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Keywords:

  • polycythaemia vera;
  • secondary polycythaemia;
  • thrombosis;
  • iron deficiency;
  • phosphatidylserine

Phosphatidylserine (PS) exposure on the erythrocyte membrane has a role in removing aged erythrocytes (Hebbel, 1991). Increased exposure of PS in erythrocytes has been postulated to play a role in the pathophysiology of sickle cell disease because of its possible effects on blood coagulation, cell adhesion, and cell clearance (Hebbel, 1991). PS exposure on the erythrocyte membrane has been known to activate clotting activity. On the other hand, polycythaemia vera is a risk factor for thrombosis. We investigated whether PS exposure on the erythrocyte membrane was increased in patients with polycythaemia vera.

Fourteen patients with polycythaemia and nine healthy subjects were studied between January and March 2010. Blood was drawn by venepuncture into evacuated tubes containing ethylenediaminetetra-acetic acid (EDTA) anticoagulant. PS exposure on erythrocytes in whole blood was measured using a modified flow cytometric assay, as previously described (Wood et al, 1996), and expressed as the percentage of annexin-V-binding erythrocytes. All study participants provided informed consent, and the study design was approved by an ethics review board at our institution.

Six of 14 patients with polycythaemia had the JAK2 V167F mutation and satisfied the 2008 World Health Organization criteria for polycythaemia vera (Vardiman et al, 2009). The remaining eight patients did not show the mutation and therefore did not meet the criteria for polycythaemia vera. These patients had factors such as smoking or lung disease and were diagnosed as having secondary polycythaemia. Healthy volunteer subjects were our hospital medical workers, who were considered to be suitable controls by routine health check. There were no significant differences in age or sex distribution among the polycythaemia vera, secondary polycythaemia, and healthy subject groups (Table I). No patients in this study had existing renal disease, such as renal failure.

Table I.   Clinical features and exposure of phosphatidylserine on the erythrocyte membrane in patients with polycythaemia vera.
 Polycythaemia vera N = 6Secondary polycythaemia N = 8Healthy subjects N = 9
  1. PS exposure on the erythrocyte membrane was measured as the percentage of annexin-V-binding erythrocyte. PS exposure on the erythrocyte membrane in patients with polycythaemia vera (JAK2 mutation+) was higher than that in both patients with secondary polycythaemia (JAK2 mutation) and healthy subjects (non-polycythaemia). We compared the differences among the polycythaemia vera group, secondary polycythaemia group, and control group using Wilcoxon analysis. Data represent the mean ± standard error. All statistical procedures were conducted using jmp Version 8.0 software (SAS Institute Inc., Cary, NC, USA), and significance was defined as < 0·05(*).

Age (years)64 ± 461 ± 458 ± 4
Gender (Male/Female)4/28/04/5
Medicine
 Aspirin620
 Hydroxycarbamide200
Haematology
 Leucocytes (×109/l)19·5 ± 0·3*7·0 ± 0·55·2 ± 0·7
 Platelets (×109/l)400 ± 80*200 ± 20220 ± 30
 MCV (fl)76 ± 3*92 ± 192 ± 3
PS (%)1·42 ± 0·46*0·49 ± 0·070·49 ± 0·03

With respect to haematological findings, leucocyte and platelet counts were significantly higher in the polycythaemia vera group compared to the secondary polycythaemia group (Table I). In addition, the mean corpuscular volume (MCV) in the polycythaemia vera group was significantly lower than that of the secondary polycythaemia group (Table I).

As shown in Table I and Fig 1, PS exposure on the erythrocyte membrane in patients with polycythaemia vera was 1·42 ± 0·46%, which was significantly higher compared to patients with secondary polycythaemia or healthy subjects. The significant increase in PS exposure on the erythrocyte membrane in patients with polycythaemia vera was not only shown in the absolute number of PS-expressing erythrocytes count (5·5 times that of healthy subjects) but also in the percentage of PS-expressing erythrocytes in the total red blood cell (RBC) count.

image

Figure 1.  Typical flow cytometric plots for the presence of phosphatidylserine (PS) on erythrocytes from a healthy subject, and patients with polycythaemia vera or secondary polycythaemia. (A) PS expression from a healthy subject (48-year-old female). PS (%): 0·4%. (B) PS expression from a patient with polycythaemia vera (74-year-old male). PS (%): 2·9%. (C) PS expression from a patient with secondary polycythaemia (62-year-old male). PS (%): 0·2%. PS exposure on the erythrocyte membrane was measured as the percentage of annexin-V-binding erythrocyte. (D) Typical flow cytometric histograms between the results in polycythaemia vera (plot1 PRV) and secondary polycythaemia (plot 2 SP).

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Cesar et al (1993) previously investigated the expression of PS on the surface of platelets in patients with myeloproliferative disease and reported that PS exposure on erythrocytes from patients with polycythaemia was similar to that of healthy subjects. Recently, it was reported that PS exposure on the erythrocyte membrane was increased in erythropoietin-producing transgenic mice (Föller et al, 2007). The current study is the first to show an increase in PS exposure on erythrocytes in patients with polycythaemia vera. In the study by Cesar et al (1993), PS in whole cells was assayed by gas chromatography. Our assay involved the measurement of PS on the erythrocyte surface. Therefore, we consider the difference in results between the current study and that of Cesar et al (1993) to be due to the different methods used.

Several other factors have been reported to influence PS exposure on the erythrocyte membrane. Medicines, such as rebabirin and α methyldopa, have been shown to increase PS exposure, however, these agents were not prescribed to any of the subjects in this study (Haynes et al, 2008; Qadri et al, 2009). On the contrary, prior to the study, some of our patients with polycythaemia vera received hydroxycarbamide or aspirin, which are reported to decrease PS exposure (Haynes et al, 2008; Qadri et al, 2009).

Iron deficiency is also reported to elevate PS exposure on the erythrocyte membrane (Kemmpe et al, 2006). In the current study, patients in the polycythaemia vera group showed a significantly lower MCV (76 ± 3 fl) compared to the secondary polycythaemia and healthy subject groups. As a detailed examination on the presence of iron deficiency was not included in our study, the possible association of iron deficiency to these results need to be further determined (Pavic et al, 2003).

In conclusion, we found that PS exposure on the erythrocyte membrane was significantly increased in patients with polycythaemia vera. This finding may serve as a key to revealing the mechanism of the increased risk of thrombosis in patients with polycythaemia vera.

References

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  2. References
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