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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Department of Pediatrics, Division of Research
Hematology, Jefferson Medical College, Thomas Jefferson University; the
Department of Medicine and Thrombosis Research, Temple University
School of Medicine, and The Marian Anderson Comprehensive Sickle Cell
Center, Philadelphia, PA.
Complex pertubations of hemostasis occur in sickle cell disease
(SCD). Although the procoagulant property of sickle erythrocytes in
vitro is tied to exposure of phosphatidylserine (PS), no study has
directly linked this PS positivity to in vivo thrombin generation. This
study was designed to determine if thrombin generation in SCD
correlates with erythrocyte PS, or whether platelets play a significant
role. PS was quantified on erythrocytes and platelets from 40 patients
with SCD (SS genotype = 25; SC genotype = 15) and 11 controls.
Markers of thrombin generation (prothrombin fragment F1.2;
thrombin-antithrombin or TAT complexes) and fibrin dissolution (D-dimer; plasmin-antiplasmin or PAP complexes) were also evaluated. Thrombin generation and activation of fibrinolysis occurred with elevations in F1.2, TAT, and D-dimer. Although numbers of both PS-positive erythrocytes and platelets were elevated, there was no
correlation between PS-positive platelets and any hemostatic markers.
In contrast, correlations were noted between PS-positive erythrocytes
and F1.2 (P < .0002), D-dimer
(P < .000002), and PAP (P < .01).
Correlations between F1.2 and D-dimer (P < .0001) demonstrated that fibrinolysis was secondary to thrombin generation. In
patients with the SC genotype, abnormalities in coagulation, although
present, were of a lesser magnitude than in SS disease. This study
suggests that the sickle erythrocyte is the cell responsible for the
thrombophilic state in SCD because associations between erythrocyte PS
and thrombin generation were observed. No such relationship with
platelet PS was noted. The use of erythrocyte PS as a surrogate marker
in trials testing new therapeutic modalities may provide insights into
the vascular complications of SCD.
(Blood. 2001;98:3228-3233) Although sickle cell disease (SCD) has played
a singular role in establishing the field of molecular medicine, this
entity remains one in which the bench to bedside translation of
biologic concepts is still very much a work in progress. One of the
complications of SCD is a thrombophilic state associated with complex
pertubations of plasma and cellular hemostatic
mechanisms.1,2 These changes include evidence for
thrombin generation,3-8 depletion of natural anticoagulants,6-10 the activation of cellular elements
including white cells11-13 and platelets,2,14
and increased levels of circulating soluble tissue factor and
microvascular endothelial cells with a tissue factor
phenotype.15-17 Numerous investigations have been
conducted to elucidate the mechanisms responsible for the prothrombotic
state. Initial studies demonstrated that sickle erythrocytes
accelerated the clotting time and stimulated prothrombinase activity.18,19 Subsequently, it was shown that the loss of normal membrane phospholipid asymmetry with the appearance of anionic
phosphatidylserine (PS) on the erythrocyte surface, promoted the
assembly of clotting factors on the cell membrane leading to the
development of a sickle erythrocyte with procoagulant
phenotype.18-20 Another link between cause and effect
would be the demonstration that in vivo thrombin generation in SCD does
in fact correlate with sickle erythrocyte membrane PS exposure and that
platelets (which also can provide the catalytic template on which
coagulation factors interact)21 would in this circumstance
be a secondary player. Our study was designed in an attempt to forge
such a link.
The use of annexin V (a calcium-dependent phospholipid-binding protein)
and flow cytometry has facilitated the demonstration of PS on cell
surfaces.22,23 We have quantified PS exposure on
erythrocytes and platelets in SCD by using this technique and have
additionally evaluated various hemostatic parameters to assess whether
thrombin generation is related to the exposure of PS on the erythrocyte
and platelet surfaces. Markers of in vivo thrombin generation included
prothrombin fragment F1.2 and thrombin-antithrombin (TAT) complexes.
D-dimer (a measure of both cross-linked fibrin formation and
degradation by plasmin) and plasmin-antiplasmin (PAP; the irreversible
complex between plasmin and its principal physiologic inhibitor
Materials
Collection of blood
Flow cytometric analyses of PS-positive erythrocytes and platelets Anticoagulated whole blood (5 µL) was incubated for 30 minutes at room temperature with 10 µL annexin V-FITC and 20 µL of either anti-CD235a-PE or anti-CD61-PE in the presence of either 2.5 mM CaCl2 or EDTA in a total volume of 100 µL adjusted with HBSS-HEPES buffer. Incubation mixtures were then diluted with 1 mL buffer containing either 2.5 mM CaCl2 or EDTA and analyzed in a Becton Dickinson Flow Cytometer (San Jose, CA) formatted for 2-color analyses as previously described.24 Data from 60 000 events, collected at a flow rate of 300 to 500 events per second, were analyzed. Red cells and platelets were separated by size and identified by their distinct immunofluorescence (CD235a-positivity for red cells or CD61-positivity for platelets). As shown in Figure 1, panels A and B, using the dot plots of CD235a-PE fluorescence and forward size scatter (FSC), CD235a-positive events (red cell-associated, region marked R1) were separated from CD235a-negative (non-red cell) events. Annexin V-positive cells in the red cell region were then determined by using the histograms of annexin V-FITC fluorescence as shown in panels C and D. Negative (gate M1) and positive histogram regions (gate M2) were set with the use of samples stained with annexin V-FITC and CD235a-PE in the presence of EDTA (Figure 1C). Nonspecific membrane immunofluorescence (gate M2, panel C) was subtracted from the respective sample fluorescence (gate M2, panel D). Data were expressed as the percentage of PS-positive red cells. PS positivity associated with platelets was assessed by using the dot plots of CD61-PE fluorescence and forward size scatter plus the respective histograms of annexin V-FITC fluorescence (Figure 2A-D) employing the steps outlined above for red cell analyses.
Analysis of markers for thrombin generation and fibrinolysis Plasma levels of hemostatic markers, including prothrombin fragment F1.2, TAT, D-dimer, PAP complexes, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1), were measured by using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Dade Behring, Hollywood, FL, and American Diagnostica, Greenwich, CT).Data analysis Statistical evaluation was performed by using the Sigmastat Statistical Package (Jandel Scientific, San Rafael, CA). All results are presented as the mean ± SD. Because analyses of the data related to PS-positive blood cells and coagulation markers showed a nonparametric distribution, significant differences between control and patients were analyzed by using the Kruskal-Wallis test. If the P value for this overall comparison was significant at P < .05, group-wise comparisons were performed, using the Mann-Whitney test. Both Pearson and Spearman correlation tests were used to determine the relationship between 2 variables. Both tests yielded similar results for the same pair of variables analyzed. Values presented for the R and P values were obtained by Pearson tests on log-transformed data.
PS-positive erythrocytes and platelets in SCD The mean level of PS-positive red cells in the controls was 0.66% ± 0.34% (± SD). Levels in the patient groups with SC and SS genotypes were 1.68% ± 1.03% and 4.12% ± 2.41%, respectively (Figure 3A). Although a moderate increase in PS-positive erythrocytes was noted in SC disease (P < .0001), marked increases were observed in patients with the SS genotype when compared with both SC patients (P < .0001) and controls (P < .0001). Levels of PS-positive platelets in controls and patients with SC and SS genotypes were 1.16% ± 0.56%, 2.52% ± 1.23%, and 2.95% ± 1.79%, respectively (Figure 3B). Although significant differences were noted between controls and both patient groups (P < .004 in SC; P < .0001 in SS), there were no interpatient group differences (P > .45).
Markers of thrombin generation Mean plasma levels of prothrombin fragment F1.2 in controls and patients with SC and SS genotypes were 0.57, 0.87, and 1.30 nM, respectively (Table 1). Although a minimal but statistically significant increase in the level of F1.2 was noted in the SC patient group (P < .004), levels were markedly elevated in SS disease when compared with either controls (P < .0001) or the SC genotype (P < .02). Mean levels of TAT were 1.6, 4.0, and 5.54 ng/mL in controls, SC disease, and SS disease, respectively. Although significant differences were noted between controls and both patient groups (P < .03 with SC; P < .0001 with SS), there were no significant differences in TAT levels between patient groups (P > .08).
Markers of fibrinolysis Mean levels of D-dimer in the controls and patients with SC and SS disease were 0.05, 0.05, and 0.17 µg/mL, respectively (Table 1). Although there were no differences in levels between controls and SC patients (P> 0.35), levels of this marker were significantly elevated in SS disease when compared with either controls (P < .006) or the SC group (P < .0001). Plasma levels of tPA in controls, SC patients, and SS patients were 3.98, 8.27, and 6.25 ng/mL, respectively. The levels of tPA were elevated in SC patients (P < .008). No significant differences were noted between SS disease and controls (P > .05). PAP and PAI-1 levels were similar in all groups evaluated (P > .5; Table 1). In the patient groups, striking positive relationships were noted between levels of D-dimer and F1.2 (R = 0.53, P < .0001) and between D-dimer and PAP (R = 0.64, P < .000002).Relationships between hemostatic markers and PS-positive erythrocytes and/or platelets in SCD Although no correlations were noted between levels of PS-positive platelets and any of the hemostatic markers evaluated, significant positive correlations occurred between levels of PS-positive red cells and plasma F1.2, D-dimer, and PAP (Table 2; Figures 4 and 5). These correlations were most striking for F1.2 (R = 0.52, P < .0002; Figure 4A) and D-dimer (R = 0.63, P < .000002; Figure 5A). A modest association also existed between PS-positive erythrocytes and levels of PAP (R = 0.37, P < .01) and levels of the thrombin inactivation marker TAT, although the latter association did not attain statistical significance (P > .09).
Longitudinal hemostatic evaluations Results of paired blood samples performed at least 1 year apart (mean = 14 months) on the 8 infants previously noted revealed interesting although nonconclusive results. Initial values for F cells (red cells containing HbF), erythrocyte, and platelet PS were 72% ± 21%, 2.7% ± 1.6%, and 1.9% ± 0.7%, respectively. Follow-up evaluations as expected revealed a fall in F cell levels to 50% ± 25% (P < .0001, paired t test), whereas PS levels on erythrocytes and platelets increased to 3.7% ± 1.7% (P = .15) and 2.8% ± 1.9% (P = .14), respectively. Evaluations of hemostatic markers also revealed a trend toward activation with elevated levels of F1.2, D-dimer, and TAT in 6 of 8 paired samples when compared with their initial values [F1.2 = 0.95 ± 0.3 versus 1.29 ± 0.58 nM (P = .17); D-dimer = 0.08 ± 0.06 versus 0.24 ± 0.33 µg/mL (P = .14); TAT = 2.78 ± 1.06 versus 3.83 ± 1.86 ng/mL (P = .29)].
The presence of PS on cell surfaces is associated with numerous pathophysiologic consequences. Externalization of PS on platelet membranes following platelet activation is critical to coagulation mechanisms and the efficient propagation of the hemostatic process.20,21 In SCD, the consequences of PS exposure on the red cell membrane include an exacerbation of anemia because of enhanced phagocytic recognition and removal,20 enhanced adhesivity to the vascular wall,25 and the expression of procoagulant activity that can promote assembly on the membrane of both tenase and prothrombinase complexes.19,20 Although studies have demonstrated the surface exposure of PS on the erythrocytes in patients with SCD,22,23 none have sought to assess the effect of this pathologic procoagulant phenotype in relation to thrombin generation and fibrinolysis. Moreover, because platelet activation occurs in SCD, an additional question that is important to address is whether PS exposure on the platelet occurs in these patients and, if so, what is the relative contribution of erythrocyte versus platelet PS to the pathophysiology of thrombin generation. Results presented elucidate a crucial role for abnormal erythrocyte PS
exposure in the thrombophilia of SCD. Striking correlations were noted
between erythrocyte PS levels and prothrombin fragment F1.2
(P < .0002; Figure 4), with a similar trend with respect to TAT levels (Table 2). Although additional evidence of abnormal platelet membrane PS exposure was noted, the absence of any
correlations between platelet PS and hemostatic markers implies that
platelet activation is not the predominant mechanism responsible for
thrombin generation. Our longitudinal paired studies (in progress at
this time) suggests an additional trend between increasing levels of erythrocyte PS and hemostatic activation in 6 of 8 infants evaluated to
date, although the numbers were too small to achieve statistical significance. The inference that red cell PS is one of the major factors controlling coagulation activation is also strengthened by a
recent study we performed in infants with SCD.24 Because HbF inhibits polymerization of sickle cell hemoglobin, we hypothesized that, in vivo sickling/desickling with consequent membrane
perturbation, procoagulant PS exposure and activation of coagulation
mechanisms would be prevented in infants with SCD by the high HbF
levels. With progression into early childhood and a decline in HbF
levels, PS-positive red cells and coagulation activation would
supervene. This scenario was proven to occur in that study with
increasing plasma F1.2 levels correlating with increasing erythrocyte
PS positivity and decreasing numbers of F cells.24 These 2 reports provide crucial evidence for the promoter effect on coagulation that results from erythrocyte PS exposure. Although acceleration of
coagulation via enhanced procoagulant activity is the hallmark of the
PS-positive erythrocyte, we must be cognizant that the trigger for
hemostatic activation in SCD is presumably related to elevated
circulating levels of tissue factor and the presence of microvascular
endothelial cells with a tissue factor phenotype that circulate in
increased numbers in these patients.15,16 This latter
finding appears to be linked to the up-regulation of endothelial
nuclear factor Our studies also address the alterations in the fibrinolytic system. No correlations were observed between platelet PS and the fibrinolytic markers assessed. A striking correlation, however, between erythrocyte PS and D-dimer levels (P < .000002; Figure 5) was noted, with a more modest one between red cell PS and PAP levels (P < .01). In addition, a correlation between F1.2 and D-dimer was observed (P < .0001) consistent with the hypothesis that activation of the fibrinolytic system and D-dimer formation is secondary to the generation of thrombin and fibrin deposition. In addition, because leucocytosis and white cell activation has been observed in SCD,11-13 it is logical to ask the question whether proteolytic activity released from these cells could induce fibrinolysis unrelated to the effect of plasmin. The correlation noted, however, between D-dimer and PAP levels (P < .000002) suggests that fibrinolysis occurs mainly because of plasmin generation and not because of white cell proteolytic activity. Comparison of hemostatic markers between children with the SS and SC
genotypes has not been previously reported. Our study documents the
same trend that has been observed in the adult8 In conclusion, we have demonstrated unequivocally the association between red cell PS exposure and thrombin generation in patients with SCD. Although activation of the fibrinolytic system was noted, it appears to be a secondary phenomenon in response to thrombin generation and fibrin deposition. With the availability of transgenic murine models and novel interventive strategies (such as antiadhesion therapy, inhibitors of cellular dehydration, and inducers of HbF other than hydroxyurea), we are on the threshold of a new era of clinical trials in SCD. We suggest that the use of erythrocyte PS as a surrogate marker in such trials could provide valuable insights into disease pathophysiology, especially in relation to the vascular complications of this disease entity.
A recent paper of Tomer et al30 requires discussion in light of our findings. The authors demonstrate that a diet rich in n-3 fatty acids, provided as fish oil, decreased hemostatic activation in a small group (n = 5) of patients with SCD. They also demonstrated a decrease in platelet activation markers and suggested that the inhibition of hemostatic activation provided by fish oil ingestion was directly linked to platelet inhibition. While markedly elevated red cell PS levels were noted at study inception, no concomitant data is provided on red cell annexin V binding (ie, erythrocyte PS levels) after therapy. An alternate explanation is that a decrease in red cell procoagulant activity with a resulting decrease in in vivo thrombin generation could have caused the observed platelet effects. Thus, the data presented are open to various interpretations, with the real possibility that the platelet effects noted were secondary rather than primary.
We thank Surekha Kulkarni and Vijender R. Viadyula, PhD, for technical assistance; Patricia O'Neal, Sandra Moss, and Miriam Gilday for their phlebotomy expertise; and Lorenzo Thomas for secretarial assistance.
Submitted June 4, 2001; accepted July 25, 2001.
Supported by grants HL51497 and 1P60HL62148 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: B. N. Yamaja Setty, Thomas Jefferson University, Department of Pediatrics, Medical College Bldg, Suite 727, 1025 Walnut St, Philadelphia, PA 19107; e-mail: yamaja.setty{at}mail.tju.edu.
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E. J. van Beers, M. C.L. Schaap, R. J. Berckmans, R. Nieuwland, A. Sturk, F. F. van Doormaal, J. C.M. Meijers, B. J. Biemond, and on behalf of the CURAMA study group* Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease Haematologica, November 1, 2009; 94(11): 1513 - 1519. [Abstract] [Full Text] [PDF]
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J. Villagra, S. Shiva, L. A. Hunter, R. F. Machado, M. T. Gladwin, and G. J. Kato Platelet activation in patients with sickle disease, hemolysis-associated pulmonary hypertension, and nitric oxide scavenging by cell-free hemoglobin Blood, September 15, 2007; 110(6): 2166 - 2172. [Abstract] [Full Text] [PDF]
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N. J. Wandersee, S. C. Olson, S. L. Holzhauer, R. G. Hoffmann, J. E. Barker, and C. A. Hillery Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis Blood, January 15, 2004; 103(2): 710 - 716. [Abstract] [Full Text] [PDF]
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L. S. Kean, E. A. Manci, J. Perry, C. Balkan, S. Coley, D. Holtzclaw, A. B. Adams, C. P. Larsen, L. L. Hsu, and D. R. Archer Chimerism and cure: hematologic and pathologic correction of murine sickle cell disease Blood, December 15, 2003; 102(13): 4582 - 4593. [Abstract] [Full Text] [PDF]
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A. S. Shet, O. Aras, K. Gupta, M. J. Hass, D. J. Rausch, N. Saba, L. Koopmeiners, N. S. Key, and R. P. Hebbel Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes Blood, October 1, 2003; 102(7): 2678 - 2683. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2-antiplasmin) were the main tests of fibrinolysis assessed.
-subunit of the human fibrinogen receptor CD61 (anti-CD61-PE, clone PM6/13), and isotypic control antibody (clone 679.1Mc7) were
obtained from Immunotec (Beckman-Coulter, Miami, FL) or Serotec (Raleigh, NC). Fluorescein isothiocyanate (FITC)-labeled annexin V was
purchased from R&D Systems (Minneapolis, MN).
80°C until assayed.
B (NF
namely, that, whereas markers of in vivo thrombin production are moderately elevated in children with SC disease, they are more marked in SS
disease. PS functions as an essential cofactor for prothrombin activation by factor Xa.