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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

Background : A pegylated interferon-alpha-induced decrease in platelet counts may become a limiting factor for continuation of therapy.

Aim : To evaluate the effect of pegylated interferon-α administration on platelet plug formation and von Willebrand factor antigen release in patients with chronic hepatitis C.

Methods : Thirty patients with chronic hepatitis C (genotype 1; fibrosis 1–3: n = 16, cirrhosis: n = 14) received a single dose of 9 MU interferon-α2a, followed by weekly administration of 180 μg of pegylated interferon-α2a/ribavirin for 48 weeks. Platelet counts, platelet function (collagen–epinephrine-induced closure time) and von Willebrand factor antigen were measured.

Results : Platelet counts and collagen–epinephrine-induced closure time decreased by 13% and 16%, respectively, 24 h after the first dose of interferon-α2a, and von Willebrand factor antigen levels increased by 31% (P < 0.01) compared with baseline. During a 48-week observation period, platelet counts decreased by a maximum of 33% (P < 0.001), von Willebrand factor antigen levels increased by 69% (P < 0.001) whereas collagen–epinephrine-induced closure time did not change. In noncirrhotic patients, the increase of von Willebrand factor antigen levels was maintained throughout therapy without a change in collagen–epinephrine-induced closure time. In contrast, in cirrhotics, von Willebrand factor antigen levels did not increase, while collagen–epinephrine-induced closure time was prolonged.

Conclusion : Single-dose interferon-α decreases platelet counts but improves platelet function, possibly by the release of von Willebrand factor antigen. Accordingly, long-term antiviral treatment had no effect on collagen–epinephrine-induced closure time, despite the decrease in platelet count in noncirrhotic patients. Such a compensation of decreased platelet counts by increased von Willebrand factor antigen level did not occur in cirrhotics.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

The administration of pegylated interferon-alpha (PEG-IFN-α) and ribavirin is standard therapy for chronic hepatitis C.1, 2 While this treatment has improved the results of antiviral therapy, it is associated with serious side-effects3, 4 like thrombocytopenia.5, 6 PEG-IFN-α reduces the platelet count by 10–50% during the first 4 weeks of therapy7 and thrombocytopenia can be a limiting factor for therapy,4–6, 8 necessitating reduction of PEG-IFN-α dose, or even discontinuation of therapy.3 The current recommended thresholds for a dose reduction are platelet counts below 50 × 109/L. Apart from the platelet counts, it is the platelet function that is crucial for the patients’ risk of bleeding.9–11 Platelet function depends on both platelet count and platelet quality and has not been investigated under PEG-IFN-α/ribavirin therapy so far. The present study evaluates the effect of IFN-α and PEG-IFN-α/ribavirin administration on the platelet function in patients with chronic hepatitis C by a platelet function analyzer 100 (PFA-100).12–15 PFA-100 simulates the process of platelet adhesion and aggregation following a vascular injury in vitro. Results are provided as ‘closure time’ [collagen–epinephrine-induced closure time (CEPI-CT)], defined as the time it takes for blood to plug the aperture of a cartridge. Closure time reflects platelet function in both patients with hereditary and acquired thrombocytopathia. PFA-100 has the potential ability to replace the bleeding time test as an effective first-line screening method for platelet dysfunction in clinical practice.16, 17 Studies show that 80% of post-surgery high-blood-loss patients have abnormal PFA-100 results.18

To explore a potential factor influencing platelet plug formation, plasma levels of the von Willebrand factor antigen (vWF-Ag) were also determined. vWF is an acute phase protein released by several cytokines including possibly IFN-α and has strong impact on platelet function.19, 20 At high shear rates, vWF acts as an important adhesive protein for both platelet adhesion and aggregation. vWF function is mediated by two platelet membrane receptors, glycoprotein (GP) Ib and GPIIb/IIIa, in a co-ordinated and synergistic manner.21 For intact adhesion of vWF to GPIb, large vWF-multimers are needed. Platelet adhesion results in activation of GPIIb/IIIa and consequently release of various platelet-activating mediators, such as adenosine diphosphate (ADP) and thromboxane A2 (TxA2). Released ADP and TxA2 stimulate platelet aggregation, finally leading to the development of a platelet plug.22

Study design

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

This study was performed in an open and prospective manner and approved by the local Ethics Committee. Written informed consent was obtained from all patients.

Patients and experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

Thirty consecutive patients with chronic hepatitis C, genotype 1, participating in an ongoing prospective, randomized, placebo-controlled, multicentre trial were included 23 [17 men and 13 women, median age: 46 years (range 33–64), fibrosis grade 1:2(N); 2:12; 3:2; 4:14]. None of the patients received antiplatelet therapy.

Briefly, patients received a single subcutaneous test dose of 9 MU IFN-α2a (RoferonA®; Roche, Vienna, Austria). Four weeks later, the patients received 180 μg of PEG-IFN-α2a (Pegasys®; Roche) once weekly, and 1000–1200 mg ribavirin (Copegus®; Roche) daily for 48 weeks. Platelet count, platelet plug formation time, and vWF-Ag plasma levels were measured at baseline, day 1, consecutively during 48 weeks of therapy and again 6 months after the end of therapy.

Blood sampling

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

Blood samples for analysis of platelet plug formation and vWF levels were collected in tubes containing 0.129 m buffered sodium citrate, equivalent to 3.8% sodium citrate (Vacuette®; Greiner Labortechnik, Vienna, Austria).

PFA-100 testing

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

The PFA-100 system utilizes disposable test cartridges where a platelet plug occludes a microscope aperture cut into a membrane coated with collagen and epinephrine (CEPI). The time required for occlusion [closure time (CT)] is indicative of platelet function and primary haemostasis capacity.15, 24 The PFA-100 measures platelet plug formation time under shear stress and is strongly dependent on vWF levels in plasma. CEPI-CT values (normal values in healthy volunteers 78–183 s, maximum value 300 s) have a 9% intra-individual day-to-day variability.25 All PFA-100 measurements were carried out within 4 h after blood sample collection. To test the validity and sensitivity of the PFA-100 device in detecting drug-induced platelet dysfunction, we analysed blood from our staff members after intake of 500 mg of acetylsalicylic acid and documented closure times >300 s.

Von Willebrand factor antigen levels

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

Plasma levels of vWF were measured with a fully automated STA analyser by using the vWF-Liatest (Diagnostica Stago, Paris, France).

Statistical methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

All statistical analyses were performed by using Statistica for Windows (Version 6.0). Data are expressed as mean and 95% confidence intervals (CI) in the text and Table 1 and as mean and standard error of mean (S.E.M.) in the figures. Follow-up data were expressed as their percentage changes from baseline values. All statistical comparisons were performed using the Wilcoxon matched pairs test for post hoc comparisons. Differences between groups were assessed by the Mann–Whitney U-test. A two-tailed P-value <0.05 was considered significant.

Table 1.  Absolute platelet counts and CEPI-CT values at different time points in patients with/without cirrhosis during combination antiviral therapy. Data are presented as mean and 95% CI
Timepoint of the therapyPlatelet counts (×109/L)CEPI-CT (s)
Fibrosis 1–3CirrhosisP-valueFibrosis 1–3CirrhosisP-value
Baseline231 (200–261)173 (149–196)0.004164 (144–185)199 (155–244)0.4
Week 2173 (142–204)133 (118–147)0.06142 (113–171)221 (177–265)0.005
Week 4162 (133–191)116 (96–136)0.03150 (126–174)224 (183–264)0.001
Week 8157 (121–192)117 (102–133)0.14168 (137–199)195 (149–241)0.27
Week 12151 (123–179)116 (94–139)0.07155 (137–172)215 (165–265)0.12
Week 24146 (118–175)117 (94–139)0.17163 (136–190)227 (179–275)0.01
Week 36145 (122–168)126 (96–157)0.2178 (149–207)229 (184–275)0.03
Week 48165 (133–197)128 (100–156)0.13186 (146–225)260 (224–297)0.01
Follow up212 (184–240)190 (161–218)0.25147 (122–173)185 (137–233)0.2

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

Sixteen patients were sustained virolologic responders, 10 patients relapsed and four patients were nonresponders. Short- and long-term effects of IFN-α, PEG-IFN-α/ribavirin on platelet counts, platelet plug formation time (CEPI-CT) and vWF are shown in Figures 1 and 2, respectively. The effects of PEG-IFN-α/ribavirin on platelet counts, CEPI-CT and vWF in patients with cirrhosis (i.e. patients with fibrosis grade 4, n = 14) and without cirrhosis (i.e. patients with fibrosis grade 1–3, n = 16) are shown in Figure 3.

image

Figure 1. Effect of a single-dose of IFN-α on platelet counts, CEPI-CT mesured by the PFA-100 (CEPI-CT) and vWF-Ag levels in patients with chronic hepatitis C (n = 30). Data are presented as mean ± S.E.M. *P < 0.01 vs. baseline.

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image

Figure 2. Effect of long term therapy with PEG-IFN-α/ribavirin on platelet counts, CEPI-CT measured by the PFA-100 and vWF-Ag levels in patients with chronic hepatitis C (n = 30). Data are presented as mean ± S.E.M. *P < 0.05 vs. baseline.

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image

Figure 3. Effect of long-term therapy with PEG-IFN-α/ribavirin on platelet counts (down), CEPI-CT (middle) and vWF-Ag levels (upper) in patients with hepatitis C-induced cirrhosis (closed, n = 14) and without cirrhosis (open, n = 16). Data are presented as mean ± S.E.M. *P < 0.05 cirrhotics vs. noncirrhotics.

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The baseline platelet counts were 206 × 109/L; 95% CI: 185–227 × 109/L, and were lower in patients with cirrhosis than in noncirrhotics (172 × 109/L; 95% CI: 149–196 × 109/L vs. 231 × 109/L; 95% CI: 200–262 × 109/L P < 0.05). Twenty-four hours after first-dose administration of IFN-α2a platelet counts decreased by a mean of 13%; 95% CI: 8–18%; P < 0.05 in all patients. During combination antiviral therapy, platelet counts decreased further and reached a maximum decrease of 33%; 95% CI: 24–42% after 24 weeks of therapy (P < 0.01). The magnitude of the decrease in platelet counts between patients with and without cirrhosis was not different during the whole study (Figure 3). At any timepoint, none of our patients had platelet counts below 50 × 109/L. Eight of our patients had platelet counts below 100 × 109/L during the study (four patients with fibrosis 1–3 and four with cirrhosis). In two patients, CEPI-CT was not prolonged despite platelet counts below 100 × 109/L (both patients with fibrosis 1–3).

The baseline CEPI-CT was 176 s (95% CI: 155–197) and was not different between patients with or without cirrhosis. Twenty-four hours after first-dose administration of IFN-α2a CEPI-CT shortened by a mean of 16% (95% CI: 2–24%). During combination antiviral therapy, CEPI-CT tended to increase but did not change significantly (P > 0.06 and P > 0.1 at week 36 and 48 of therapy, respectively). In cirrhotic patients, CEPI-CT was prolonged by 29%, 95% CI: 4–54%; 28%, 95% CI: 4–56%; and 46%, 95% CI: 12–80% after 24, 36 and 48 weeks of therapy, respectively (Figure 3; P < 0.05), whereas it did not change in patients with fibrosis grade 1–3 (Figure 3; P > 0.05). CEPI-CT was significantly prolonged in cirrhotic when compared with noncirrhotic patients after week 24 of therapy (P < 0.05).

The baseline vWF was 202% (95% CI: 166–238%) and was higher in patients with cirrhosis (261%; 95% CI: 196–327%) than in those without (153%; 95% CI: 122–184%) (P < 0.05). Twenty-four hours after first-dose administration of IFN-α2a, vWF-Ag increased by 31% (95% CI: 24–41%) (P < 0.01). During 48 weeks of combination therapy, plasma levels of vWF increased by a maximum of 69% (P < 0.001) (95% CI: 40–98%) after 4 weeks of therapy. In patients with fibrosis grade 1–3, vWF increased throughout the whole study period with a maximum of 89% at week 12 of therapy (95% CI: 24–154%; P < 0.001). In patients with cirrhosis, vWF increased only initially by 25% (95% CI: 7–43%) and 34% (95% CI: 9–59%) (P < 0.05) at week 2 and 4 of therapy, respectively. After week 4, vWF did not change in patients with cirrhosis compared with baseline (P > 0.05; Figure 3).

Six months after the end of the therapy, platelet counts returned to baseline levels (202 × 109/L; 95% CI: 184–220 × 109/L), platelet plug formation returned to baseline (162 s; 95% CI: 139–185 s) and vWF levels decreased to a mean of 147%; 95% CI: 120–175% (P < 0.01 compared with baseline).

Collagen–epinephrine-induced closure time did not correlate with platelet counts before and after chronic PEG-IFN-α therapy in cirrhotic and noncirrhotic patients. Furthermore, there was no correlation between the individual values of CEPI-CT change vs. platelet count change during and after chronic PEG-IFN-α therapy (Figure 4). vWF did not correlate with the platelet plug formation. On the day after the test dose IFN-α administration, vWF inversely correlated with platelet plug formation (R = −0.57, P < 0.05). No correlation was observed during the rest of the study. As expected, both haemoglobin and leucocytes decreased on treatment. There was no significant difference between cirrhotic and noncirrhotic patients at any given time point (data not shown). We did not find any significant correlation between CEPI-CT and haematocrits and leucocyte counts before, during and after chronic PEG-IFN-α therapy.

image

Figure 4. No correlation was found between the individual values of CEPI-CT change vs. platelet count change after 24 and 48 weeks of long-term PEG-IFN-α therapy.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References

The results of this study indicate that continuation of IFN-α/PEG-IFN-α therapy at full dose appears to be safe even in the presence of moderate thrombocytopenia in noncirrhotic patients. The decrease in platelet counts is compensated by an increase in vWF levels, which improves platelet function as measured by the Food and Drug Administration (FDA)-approved PFA-100 device. This is the first study exploring potential mechanisms accounting for this phenomenon. In noncirrhotic patients, after initial increase, vWF plasma levels remained elevated on continued treatment with pegylated IFN-α/ribavirin and CEPI-CT did not change throughout therapy. In contrast, in cirrhotic patients, vWF plasma levels were already increased at baseline, slightly increased initially but returned to baseline during further therapy, while CEPI-CT was significantly prolonged compared with noncirrhotics during the second half of therapy. Thus, the concomitant increase in vWF levels may partly compensate CEPI-CT in spite of thrombocytopenia in noncirrhotic patients. Six months after the end of therapy, platelet counts and CEPI-CT values returned to baseline, and vWF decreased compared with baseline and reached mean values similar to those seen in healthy volunteers.25

The mechanism by which pegylated IFN-α/ribavirin increases vWF is unknown. It may be mediated by IFN-α induced tumour necrosis factor-alpha (TNF-α) release.26, 27 Likewise, lipopolysaccharide (LPS)-induced TNF-α in healthy volunteers was associated with increased vWF and thereby improved platelet function as measured by PFA-100.20, 28 In a previous study, IFN-α normalized decreased vWF in patients with essential thrombocytosis.29 A correlation between vWF and CEPI-CT measured by PFA-100 was reported in healthy subjects.20, 25 However, such a correlation was not observed in our patients with chronic hepatitis C. This lack of correlation may be explained by the increase of baseline vWF, like in other chronic inflammatory diseases.30, 31

In cirrhotic patients, vWF levels were already higher at baseline compared with noncirrhotic patients. This increase may reflect a compensatory release of vWF for thrombocytopenia and other haemostatic problems associated with cirrhosis.32–34 It may be triggered by the chronic inflammation itself, by endotoxaemia, or reduced cleavage of vWF by metalloprotease.35 Apparently, PEG-IFN-α/ribavirin was not able to increase vWF levels beyond the already elevated baseline levels in cirrhotic patients. Consequently, PEG-IFN-α/ribavirin induced thrombocytopenia leads to prolonged CEPI-CT in cirrhotic patients. Patients with cirrhosis had a lower baseline platelet count. Despite the decrease in platelet count being similar in patients with and without cirrhosis, platelet counts were lower in patients with cirrhosis only up to week 4 but did not differ significantly anymore thereafter. As platelet counts did not correlate with CEPI-CT results before and after long-term PEG-IFN-α therapy in cirrhotic and noncirrhotic patients and no correlation was found between the individual values of CEPI-CT change vs platelet count change during and after chronic PEG-IFN-α therapy, we conclude that increased PFA-100 CT are not a direct result of decreased platelet counts in our patients. Also, platelet counts below 100 × 109/L were not not always associated with CEPI-CT prolongation. As no patient had a drop in platelet count below 50 × 109/L, we cannot draw any firm conclusion with regard to changes in bleeding tendency in the patients with the presumably greatest propensity to bleed. Additional studies correlating bleeding complications under PEG-IFN-α therapy with CEPI-CT in patients with very low platelet counts would be needed to clarify the clinical significance of these findings.

The role of ribavirin on platelet function is yet unknown but ribavirin did not increase PEG-IFN-α induced thrombocytopenia.3, 4 On the contrary, it seems to counteract it indirectly through a compensatory increase in the endogenous EPO (erythropoietin)-levels resulting from ribavirin-induced anaemia.7 Moreover, EPO has been shown to stimulate platelet function in healthy humans and patients with liver cirrhosis.36, 37

In conclusion, single-dose IFN-α decreases platelet counts but improves platelet function, possibly by release of vWF-Ag. Accordingly, long-term antiviral treatment had no negative effect on CEPI-CT, despite the decrease in platelet count in noncirrhotic patients. Such a compensation of decreased platelet counts by increased vWF-Ag did not occur in cirrhotics. Additional studies in patients with platelet counts <50 × 109/L are needed to clarify the clinical importance of this test in patients with very low platelet counts in an effort to determine the true thresholds for safe application of PEG-IFN in patients with thrombocytopenia.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods, Patients, Materials
  5. Study design
  6. Patients and experimental procedures
  7. Blood sampling
  8. PFA-100 testing
  9. Von Willebrand factor antigen levels
  10. Statistical methods
  11. Results
  12. Discussion
  13. Acknowledgement
  14. References
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    Homoncik M, Jilma-Stohlawetz P, Schmid M, Ferlitsch A, Peck-Radosavljevic M. Erythropoietin increases platelet reactivity and platelet counts in patients with alcoholic liver cirrhosis. A randomized, double blind, placebo controlled study. Aliment Pharmacol Ther 2004; 15: 43743.