The contribution of hypercoagulability to the pathophysiology of sickle cell disease (SCD) remains poorly defined. We sought to evaluate the association of markers of coagulation and platelet activation with specific clinical complications and laboratory variables in patients with SCD.
Design and Methods
Plasma markers of coagulation activation (D-dimer and TAT), platelet activation (soluble CD40 ligand), microparticle-associated tissue factor (MPTF) procoagulant activity and other laboratory variables were obtained in a cohort of patients with SCD. Tricuspid regurgitant jet velocity was determined by Doppler echocardiography and the presence/history of clinical complications was ascertained at the time of evaluation, combined with a detailed review of the medical records.
No significant differences in the levels of D-dimer, TAT, soluble CD40 ligand, and MPTF procoagulant activity were observed between patients in the SS/SD/Sβ0 thalassemia and SC/Sβ+ thalassemia groups. Both TAT and D-dimer were significantly correlated with measures of hemolysis (lactate dehydrogenase, indirect bilirubin and hemoglobin) and soluble vascular cell adhesion molecule-1. In patients in the SS/SD/Sβ0 thalassemia group, D-dimer was associated with a history of stroke (p = 0.049), TAT was associated with a history of retinopathy (p = 0.0176), and CD40 ligand was associated with the frequency of pain episodes (p = 0.039). In multivariate analyses, D-dimer was associated with reticulocyte count, lactate dehydrogenase, NT-proBNP and history of stroke; soluble CD40 ligand was associated with WBC count and platelet count; and MPTF procoagulant activity was associated with hemoglobin and history of acute chest syndrome.
This study supports the association of coagulation activation with hemolysis in SCD. The association of D-dimer with a history of stroke suggests that coagulation activation may contribute to the pathophysiology of stroke in clinically severe forms of SCD. More research is needed to evaluate the contribution of coagulation and platelet activation to clinical complications in SCD.
Citation: Ataga KI, Brittain JE, Desai P, May R, Jones S, Delaney J, et al. (2012) Association of Coagulation Activation with Clinical Complications in Sickle Cell Disease. PLoS ONE 7(1): e29786. doi:10.1371/journal.pone.0029786
Editor: Dimas Tadeu Covas, University of Sao Paulo – USP, Brazil
Received: September 19, 2011; Accepted: December 5, 2011; Published: January 11, 2012
Copyright: © 2012 Ataga et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by NIH grants HL79915, HL091265, and HL094592. Support for this work was also provided by an award from the North Carolina State Sickle Cell Program. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Patients with sickle cell disease (SCD) have an increased risk of stroke, pulmonary hypertension, avascular necrosis of large joints, and pregnancy-related complications, all of which may be due, at least in part, to thrombotic vascular occlusion . They also appear to have a higher risk of pulmonary embolism as well as pregnancy-related venous thromboembolism compared with race-matched controls without SCD , . In addition to these thrombotic complications, patients with SCD manifest alterations in platelet function, as well as changes in the procoagulant, anticoagulant and fibrinolytic systems in the direction of a procoagulant phenotype . Furthermore, tissue factor (TF) antigen and TF procoagulant activity are reported to be elevated in the circulation of SCD patients when compared with normal controls –. As a result of these findings, SCD is frequently referred to as a “hypercoagulable state.”
Despite the abundant laboratory evidence of hypercoagulability observed in these patients, it still remains uncertain whether the observed platelet activation, as well as increased thrombin and fibrin generation contributes to the vascular occlusive episodes that characterize SCD or are rather simple epiphenomena.
In the present study, we sought to evaluate the association of markers of coagulation and platelet activation with specific SCD-related clinical complications and laboratory variables in a cohort of patients followed at an adult sickle cell clinic. In addition, we evaluated the relationship between microparticle-associated tissue factor procoagulant activity, plasma markers of coagulation activation and platelet activation.
Patients and Study Design
The study subjects represent a cohort of patients followed at the Adult Sickle Cell Clinic at the University of North Carolina (UNC), Chapel Hill. The data were collected as part of an ongoing study to investigate the pathophysiology of pulmonary hypertension in SCD. Sixty four patients with SCD were included in the analyses that assessed the associations of plasma markers of coagulation and platelet activation with both SCD-related clinical complications and laboratory variables. Patients with SCD were assessed while in the non-crisis, “steady state” (no current acute painful episode or illness and none requiring medical intervention for at least 1 week); had not experienced an episode of acute chest syndrome in the 4 weeks preceding enrollment; and had no clinical evidence of congestive heart failure. None of the patients was on chronic red blood cell transfusion therapy. This study was approved by the Institutional Review Board at UNC, Chapel Hill and all subjects gave written informed consent to participate in accordance with the Declaration of Helsinki.
Blood Sample Collection and Preparation.
The samples from the study group were obtained during research visits while in the non-crisis, “steady state.” Blood samples were obtained via venipuncture using a 21-gauge needle and drawn into citrate-containing tubes. Plasma samples were prepared in a manner that minimized platelet activation as previously described . The plasma samples were aliquoted and frozen immediately at −80°C for subsequent analysis.
Microparticle-Associated Tissue Factor (MPTF) Procoagulant Activity.
Platelet-free plasma was obtained from citrated whole blood by centrifugation at 1,500 g for 15 minutes, followed by a clearing spin of 13,000 g for 2 minutes. Microparticles were isolated from platelet-free plasma by centrifugation at 21,000 g for 30 minutes. The pellet was re-suspended in HBSA (20 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mg/ml bovine serum albumin) and re-centrifuged at 20,000 g for 30 minutes, before the final re-suspension in 250 µL HBSA, and MPTF procoagulant activity was measured as previously described using a chromogenic assay , .
Plasma Markers of Thrombin Generation, Platelet Activation and Endothelial Activation
Quantification of D-dimer (DiaPharma, Westchester, OH), thrombin-antithrombin (TAT) complexes (Dade Behring, Marburg, Germany), soluble CD40 ligand (R&D systems, Minneapolis, MN) and human soluble vascular cell adhesion molecule-1 (soluble VCAM-1) (R&D systems, Minneapolis, MN) was accomplished using commercially available ELISA kits. Samples were assayed in duplicate and according to manufacturer's instructions. Measurements of N-terminal pro-brain natriuretic peptide (NT-proBNP) and other routine laboratory studies were performed by the McClendon Clinical Laboratory at UNC Hospitals.
Sickle Cell Disease-Related Clinical Complications
The presence or history of clinical complications in SCD patients was ascertained from a history at the time of evaluation, combined with a detailed review of the medical records. Acute pain episodes (or crises), acute chest syndrome, stroke and other SCD-related complications were defined using standard definitions –. Based on a modification of the definition from both the Cooperative Study of Sickle Cell Disease  and the Multicenter Study of Hydroxyurea , acute pain episodes referred to episodes that required a visit to a medical facility for acute sickling-related pain for which treatment with a parenterally administered analgesic was needed. Tricuspid regurgitant jet velocity was measured by Doppler echocardiography as previously described .
Wilcoxon rank sum tests were used to compare distributions of continuous variables in two groups. Fisher's exact tests were used to compare categorical variables. Spearman rank correlations were used to identify associations between markers of coagulation activation and specified variables (α = 0.05). Reported p-values are considered ‘nominal’ and are for individual tests, unadjusted for multiple comparisons because of the exploratory nature of this study. Multivariate analyses were performed using multiple regression to assess the associations between clinical and laboratory variables that were found to be associated with markers of coagulation and platelet activation (TAT, D-dimer, soluble CD40 ligand and MPTF procoagulant activity) in univariate analyses (P<0·15). Backwards elimination using Akaike's Information Criterion (AIC) was used to select the best model. Statistical analyses were performed using R statistical software (www.r-project.org).
Demographic and Laboratory Characteristics
Sixty four patients with SCD (SS: 47; SC: 7; Sβ0 thalassemia: 4; Sβ+ thalassemia: 5; and SD: 1) were evaluated. Due to the clinical heterogeneity of SCD, the patients were arbitrarily categorized into 2 groups based on presumed disease severity - SS/SD/Sβ0 thalassemia and SC/Sβ+ thalassemia. The demographic and baseline laboratory characteristics of all the study subjects are shown in Table 1. As expected, patients in the SS/SD/Sβ0 thalassemia group had significantly lower hemoglobin values (8.9 g/dL vs. 10.65 g/dL, p<0.0001), but higher platelet counts (412×109/L vs. 320.5×109/L; p = 0.0058), reticulocyte counts (6.6% vs. 3.25%; p = 0.0011), total bilirubin (2.2 mg/dL vs. 0.95 mg/dL; p<0.0001), indirect bilirubin (2.11 mg/dL vs. 0.86 mg/dL; p<0.0001) and lactate dehydrogenase (894 U/L vs. 641 U/L; p = 0.0011) than patients in the SC/Sβ+ thalassemia group. No significant differences were observed in the WBC count or in the level of soluble VCAM-1 when patients in both groups were compared.
Correlation of Markers of Coagulation and Platelet Activation with Laboratory Variables
No significant differences in the levels of D-dimer (1424 ng/mL; interquartile range [IQR], 745–2536 ng/mL vs. 1533.4 ng/mL; IQR, 591–2006 ng/mL, p = 0.47), TAT (5.63 ng/L; IQR, 3.62–8.9 ng/L vs. 4.86 ng/L; IQR, 3.91–6.19 ng/L, p = 0.59), soluble CD40 ligand (0.453 ng/mL; IQR, 0.339–0.60 ng/mL vs. 0.409 ng/mL; IQR, 0.201–0.60 ng/mL, p = 0.24), and MPTF procoagulant activity (0.145 pg/mL, IQR 0.094–0.265 pg/mL, vs. 0.115 pg/mL, IQR 0.056–0.173 pg/mL, p = 0.17) were observed when patients in the SS/SD/Sβ0 thalassemia group were compared with patients in the SC/Sβ+ thalassemia group. We observed a significant correlation between TAT and D-dimer (r = 0.66; p<0.0001) (Figure 1). However, no correlations were observed between TAT and soluble CD40 ligand (r = 0.016, p = 0.91) or between TAT and MPTF procoagulant activity (r = 0.064, p = 0.63). Similarly, no correlations were observed between D-dimer and soluble CD40 ligand (r = −0.17; p = 0.19) or between D-dimer and MPTF procoagulant activity (r = 0.11, p = 0.40).
Plasma TAT is significantly correlated with plasma D-dimer in our study cohort (r = 0.66; p<0.0001).
The plasma markers of coagulation activation, TAT and D-dimer, were associated with laboratory measures of hemolysis (Table 2). There was a correlation between TAT and lactate dehydrogenase (r = 0.57, p<0.0001) (Figure 2), with borderline correlations between TAT and total bilirubin (r = 0.25; p = 0.054), indirect bilirubin (r = 0.26, p = 0.051), and hemoglobin (r = −0.24; p = 0.071). TAT was also correlated with the absolute monocyte count (r = 0.27; p = 0.035) and NT-proBNP (r = 0.35; p = 0.005). Similarly, D-dimer was correlated with lactate dehydrogenase (r = 0.56; p<0.0001) (Figure 3), indirect bilirubin (r = 0.26; p = 0.048), hemoglobin (r = −0.32; p = 0.012), NT proBNP (r = 0.42; p<0.0001), with borderline correlations with absolute monocyte count (r = 0.23; p = 0.074). Both TAT (r = 0.37; p = 0.004) and D-dimer (r = 0.49; p<0.0001) (Figure 4) were also correlated with soluble VCAM-1.
TAT is correlated with lactate dehydrogenase in our study cohort (r = 0.57, p<0.0001).
D-dimer is correlated with lactate dehydrogenase in our study cohort (r = 0.56; p<0.0001).
D-dimer is correlated with soluble VCAM-1 in our study cohort (r = 0.49; p<0.0001).
Soluble CD40 ligand was correlated with WBC count (r = 0.30; p = 0.018) and platelet count (r = 0.40; p = 0.0014), but was not correlated with lactate dehydrogenase, total bilirubin, indirect bilirubin or hemoglobin (Table 3). No correlations were observed between MPTF procoagulant activity and measures of hemolysis or other measured laboratory variables.
When the analyses were limited to only patients in the SS/SD/Sβ0 thalassemia group, there was a significant correlation between TAT and lactate dehydrogenase (r = 0.62, p<0.0001), indirect bilirubin (r = 0.296, p = 0.044), and NT-proBNP (r = 0.32, p = 0.026), with borderline correlation with total bilirubin (r = 0.28, p = 0.053) and absolute monocyte count (r = 0.25, p = 0.089). Similarly, we observed significant correlations between D-dimer and lactate dehydrogenase (r = 0.60, p<0.0001), hemoglobin (r = −0.29, p = 0.039), fetal hemoglobin (r = −0.31, p = 0.035), and NT-proBNP (r = 0.49, p<0.0001), with borderline correlations with platelet count (r = −0.28, p = 0.053), total bilirubin (r = 0.24, p = 0.093) and indirect bilirubin (r = 0.26, p = 0.07). Soluble CD40 ligand was correlated with platelet count (r = 0.41, p = 0.003), but not correlated with lactate dehydrogenase, total bilirubin, indirect bilirubin or hemoglobin. MPTF procoagulant activity was correlated with hemoglobin (r = 0.31, p = 0.034), but not correlated with measures of hemolysis or other measured laboratory variables.
Association of Measures of Coagulation and Platelet Activation with Clinical Complications.
The level of TAT was significantly higher in patients with a history of retinopathy compared to those without this complication (6.05 ng/L; IQR, 4.91, 10.98 ng/L vs. 4.52 ng/L; IQR, 3.22, 7.33 ng/L, p = 0.023) (Table 4). In addition, the level of TAT was lower in patients on hydroxyurea therapy compared to those not on such therapy (4.39 ng/L; IQR, 3.25, 8.77 ng/L vs. 6.5 ng/L; IQR, 5.08, 8.76 ng/L, p = 0.044). D-dimer appeared to be associated with a history of stroke (3003.1 ng/mL [FEU]; IQR, 1513, 3067 ng/mL [FEU] vs. 1399.5 ng/mL [FEU]; 648.8, 2217 ng/mL [FEU], p = 0.062), although the difference was not statistically significant. When those patients with a measurable tricuspid regurgitant jet velocity were evaluated (N = 43), there was no correlation between D-dimer and tricuspid regurgitant jet velocity (r = 0.25, p = 0.10). There was an association between MPTF procoagulant activity and a history of acute chest syndrome (0.1445 pg/mL; IQR, 0.11, 0.268 pg/mL vs. 0.07 pg/mL; IQR, 0, 0.149 pg/mL, p = 0.045) (Table 5). Finally, soluble CD40 ligand appeared to be associated with the frequency of pain episodes (<3 episodes vs. ≥3 episodes in the past year: 0.406 ng/mL; IQR, 0.428, 0.677 ng/mL vs. 0.498 ng/mL; IQR, 0.292, 0.579 ng/mL, p = 0.058), although the difference was not statistically significant.
When the analyses were limited to patients in the SS/SD/Sβ0 thalassemia group, we observed associations between D-dimer and a history of stroke (3035.2 ng/mL [FEU]; IQR 2559, 3494 vs. 1269.6 ng/mL [FEU]; IQR 688.4, 2347, p = 0.049), TAT and history of retinopathy (7.145 ng/L; IQR 5.225, 12.01vs. 4.465 ng/L; IQR 3.215, 7.332, p = 0.018), and soluble CD40 ligand and frequency of pain episodes (<3 episodes vs. ≥3 episodes in the past year: 0.398 ng/mL; IQR 0.308, 0.568 ng/mL vs. 0.543 ng/mL; IQR 0.434, 0.730 ng/mL, p = 0.039), but the previously observed association between MPTF procoagulant activity and acute chest syndrome was no longer significant (0.148 pg/mL; IQR 0.115, 0.274 pg/mL vs. 0.094 pg/mL; IQR 0.047, 0.155 pg/mL, p = 0.32). There appeared to be a correlation between tricuspid regurgitant jet velocity and D-dimer (r = 0.3, p = 0.067), although this was not statistically significant.
Clinical and laboratory variables were selected for use in the multivariate model via a backwards elimination procedure using AIC as the fit criterion. The clinical variables were history of stroke, retinopathy, avascular necrosis, history of leg ulcers, and history of acute chest syndrome. The laboratory variables were lactate dehydrogenase, hemoglobin, reticulocyte count, white blood cell count, absolute neutrophil count, platelet count, total bilirubin, direct bilirubin, and NT-proBNP (Table 6). In this multivariate model, D-Dimer was associated with reticulocyte count (estimate: 92.56, p = 0.04), lactate dehydrogenase (estimate: 1.22, p = 0.0002), NT-proBNP (estimate: 0.44, p<0.0001) and history of stroke (estimate: 984.01, p = 0.051). This means that for continuous variables such as lactate dehydrogenase, we expect an increase in D-dimer by 1.22 units for every 1 unit increase in lactate dehydrogenase. For binary responses such as history of stroke, the estimated value of 984.01 indicates that having a history of stroke is predicted to result in an increase in D-dimer of 984.01 ng/mL [FEU] compared to no history of stroke. Soluble CD40 ligand was associated with WBC count (estimate: 0.04, p = 0.05), and platelet count (estimate: 0.001, p = 0.0016). Finally, MPTF procoagulant activity was associated with hemoglobin (estimate: 0.024, p = 0.023), and history of acute chest syndrome (estimate: 0.103, p = 0.029).
Although SCD is characterized by hypercoagulability , , the contribution of coagulation and platelet activation to disease pathophysiology remains poorly defined. Plasma levels of TAT, prothrombin fragment 1.2 and D-dimer are increased in patients with SCD during the non-crisis, “steady state” and are further increased during acute pain episodes . D-dimer levels are reported to correlate with the frequency of pain episodes measured during the following year  as well as the interval for development of pain episodes, suggesting that coagulation activation may contribute to vaso-occlusion in SCD. Platelets are also activated in SCD during the non-crisis, “steady state,” with further activation during acute pain episodes , . In addition, platelet activation, marked by activated fibrinogen receptor, has been reported to correlate with echocardiography-derived tricuspid jet regurgitant velocity suggesting an association with pulmonary hypertension in SCD .
In our present cross-sectional study, we observed no differences in markers of coagulation and platelet activation when patients in the SS/SD/Sβ0 thalassemia group were compared to patients in the SC/Sβ+ thalassemia group. Patients with sickle cell anemia (HbSS) have previously been reported to have higher plasma levels of markers of coagulation activation compared to patients with HbSC disease . The reason for the absence of a difference between the groups in our study is uncertain, but may be related to the arbitrary grouping of the various genotypes based on the presumed severity of disease. Furthermore, as patients with more “severe” genotypes are more likely to be treated with hydroxyurea, it is possible that the lack of a difference may be due to a decrease in disease severity following hydroxyurea therapy. This presumption is supported by our finding of an association between TAT and hydroxyurea use on univariate analysis. In addition, hydroxyurea has previously been reported to decrease D-dimer levels in patients with SCD , suggesting that this drug may decrease the coagulation activation observed in SCD.
As expected, we observed significant correlations between TAT and D-dimer. This is consistent with previous reports in SCD . Similar to previous publications by our group and others , , our finding of correlations between both TAT and D-dimer with measures of hemolysis extends and confirms the contribution of hemolysis to coagulation activation in SCD. Heme, an inflammatory mediator and a product of hemolysis, has been shown to induce TF expression on the surface of both macrovascular and microvascular endothelial cells in a concentration-dependent manner . In addition, heme produces an upregulation of the expression of TF mRNA, TF protein, and TF procoagulant activity in endothelial cells in a time-dependent manner, effects that may be mediated, at least in part, by the transcription factor, NFkappaB. Thus, heme-induced endothelial TF expression may provide a pathophysiologic link between hemolysis and the coagulation activation in SCD. Unlike a previous report that suggested an association between hemolysis and platelet activation , no correlation was observed between soluble CD40 ligand and measures of hemolysis in our study. Surprisingly, no correlations were observed between the measured plasma markers of coagulation activation (TAT and D-dimer) with either soluble CD40 ligand or MPTF procoagulant activity in our study. As CD40 ligand is known to induce the expression of TF in SCD , the reason for the lack of association between the plasma markers of coagulation activation and soluble CD40 ligand is uncertain. Furthermore, although MPTF procoagulant activity has previously been reported to correlate with plasma markers of coagulation activation in SCD , a recent report showed that while whole blood TF procoagulant activity was correlated with TAT, D-dimer, as well as markers of hemolysis, inflammation and endothelial activation, no such correlations were observed between MPTF procoagulant activity and any of these markers . As a result, the contribution of MPTF procoagulant activity to coagulation activation in SCD remains uncertain.
The correlation of both TAT and D-dimer with soluble VCAM-1 suggests a relationship between activation of the coagulation system and endothelial activation in SCD. Furthermore, the correlation of TAT (and possibly D-dimer) with the absolute monocyte count suggests that monocytes may play a role in the coagulation activation in SCD. Monocytes are activated , abnormally express TF  and are a source of TF-positive microparticles  in SCD. There is increasing evidence of a crosstalk between coagulation activation and inflammation . SCD is often referred to as a chronic inflammatory disease ,  and circulating endothelial cells in SCD patients are reported to express TF . While the observed association between plasma markers of coagulation activation and measures of inflammation in our study does not prove causality, the inflammatory state in SCD may indeed contribute to hypercoagulability in SCD. Studies in transgenic SCD mice demonstrate that the induction of ischemia-reperfusion injury by exposure of the mild sickle-cell phenotype, NY1DD mice, to a hypoxic environment followed by a return to ambient air resulted in increased TF expression in the pulmonary veins .
The association between D-dimer and a history of stroke suggests that coagulation activation may contribute to the pathogenesis of stroke in SCD. Although the pathophysiology of stroke in SCD is complex, large vessel arterial obstruction with superimposed thrombosis is commonly observed in patients with thrombotic stroke . Despite the absence of a significant association between coagulation activation and echocardiography-derived tricuspid regurgitant jet velocity in our study, autopsy series show the presence of in situ thrombosis in the pulmonary vasculature of patients with SCD-related pulmonary hypertension . A recent study evaluating the relative role played by cells and plasma in the hypercoagulability observed in β thalassemia patients showed evidence of hypercoagulability based on thromboelastometry evaluation, but thrombin generation determined in platelet-poor plasma was not significantly different from healthy individuals . With the known contribution of red blood cells and other cellular elements to hypercoagulability in SCD, this finding suggests that much like in β thalassemia, evaluation of whole blood may be required to accurately demonstrate associations between coagulation activation and clinical complications in SCD. The absence of an association between coagulation activation and echocardiography-derived tricuspid regurgitant jet velocity in our study may also reflect the small number of patients with catheterization-proven pulmonary hypertension in our cohort. Indeed, a recent multicenter study found no evidence of pulmonary hypertension following right heart catheterization in 72 of 96 patients with elevated tricuspid regurgitant jet velocities on echocardiography, suggesting that Doppler echocardiography may overestimate the prevalence of this condition .
The association between soluble CD40 ligand and the frequency of pain episodes in the past year in the SS/SD/Sβ0 thalassemia group suggests that platelet activation may contribute to the pathogenesis of acute pain episodes in SCD. This finding is consistent with a previous report showing a correlation between platelet procoagulant activity assessed by annexin V binding and the frequency of pain episodes measured during the following year . Platelet-derived CD40 ligand has been shown to induce tissue factor expression, endothelial cell expression of ICAM-1 and B-cell proliferation in SCD  and as such may play a role in disease pathophysiology.
Our study has several limitations. The patients were recruited from a specialty clinic at a tertiary care medical center, and may not represent all patients with SCD. The study population is relatively small, while the number of comparisons made is relatively large. We have reported only nominal p-values for each statistical test, with no adjustment made for multiple comparisons, reflecting the exploratory nature of this study. As with all cross-sectional studies, this analysis demonstrates associations, but cannot prove causation.
In summary, our study supports and extends the association of coagulation activation with hemolysis in SCD. TAT was associated with D-dimer, but these markers of coagulation activation were not associated with soluble CD40 ligand or MPTF procoagulant activity. We also show that D-dimer is associated with a history of stroke in patients with clinically severe forms of SCD. More research is needed to evaluate the pathophysiology of coagulation and platelet activation and their contribution to clinical complications in SCD.
The authors thank Ms. Melissa Caughey, MPH for help with the echocardiographic studies.
Conceived and designed the experiments: KIA JEB. Performed the experiments: JEB JD. Analyzed the data: KIA RM. Contributed reagents/materials/analysis tools: JEB AH NSK. Wrote the paper: KIA JEB PD RM SJ JD DS AH NSK.
- 1. Ataga KI, Key NS (2007) Hypercoagulability in sickle cell disease: new approaches to an old problem. Hematology Am Soc Hematol Educ Program 91–96.
- 2. Stein PD, Beemath A, Meyers FA, Skaf E, Olson RE (2006) Deep venous thrombosis and pulmonary embolism in hospitalized patients with sickle cell disease. Am J Med 119: 897.e7–e11.
- 3. James AH, Jamison MG, Brancazio LR, Myers ER (2006) Venous thromboembolism during pregnancy and the postpartum period: incidence, risk factors and mortality. Am J Obst Gynecol 194: 1311–1315.
- 4. Francis RB (1991) Platelets, coagulation, and fibrinolysis in sickle cell disease: their possible role in vascular occlusion. Blood Coagul Fibrinolysis 2: 341–353.
- 5. Key NS, Slungaard A, Dandelet L, Nelson SC, Moertel C, et al. (1998) Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease. Blood 91: 4216–4223.
- 6. Mohan JS, Lip GYH, Wright J, Bareford D, Blann AD (2005) Plasma levels of tissue factor and soluble E-selectin in sickle cell disease: relationship to genotype and to inflammation. Blood Coagul Fibrinolysis 16: 209–214.
- 7. Lee SP, Ataga KI, Orringer EP, Parise LV (2006) Biologically active CD40 ligand is elevated in sickle cell disease: potential role for platelet-mediated inflammation. Arterioscler Thromb Vasc 26: 1626–1631.
- 8. Brittain JE, Hulkower B, Jones SK, Strayhorn D, De Castro L, et al. (2010) Placenta growth factor in sickle cell disease: association with hemolysis and inflammation. Blood 115: 2014–2020.
- 9. Manly DA, Wang J, Glover SL, Kasthuri R, Liebman HA, et al. (2009) Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism. Thromb Res 125: 511–512.
- 10. Khorana AA, Francis CW, Menzies KE, Wang JG, Hyrien O, et al. (2008) Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer. J Thromb Haemost 6: 1983–1985.
- 11. Platt OS, Thorington BD, Brambilla DJ, Milner PF, Rosse WF, et al. (1991) Pain in sickle cell disease. Rates and risk factors. N Engl J Med 325: 11–16.
- 12. Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, et al. (1995) Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med 332: 1317–1322.
- 13. Vichinsky EP, Neumayr LD, Earles AN, Williams R, Lennette ET, et al. (2000) Causes and outcomes of the acute chest syndrome in sickle cell disease. National Acute Chest Syndrome Study Group. N Engl J Med 342: 1855–1865.
- 14. Ohene-Frempong K, Weiner SJ, Sleeper LA, Miller ST, Embury S, et al. (1998) Cerebrovascular accidents in sickle cell disease: rates and risk factors. Blood 91: 288–294.
- 15. Ataga KI, Moore CG, Hillery CA, Jones S, Whinna HC, et al. (2008) Coagulation activation and inflammation in sickle cell disease-association pulmonary hypertension. Haematologica 93: 20–26.
- 16. Tomer A, Harker LA, Kasey S, Eckman JR (2001) Thrombogenesis in sickle cell disease. J Lab Clin Med 137: 398–407.
- 17. Villagra J, Shiva S, Hunter LA, Machado RF, Gladwin MT, et al. (2007) Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension and nitric oxide scavenging by cell-free hemoglobin. Blood 110: 2166–72.
- 18. Westerman MP, Green D, Gilman-Sachs A, Beaman K, Freels S, et al. Antiphospholipid antibodies, proteins C and S, and coagulation changes in sickle cell disease. J Lab Clin Med 134: 352–62.
- 19. Orringer EP, Jones S, Strayhorn D, Hoffman E, Parker J, et al. (1996) The Effect of Hydroxyurea (HU) Administration on Circulating D-Dimer Levels in Patients with Sickle Cell Anemia. Blood 88: 496a.
- 20. van Beers EJ, Spronk HM, Ten Cate H, Duits AJ, Brandjes DP, et al. (2008) No association of the hypercoagulable state with sickle cell disease related pulmonary hypertension. Haematologica 93: e42–44.
- 21. Setty BN, Betal SG, Zhang J, Stuart MJ (2008) Heme induces endothelial tissue factor expression: potential role in hemostatic activation in patients with hemolytic anemia. J Thromb Haemost 6: 2202–2209.
- 22. Shet AS, Aras O, Gupta K, Hass MJ, Rausch DJ, et al. (2003) Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood 102: 2678–2683.
- 23. Setty Y, Key NS, Rao AK, Krishnan S, Gayen-Betal S, et al. (2010) Tissue factor procoagulant activity and coagulation activation in sickle cell disease: Relationship with biomarkers of hemolysis and inflammation. Blood 116: 4210A.
- 24. Belcher JD, Marker PH, Weber JP, Hebbel RP, Vercellotti GM (2000) Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion. Blood 96: 2451–2459.
- 25. Levi M, ten Cate H, van der Poll T (2002) Endothelium: Interface between coagulation and inflammation. Crit Care Med 30: S220–224.
- 26. Platt OS (2000) Sickle cell anemia as an inflammatory disease. J Clin Invest 106: 337–338.
- 27. Hebbel RP, Osarogiagbon R, Kaul D (2004) The endothelial biology of sickle cell disease: inflammation and a chronic vasculopathy. Microcirculation 11: 129–51.
- 28. Solovey A, Gui L, Key NS, Hebbel RP (1998) Tissue factor expression by endothelial cells in sickle cell anemia. J Clin Invest 101: 1899–1904.
- 29. Solovey A, Kollander R, Shet A, Milbauer LC, Choong S, et al. (2004) Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin. Blood 104: 840–846.
- 30. Prengler M, Pavlakis SG, Prohovnik I, Adams RJ (2002) Sickle cell disease: the neurological complications. Ann Neurol 51: 543–552.
- 31. Adedeji MO, Cespedes J, Allen K, Subramony C, Hughson MD (2001) Pulmonary thrombotic arteriopathy in patients with sickle cell disease. Arch Pathol Lab Med 125: 1436–1441.
- 32. Tripodi A, Cappellini MD, Chantarangkul V, Padovan L, Fasulo MR, et al. (2009) Hypercoagulability in splenectomized thalassemic patients detected by whole-blood thromboelastometry, but not by thrombin generation in platelet-poor plasma. Haematologica 94: 1520–1527.
- 33. Parent F, Bachir D, Inamo J, Lionnet F, Driss F, et al. (2011) A hemodynamic study of pulmonary hypertension in sickle cell disease. N Engl J Med 365: 44–53.