|Year : 2013 | Volume
| Issue : 4 | Page : 131-136
Assessment of some plasma fibrinolytic proteins in sickle cell anemia patients in steady state and in vaso-occlusive crises
Timothy Ekwere1, Sunday Paul Ogunro2, Alani S Akanmu3
1 Department of Haematology, College of Health Sciences, University of Uyo, Uyo, Akwa-Ibom State, Nigeria
2 Department of Chemical Pathology, College of Health Science, Ladoke Akintola University of Technology, Osogbo, Nigeria
3 Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Lagos State, Nigeria
|Date of Web Publication||26-Feb-2014|
Sunday Paul Ogunro
Department of Chemical Pathology, College of Health Science, Ladoke Akintola University of Technology, Osogbo
Source of Support: None, Conflict of Interest: None
Background: The clinical manifestations of vaso-occlusion result from a dynamic combinations of abnormalities in hemoglobin (Hb) structure and functions, red blood cell membrane integrity, erythrocyte density, endothelial activation, microvascular tone, inflammatory mediators and coagulation factors.
Objective: The objective of the following study is to determine the changes in plasma concentration of fibrinolytic proteins among sickle cell anemia (SCA) patients in two clinical states; steady state and vaso-occlusive crises (VOC) and compare with HbAA controls and also to determine if any, the clinical relevance of these proteins in evaluating these patients in the different clinical state.
Materials and Methods: A total of 25 (14M: 11F) HbSS subjects in VOC, 24 (13M: 11F) HbSS subjects in a steady state and 30 (17M: 13F) healthy HbAA volunteers matched for age and sex with the subjects were recruited for the study. Hematological parameters, i.e., full blood count, fibrinolytic proteins concentration including plasma concentration of D-dimer, plasminogen, fibrinogen (FBG), tissue plasminogen activator (tPA) and fibrinopetide-A were determined.
Result: Plasma fibrinopeptide A (FPA) concentration of 680.99 411.37 ng/ml for SCA subjects in VOC and 449.67 310.01 ng/ml in steady state subjects were significantly increased (P < 0.001) compared with 163.52 86.26 ng/ml for HbAA controls. However, there were no statistically significant changes in the plasma concentrations of D-dimer, FBG, plasminogen and tPA among the subjects in any of the clinical states (steady state and VOC) and controls studied.
Conclusion: The significant increase in FPA concentration observed among subjects compared to controls may have been confounded by hyperleucocytosis especially polymorphonuclear leucocytes commonly associated with SCA. Thus, the clinical relevance of these plasma proteins in evaluating these patients in either of the clinical states of this disorder is doubtful.
Keywords: D-dimer, fibrinogen, plasminogen, sickle cell anemia, tissue plasminogen activator fibrinopeptide A
|How to cite this article:|
Ekwere T, Ogunro SP, Akanmu AS. Assessment of some plasma fibrinolytic proteins in sickle cell anemia patients in steady state and in vaso-occlusive crises. J Appl Hematol 2013;4:131-6
|How to cite this URL:|
Ekwere T, Ogunro SP, Akanmu AS. Assessment of some plasma fibrinolytic proteins in sickle cell anemia patients in steady state and in vaso-occlusive crises. J Appl Hematol [serial online] 2013 [cited 2018 Jan 17];4:131-6. Available from: http://www.jahjournal.org/text.asp?2013/4/4/131/127895
| Introduction|| |
Patients with sickle cell anemia (SCA) suffer from a variety of clinical events associated with small and large vessel occlusion, including vaso-occlusive painful episodes, strokes and acute chest syndrome. Such events may be related to the well-described complex derangements of plasma and cellular hemostatic mechanisms that occur in SCD, which may impact a thrombogenic tendency to this disorder.  Reported changes in the hemostatic system in SCA patients include increases in thrombin generation and activation of fibrinolysis.  These changes are seen in SCA both in steady state and in vaso-occlusive crises (VOC). , Some documented abnormalities in fibrinolytic system in SCA include reduced plasminogen concentration,  elevated D-dimer , and defective release of tissue plasminogen activator (tPA). 
However, whether the activation of blood coagulation and fibrinolysis is contributory to the occurrence of VOC in SCA is unknown and therefore the clinical relevance of these fibrinolytic proteins in the different disease state (steady state and VOC) has not been fully ascertained.
Therefore, the objectives of this study is to determine the changes in plasma concentration of fibrinolytic proteins in SCA patients in steady state and in VOC and compare with hemoglobin (HbAA) controls and also to determine if any, the clinical relevance of these proteins in evaluating these patients in the different clinical state.
| Materials and Methods|| |
The protocol of the study was approved by the Ethical Committee of Lagos University Teaching Hospital (LUTH), Lagos Nigeria.
A total of 25 (14 males and 11 females) HbSS subjects in VOC and 25 (14 males and 11 females) HbSS subjects in steady state were recruited from the Adult and Pediatrics Hematology Out-patient Clinics as well as the Adult and Pediatrics Emergency Department of LUTH. Twenty-five (14 males and 11 females) healthy HbAA volunteers matched for age and sex with the subjects, were used as controls in this study. All the subjects and controls were between the ages of 10 and 40 years. Recruitment of subjects lasted 4 months from February 2012 to May 2012.
Steady state was defined as SCA patient who is crises free for a period not <2-weeks,  while VOC was defined as multiple site of skeletal and other pain that last for at least 12 h. 
All subjects who signed an informed consent were recruited into the study. For the children, consent was obtained from their parents or care giver.
All subjects who refuse to give or sign an informed consent, pregnant female subjects, SCA and control subjects on medication such as anticoagulant, contraceptive pills, or anti-aggregant, e.g., aspirin which could impact on hemostasis and subjects who have had blood transfusion <3 weeks previously were excluded from the study.
Specimen Collection and Preparation
A volume of 5 ml of free flowing venous blood were obtained from each subject using a pre-coated ethylenediaminetetraacetic acid (EDTA) vacutainer blood collection tubes. The sampled was mixed gently and 2 ml of it was dispensed into a plane tube. This was used for full blood count. The analysis was performed within 2 h of sample collection.
The remaining 3 ml of the EDTA blood sample was spurn at 2000g for 15 min at room temperature within 30 min of sample collection to obtain plasma. The plasma was separated and transferred into plain sterile cryotubes in aliquot using polypropylene transfer pipette and stored at −80°C until analysis done. These samples were used for the assays of fibrinolytic proteins (D-dimer, fibrinogen [FBG], plasminogen, tPA and fibrinopeptide A [FPA]). Sample preparation was done in accordance with the manufacturer's instructions using either EDTA plasma or citrated plasma for the assay of the fibrinolytic proteins. The former was used in this assay.
All samples collected were labeled with a serial number allocated to each subject including the controls.
Full blood count was carried out on EDTA anticogulated blood sample using the Sysmex KX 31 Hematology auto-analyzer.
Plasma concentration of D-dimer was estimated using a commercial assay kit-Human D-dimer enzyme-linked immunosorbent assay (ELISA) kit manufactured by Cusabio Biotech Co.; Ltd., China with Lot # P08062568. The test procedure was carried out as recommended by the manufacturer. The test procedures were as follows.
Hundred microliters (100 μl) of D-dimer standard solution, blank and the test samples were each dispensed into appropriate microtiter well. This was covered with adhesive strip and incubated for 2-h at 37°C, thereafter the liquid from each well was removed. A volume of 100 μl of biotin-antibody working solution was added to each well and mixed gently until the solution becomes uniform. Each well was aspirated and washed with 200 μl of wash buffer. The process was repeated thrice and the wash buffer completely drained. 100 μl of horseradish peroxidase-avidin working solution was added to each well. The microtiter plate was covered with a new adhesive tape and incubated again for 1-h at 37°C. Aspiration and washing was again repeated, this was done 5 times. 90 μl of trimedoxime bromide (3, 3, 5, 5-tetramethyl-benzidine) was then added to each well, mixed gently and incubated for 10-min at 37°C in the dark to allow for maximum color development. The enzyme reaction was stopped by addition of 50 μl of the stop solution, i.e., dilute sulfuric acid to each well. The optical density or absorbance of each well was determined within 30-min using a microplate reader set at 450 nm. A calibration curve was plotted using the standard D-dimer concentrations on the X-axis and the corresponding mean absorbance on the Y-axis; the D-dimer concentrations of the test plasma were derived from the standard curve.
Plasma concentration of FPA was estimated using a commercial assay kit Human FPA ELISA Kit manufactured by Cusabio Biotech Co.; Ltd., China with Lot # P13062554. The ELISA test procedures as well as determination of FPA concentrations of the test plasma were as for D-dimer test assay.
Plasma concentration of FBG was estimated using a commercial assay kit-Assay Max Human FBG ELISA Kit manufactured by Assay Pro, 41 Triad South Drive St. Charles, MO 63304, USA with Lot # 0925102.
The ELISA test procedures as well as determination of FBG concentrations of the test plasma were as for D-dimer test assay.
Plasma concentration of plasminogen was estimated using a commercial assay kit-Assay Max Human plasminogen ELISA Kit also manufactured by Assay Pro, 41 Triad South Drive St., Charles MO 63304, USA with Lot #12521030. The ELISA test procedures as well as determination of Plasminogen concentrations of the test plasma were as for D-dimer test assay.
Plasma concentration of tPA was estimated using a commercial assay kit-Assay Max Human tPA ELISA Kit manufactured by Assay Pro, 41 Triad South Drive St. Charles, MO 63304, USA; with Lot # 03701103. The ELISA test procedure as well as determination of tPA concentration of the test plasma were as for D-dimer test assay.
The fibrinolytic protein assay was carried out by the same person and blinded as to which sample belong to the different study group, this is to allow for uniformity. All analysis was carried out at room temperature and the manufacturer's assay protocol was strictly adhered to.
Statistical analysis was performed with SPSS software version 10. All data were expressed as mean ± standard deviation. Statistical significance was analyzed with the paired Student's t-test. Analysis of co-variance was used as appropriate to control for confounders. Correlations were performed by Pearson's method. P < 0.05 was considered to be statistically significant in all comparison.
| Results|| |
The mean ages of subjects in the different arms of the study were 21.24 ± 8.69, 21.20 ± 7.66 and 24.67 ± 8.15 for VOC, steady state and control group respectively. The mean ages were not significantly different from each P = 0.192 [Table 1].
|Table 1: Age and sex distribution of subjects and controls according to clinical states|
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Age and sex were further controlled for using analysis of covariance to verify their effect as confounders on the measured fibrinolytic proteins. The result showed that age and sex did not act as confounders on the measured proteins either among the subjects studied or the control group [Table 2].
|Table 2: The P values after adjusting for age and sex as confounders using ANCOVA|
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The mean Hb concentration of HbAA controls was 13.01 ± 1.04 g/dl. The mean Hb concentration of subjects in VOC (6.22 ± 1.75 g/dl) was found to be significantly lower than subjects in steady state (7.42 ± 1.36 g/dl) P = 0.003 [Table 3].
|Table 3: Comparison of mean hematological parameters of subjects and control|
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Hematocrit values follows the same pattern as the Hb concentration with P = 0.002 [Table 3].
Subjects in VOC had a significantly higher mean white blood cells (19.44 ± 14.88 × 10 9 /L) compared with steady state subjects (11.84 ± 7.67 × 10 9 /L) P = 0.005; and the controls (5.15 ± 1.24 × 10) P = 0.000 [Table 3].
The mean platelets count of subjects in VOC (292.72 ± 148.57 × 10 9 /L) is significantly higher than those of the control subjects (173.44 ± 59.89 × 10 9 /L) but significantly lower when compared with steady state subjects (382.64 ± 211.12 × 10 9 /L). P =0.004 and 0.036 respectively [Table 3].
The mean D-dimer concentration of subjects in VOC (45.92 ± 37.29 ng/ml) and steady state (51.72 ± 34.12) were higher than that of the controls (37.25 ± 34.85), but this differences were not statistically significant. P =0.425 and 0.175 respectively. Furthermore, subjects in steady state had a higher concentration of D-dimer compared with those in VOC. This differences was also not statistically significant P = 0.589.
Similarly, plasma concentrations of plasminogen, FBG and tPA did not show any statistically significant differences when compared between the subjects (VOC and steady state) and also between the subjects and controls [Table 4].
|Table 4: Comparison of mean fibrinolytic proteins concentration among subjects and controls|
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However, the mean FPA plasma concentrations of 680.99 ± 411.37 ng/ml for subjects in VOC and 449.67 ± 310.01 ng/ml for steady state subjects were significantly increased compared to 163.52 ± 86.26 ng/ml for the controls P = 0.001.
| Discussion|| |
Abnormalities of fibrinolysis and coagulation in SCA have been described in several studies. ,,,,
Although some studies have reported a decreased fibrinolysis in SCA patients both in steady state and in VOC, ,, others have reported enhanced fibrinolysis in this disorder,  whilst still others found no evidence of impairment of fibrinolysis in SCA in both state of the disorder. ,
The result of this study did not show significant increase of fibrinolysis in SCA subjects, either in steady state or in VOC. This is in keeping with studies of Francis  and Gordon et al. 
D-dimers are specific plasmin cleavage products of fibrin present in excess of normal in conditions such as disseminated intravascular coagulation, deep venous thrombosis and thrombo-embolism.
Although high D-dimer levels does not necessarily imply thrombosis since numerous situations may contribute to its increase in plasma, e.g., inflammation, infection or surgery. Normal levels exclude thrombosis with a high probability and the negative predictive value is often >90%. 
Among the subjects studied in VOC, this study showed no significant increase in D-dimer levels. This result contrasts those of previous studies which documented elevated D-dimers concentration in this state of the disorder. ,,] Furthermore, there was no significant difference between the D-dimer concentration of steady state subjects and controls even though the SCA subjects (either in steady state and VOC) had a higher plasma concentration of D-dimer than the HbAA controls. This observation may probably be due to the small sample size of subjects used in the study.
The mean plasma FBG level showed no significant difference between subjects in VOC (2.07 ± 0.50 mg/ml) and steady state (2.09 ± 0.84 mg/ml), P = 0.882. No significant difference was also observed among the controls (1.94 ± 0.78 mg/ml) even though the levels of FBG was numerically higher among the SCA subjects compared with the controls.
This finding is similar to that of the study by Nsiri et al.,  who also reported no significant difference in FBG concentration between HbAA controls and HbSS subjects in VOC and steady state, but contrasts with the findings of Buseri et al.  and Famodu  who both reported a higher level of FBG concentration in HbSS subjects in steady state than in the HbAA controls. Famodu  also reported a further increase in this protein during painful crisis.
FBG is an acute phase reactants, hence the increased level observed in these individuals may be due to a reactive process in response to chronic hemolytic states that characterize SCA. However, this was not observed in this study.
tPA is the most important activator of plasminogen in vivo. It released from vascular endothelium is stimulated by several factors including trauma, venous occlusion, exercise etc. The balance between endothelial production of tPA and its specific inhibitor plasminogen activator inhibitor-1 (PAI-1) determines the overall endogenous fibrinolytic activity.
This study demonstrated no significant difference between the tPA concentration of subjects in VOC (3.26 ± 5.62 ng/ml) and steady state (1.55 ± 3.73 ng/ml) and between controls (1.75 ± 4.52 ng/ml) and VOC, P = 0.201 and 0.249 respectively. Similarly, no significant difference was observed in the tPA concentration between steady state subjects and controls, P = 0.877. This finding is in agreement with that of Francis  and Nsiri et al.  The former reported no significant difference in the tPA concentration of subjects in VOC and steady states after a standardized stimulus of tPA release from endothelial cells using the venous occlusion test at 100 mmHg for 10 min, whereas the later also reported no significant difference in the tPA concentration between HbSS subjects and controls, but also reported a high level of PAI-1 among subjects compared with the controls. However, Phillips et al.  in their own study reported a decrease in the releasable tPA in sickle cell subjects and an increase in the level of PAI-1. 
The findings from this study suggests no impairment in the endothelial release of tPA probably due to a high plasma levels of PAI-1 among SCA subject as reported in the studies above.
This study also demonstrated no significant difference in the plasma plasminogen level between the subjects in VOC (60.31 ± 63.90 ng/ml) and steady state (58.34 ± 24.99 ng/ml), P = 0.864. There was also no significant difference in plasma plasminogen concentration between controls (60.67 ± 18.82 ng/ml) and subjects in VOC and steady state, P = 0.975 and 0.833 respectively, even though a relative reduction in plasma concentration of plasminogen was observed among the subjects compared with controls. This may be as a result of the non-impairment in endothelial release of tPA as shown in this study, as well as the high basal level of PAI-1 in SCA subjects , as reported in other studies. High plasma concentration of PAI-1 may result in decreased fibrinolysis.
It has been reported that patients in VOC have evidence of intravascular coagulation since FPA levels are elevated in sickle cell crises. 
In this study a significant increase in FPA was observed among sickle cell subjects during VOC and steady state. This finding is similar to the study by Leichtman and Brewer  that also found elevated FPA among SCA subject in VOC, but contrasts their finding among steady state subjects who had FPA concentration similar to that of the HbAA controls.
FPA is a specific product of FBG cleavage by thrombin,  thus its elevation in SCA patients is thought to reflect the action of thrombin on FBG. However, the plasma FBG concentration recorded in this study was not significantly increased among the subjects. Thus, other factor(s) may have contributed to the significant increase in the FPA concentration among the SCA subjects.
Hyperleucoytosis is almost constant in SCA patients and a high polymorphonuclear leucocyte (PMNL) is a poor prognostic feature.  In the study by Greabu and Olinescu,  they showed that chemiluminescence emission produced by zymosan activated PMNL has a direct relationship with plasma concentration of FBG, FPA or fibrinogen degradation products (FDP) and that FBG as an independent risk factor in coronary heart disease is related to the level of activated PMNL in plasma. They suggested that activated plasma PMNL may contribute to elevated plasma level of FPA or FDP as a consequence of enhanced enzyme-dependent degradation of FBG. 
Hence, since no significant increase was observed in the FBG concentration of the subjects studied, the action of thrombin alone on FBG may not completely account for the elevated FPA levels observed among the subjects in this study. Perhaps the hyperleucoytosis particularly activated PMNL may have also contributed to this observed increase.
| Conclusion|| |
The plasma concentrations of fibrinolytic proteins among SCA patients either in the steady state or during VOC are not significantly different from that of HbAA controls. The significant increase in FPA concentration observed among subjects compared to controls may have been confounded by hyperleucocytosis especially PMNLs commonly associated with SCA. Thus, the clinical relevance of these plasma proteins in evaluating these patients in either of the clinical states of this disorder is doubtful.
Limitation of this Study
A few of the subjects in VOC could have been followed-up for a short time after the crises event to indicate time relations and variation in the clinical manifestation and fibrinolytic proteins concentrations.
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[Table 1], [Table 2], [Table 3], [Table 4]