of negatively charged phosphatidylserine on their surface.

In the current study,it is noticed that splenectomy has a significant effect on the concentration of D-dimer and factor-VIII activity with no significant difference in FIX level, increased activity of factor VIII is considered as a common cause of hypercoagulability [22]. Low fibrinogen level was found in our β-TI patients as well as in other studies. Fayed et al. [1] reported increased thrombotic risk in splenectomized β-thalassemia patients due to the presence of a low-grade consumptive coagulopathy in those patients when compared with those with intact spleen.

Numerous studies showed higher levels of circulating PMPs in thalassemic patients in comparison with controls [12,23]. MPs were derived mainly from damaged platelets and RBCs and the high levels of these MPs were correlated with the increased platelet and with the increased thrombotic risk measured by prothrombinase-complex assay. Also, Tantawy et al. [24] reported higher PMPs in S patients than NS patients and they suggested that their levels were elevated in relation to pulmonary hypertension and history of thrombosis, while Youssry et al. [13] reported a higher percentage of PMPs in thalassemic patients with thromboembolic event. Kheansaard et al. [11] study showed that the PMPs and MPs derived from RBCs of β-thalassemia patients were higher than controls and this was more marked in S than NS patients.

Recent studies [11] showed higher levels of endothelial-cell activation markers in human umbilical-vein endothelial cells when treated with splenectomized β-thalassemia patient MPs in comparison with NS patient MPs or that from controls. The increased endothelial-cell activation ultimately leads to increased monocyte–endothelial-cell adhesion. This finding suggests that MPs play an important role in thrombosis and vascular dysfunction in β-thalassemia/HbE disease, especially in splenectomized cases. Increased PMPs are associated with thrombotic risk in β-TI patients as a result of phosphatidylserine exposure on the surface of the abnormal RBCs in thalassemia patients, which is associated with platelet activation by mechanisms involved in its function in hemostasis followed by the generation of PMPs [24,25].

We noticed a significant positive correlation between PMPs and platelet count in S patients that may indicate chronic platelet activation and thrombotic tendency in thalassemia patients.

Our study showed no statistically significant correlation between PMPs and other hemostatic parameters, including PT, PC, aPTT, FVIII, FIX, and fibrinogen, but there was a positive correlation with D-dimer. No similar studies were done in this respect, and further researches are recommended to confirm our results.

Conclusion

The thrombotic risk in β-TI patients, particularly after splenectomy, might be related to increased circulating PMPs, which may be associated with platelet activation, platelet aggregation, and hence thrombus formation. FVIII and D-dimer level were increased in β-TI patients, particularly after splenectomy, but not correlated with PMPs, this may attributed to a limited number of patients or the duration of the study.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.

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