The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 38  |  Issue : 2  |  Page : 63--67

Evaluation of platelet dysfunction in viral liver cirrhosis ( relationship to disease severity)


Manal F. Ghozlan1, Abeer A. Saad1, Deena S. Eissa1, Heba M. Abdella2,  
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Correspondence Address:
Deena S. Eissa
MD, Department of Clinical Pathology, Ain Shams University Hospitals, Ramses St., Abbasia, Cairo 11566
Egypt

Abstract

Background

Hepatitis C virus (HCV) is a major health problem in Egypt, which has the highest HCV prevalence worldwide. Cirrhotic patients are known to have acquired platelet hypofunction; however, HCV-induced inflammatory and immunological phenomena are thought to be responsible for in-vivo platelet activation. This study aimed to evaluate the platelet function in patients with HCV-induced liver cirrhosis and also to study the relationship of platelet function to disease severity and various clinical/laboratory findings in cirrhotic patients.

Materials and methods

Evaluation of platelet function was carried out on 50 patients with HCV-induced liver cirrhosis using the platelet function analyzer-100 (PFA-100), compared with 30 healthy volunteers.

Results

The study demonstrated the presence of platelet hypofunction reflected by the prolonged PFA-100 closure time in patients of Child–Pugh stage B compared with those of stage A and C, in moderate compared with negative/mild and severe ascites, and in grade II esophageal varices compared with other grades. Patients with Child–Pugh stage B and C, severe ascites, and stage III/IV esophageal varices had significantly lower platelet count/spleen size ratios.

Conclusion

Chronic HCV-induced liver cirrhosis is implicated in alterations of primary hemostasis, with platelet hypofunction being more evident during relatively earlier disease stages compared with the later ones. PFA-100 closure time and platelet count/spleen size ratio could be used as noninvasive predictors of the grades of esophageal varices in cirrhotic patients. The simplicity of the PFA-100 could facilitate its use as a convenient primary screening test to detect platelet dysfunction in HCV-induced liver cirrhosis.




How to cite this article:
Ghozlan MF, Saad AA, Eissa DS, Abdella HM. Evaluation of platelet dysfunction in viral liver cirrhosis ( relationship to disease severity).Egypt J Haematol 2013;38:63-67


How to cite this URL:
Ghozlan MF, Saad AA, Eissa DS, Abdella HM. Evaluation of platelet dysfunction in viral liver cirrhosis ( relationship to disease severity). Egypt J Haematol [serial online] 2013 [cited 2020 Mar 28 ];38:63-67
Available from: http://www.ehj.eg.net/text.asp?2013/38/2/63/134790


Full Text

 Introduction



Egypt has the highest hepatitis C virus (HCV) prevalence worldwide, with an estimated overall prevalence of 21.9% among adults 1. Portal hypertension and esophageal varices are the most common and dangerous complications of liver cirrhosis, with variceal bleeding representing the most substantial morbidity and mortality 2,3. Cirrhotic patients are known to have acquired platelet dysfunction, which is proposed to be implicated in the development of variceal bleeding 4. Several steps of platelet activation were shown to be impaired, attributed to both intrinsic and extrinsic defects resulting in platelet hypofunction; however, the underlying mechanisms remain speculative, possibly due to the multifactorial origin of cirrhosis 5.

Various complex defects of platelet function have been described in cirrhotic patients, including impaired thromboxane A2 (TxA2) synthesis, defective signal transduction, storage pool defects, and quantitative deficiency of the platelet glycoprotein Ib (GPIb) receptor 6. In contrast, HCV-induced inflammatory and immunological phenomena in the liver tissues are assumed to be directly responsible for in-vivo platelet activation 5. Accordingly, activated platelets are considered to play a significant role in the pathogenesis of liver damage by stimulating fibrogenesis and mitogenesis of Ito liver cells 7. Presently, platelets are increasingly appreciated as inflammatory players secreting chemotactic factors and mediating cell-to-cell interactions 5.

Generally, the role of platelets in the pathophysiology of viral diseases, such as inflammation, vasculogenesis, and tissue repair, might lead to a relatively better understanding of platelet function disorders in viral liver cirrhosis 5. Nevertheless, the relationship of abnormal platelet function with disease severity and the clinical and laboratory findings associated with viral liver cirrhosis is still largely enigmatic, awaiting elucidation.

Therefore, the present study aimed to evaluate the platelet function in patients with HCV-induced liver cirrhosis using the platelet function analyzer-100 (PFA-100) and also to study the relationship of platelet function with disease severity and various clinical and laboratory findings in cirrhotic patients.

 Materials and methods



Materials

This study was carried out on 50 patients with HCV-induced liver cirrhosis admitted to the Department of Tropical Medicine of Ain Shams University Hospitals from December 2010 to May 2012; 24 were men and 26 were women, with a male to female ratio of 1 : 1.1. Their ages ranged from 33–64 years (mean±SD, 50.5±9.6 years). Thirty age-matched and sex-matched healthy volunteers were enrolled as the control group. The exclusion criteria included patients with platelet counts of less than 100 000/μl, hematocrit levels of less than 35%, acute illness, fever, or malignancy. A written informed consent was obtained from all participants, and the study was approved by the local research ethical committee.

All patients were subjected to the following: (i) full history taking; (ii) thorough clinical examination with special stress on the liver and spleen size, presence of ascites and encephalopathy, together with the manifestations of a bleeding tendency; (iii) imaging studies such as abdominal ultrasound for the assessment of the liver/spleen size and ascites; (iv) upper gastrointestinal tract endoscopy for detection of the presence and grading of esophageal varices; (v) routine laboratory investigations that included a complete blood count (Sysmex KX-21N; Sysmex Corporation, Chuo-ku, Japan), liver profile [aspartate aminotransferase (AST), alanine aminotransferase (ALT), total/direct bilirubin, albumin] (Beckman Coulter Inc., Fullerton, California, USA), prothrombin time (PT), and international normalized ratio (Sysmex CA-1500; Sysmex Corporation); and (vi) evaluation of platelet function, which was performed for all enrolled patients and controls using the PFA-100 (Dade Behring, Schwalbach, Germany).

The patients were classified according to the following: (i) Child–Pugh system into Child stage A, B, and C 8; (ii) severity of ascites into negative/mild, moderate, and severe 9; and (iii) grades of esophageal varices into grades I–IV 10. The platelet count/spleen size ratio was used to normalize the platelet count to splenic sequestration, because the use of a platelet count alone may be misleading and cannot be solely attributed to the liver condition 11. The main demographic and clinicopathologic characteristics of the studied patients are described in [Table 1].{Table 1}

Methods

Sampling

Peripheral blood was drawn directly into an evacuated plastic tube containing 3.2% buffered sodium citrate (one part anticoagulant to nine parts blood), followed by proper mixing by gently inverting the tube three to four times. The hemolyzed blood samples were discarded. The samples were stored undisturbed at room temperature (+16 to +26°C) and were stable for up to 4 h.

Evaluation of platelet function by the PFA-100 using a collagen/epinephrine cartridge

The PFA-100 system allows for rapid evaluation of platelet function on small samples of citrated whole blood. The PFA-100 creates an artificial vessel consisting of a sample reservoir, a capillary, and a biologically active membrane having a central aperture coated with collagen/epinephrine (Col/Epi), in which the process of platelet adhesion and aggregation after vascular injury is simulated in vitro. The application of a constant negative pressure aspirates the anticoagulated whole blood of the sample from the reservoir through the capillary and aperture, exposing the platelets to high shear flow conditions. The PFA-100 measures the time needed for the platelet plug to form at the aperture after activation of the platelets by Col/Epi, gradually diminishing and finally arresting the blood flow. The results are reported by the instrument in terms of ‘closure time’ (CT) in seconds. A CT is an indicator of platelet function in the analyzed whole blood sample. A normal CT for Col/Epi ranges from 84–160 s 12.

Statistical analysis

The data were coded, entered, and processed using Statistical Program for Social Science, version 15 (SPSS Inc., Chicago, Illinois, USA) on a Windows 7 operating system. The qualitative variables were expressed as number and percentage. The quantitative variables were described in terms of mean±SD or median and range, as appropriate. The analysis of variance and post-hoc test were used to compare the mean CT of PFA-100 and the mean platelet count/spleen size ratio with the Child–Pugh stages, severity of ascites, and grades of esophageal varices. Correlation studies were performed using the Pearson correlation test. P values less than 0.05 and those less than 0.01 were considered significant and highly significant, respectively, in all analyses.

 Results



Comparison between patients and controls as regards the PFA-100 CT

As shown in [Figure 1], the PFA-100 CT was significantly lower in the control group (119±21.4 s) compared with the patient subgroups (193.7±87.1, 227±63.4, 195.8±51 s in Child–Pugh stage A, B, and C, respectively) (overall P<0.001). A significant difference was also observed when comparing the control group with the patients belonging to each of the three Child–Pugh stages separately (P<0.001).{Figure 1}

PFA-100 CT in relation to clinical data of the studied patients

Although the mean PFA-100 CT was more prolonged in patients of Child–Pugh stage B compared with those of stage A and C, the difference did not reach statistical significance (P=0.2). Patients with moderate ascites had the highest PFA-100 CT among those with other grades (P<0.001); they also had a significantly prolonged CT compared with the negative/mild (P=0.002) and severe cases (P<0.001), with an insignificant difference detected between the latter two groups (P=0.2). Patients with grade II esophageal varices had the highest PFA-100 CT compared with those with other grades (P<0.001); they were followed by grade III, grade IV, and finally grade I patients. Moreover, patients with grade II esophageal varices had the highest CT when compared with patients in each of the other grades (P<0.001) [Table 2].{Table 2}

Platelet count/spleen size ratio in relation to clinical data of the studied patients

The platelet count/spleen size ratio was significantly related to the Child–Pugh stages, severity of ascites, and grade of esophageal varices (P<0.001). Patients of Child–Pugh stage B and C showed a significantly lower platelet count/spleen size ratio compared with those of stage A (P<0.001), with an insignificant difference detected between those of stage B and C (P=0.5). A significantly lower ratio was reported in patients with severe ascites compared with those with negative/mild (P<0.001) and moderate (P=0.006) ascites, whereas an insignificant difference was observed between the latter two groups (P=0.6). Moreover, patients with grade III and IV esophageal varices showed significantly lower ratios when compared with those of grade I and II (P<0.05). Neither comparison of stage I with stage II, nor comparison of stage III with stage IV showed a significant difference (P>0.05) [Table 2].

Correlation of the PFA-100 CT and platelet count/spleen size ratio with laboratory data of the studied patients

A significant positive correlation was found between the PFA-100 CT and platelet count/spleen size ratio (r=0.35; P=0.01). The PFA-100 CT revealed significant negative correlations with AST and ALT (P=0.01). In contrast, the PFA-100 CT was not correlated with the levels of total bilirubin and albumin, or PT (P>0.05). However, studying the correlation between PFA-100 CT and albumin in patients belonging to different Child–Pugh stages of liver cirrhosis revealed a significant negative correlation only in stage C patients (r=−0.623; P=0.006). The platelet count/spleen size ratio revealed a significant negative correlation with the total bilirubin level (P=0.02) and a positive correlation with the albumin level (P<0.001), whereas it failed to correlate with AST, ALT, and PT (P>0.05) [Table 3].{Table 3}

 Discussion



Although platelet dysfunction in viral liver cirrhosis is a subject of several interesting research studies, yet it remains largely unclear.

The current study demonstrated the presence of platelet hypofunction, reflected by the prolonged PFA-100 CT, in patients with Child–Pugh stage A, B, and C liver cirrhosis compared with controls. Interestingly, the prolongation in PFA-100 CT was more pronounced in patients of Child–Pugh stage B compared with those of stage A and C, although the difference did not reach statistical significance. Moreover, patients with moderate ascites had the highest PFA-100 CT compared with those with negative/mild or severe ascites. Similarly, patients with grade II esophageal varices had the highest PFA-100 CT, followed by those with grade III, grade IV, and finally grade I. Significant negative correlations between the platelet function denoted by PFA-100 CT and both AST and ALT were detected, whereas the correlation with albumin levels was significant only in patients of Child–Pugh stage C. These findings were comparable with those of previous studies 4,13–16, despite being carried out on patients with cirrhotic liver diseases with various etiologies.

The platelet dysfunction and hypoaggregability seen in liver cirrhosis have been attributed to many factors including decreased TxA2/B2 production; decreased inositol-3-phosphate (IP3)-mediated cytosolic calcium increase; decreased platelet GPIb receptor expression; and storage pool defects, namely decreased ATP and serotonin levels in the dense bodies and decreased β-thromboglobulin (βTG), platelet factor-4 (PF4), and P-selectin levels in the α granules that limit the effect of granule release on the aggregation process 5,6. A possible underlying mechanism of ‘platelet exhaustion’ was proposed, because platelets are faced with the portal hyperdynamic circulation, which causes their damage during intravascular activation and leads to their subsequent hypofunction on in-vitro testing 5. Moreover, the platelet intrinsic inhibitory pathways are upregulated in liver cirrhosis, with an increase in the two main inhibitory messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) 17, together with a reduced transmembrane signaling in cirrhotic platelets after stimulation with thrombin or collagen 6. Defective transmembrane signaling (and secondarily, a decrease in the intracellular messengers) is thought to be the most important factor in cirrhotic thrombocytopathy 17. In addition, increase in the negative plasma factors hindering platelet activation such as fibrin(ogen)-degradation products (FDPs), D-dimers, nitric oxide, apolipoprotein-E (Apo-E), and bile salts has been reported in cirrhosis 5,18,19.

In contrast, the delineated equilibrium in cirrhotic patients to hemorrhagic tendency has been explained by evidences for a paradoxical in-vivo platelet activation resulting from HCV-induced increased serum concentrations of βTG, PF4, and soluble P-selectin. Therefore, inflammatory players of HCV infection are assumed to be directly responsible for platelet activation 20,21. These findings illustrate that platelet function might not only be affected by the clinical stages of liver cirrhosis but also by HCV RNA viremia 21. Likewise, the upregulation of the von Willebrand factor (vWF) in cirrhotic patients is reported as one of the positive promoters of platelet activation. Despite the reduced number of the more active high-molecular-weight vWF multimers, as well as the reduced collagen binding capacity and relative ristocetin activity in cirrhosis, it is likely that the quantitative increase exceeds the qualitative defect 22,23.

In agreement with other studies 24–28, the platelet count/spleen size ratio showed a significant relation with the Child–Pugh stages, severity of ascites, and grades of esophageal varices. Patients with Child–Pugh stage B and C, severe ascites, and stage III and IV esophageal varices had significantly lower platelet count/spleen size ratios. A significant positive correlation was found between the platelet count/spleen size ratio and PFA-100 CT and albumin, whereas a significant negative correlation was detected with total bilirubin levels. In contrast, the disagreement in some studies 29,30 might be attributed to the different etiologies of liver cirrhosis in the studied populations.

 Conclusion



Chronic HCV-induced liver cirrhosis is implicated in alterations of primary hemostasis (platelet adhesion, activation, and aggregation), with platelet hypofunction being more evident during relatively earlier disease stages compared with the later ones. The PFA-100 CT and platelet count/spleen size ratio could be used as noninvasive predictors of the grades of esophageal varices in cirrhotic patients. The simplicity of PFA-100 could facilitate its use as a convenient primary screening test to detect platelet dysfunction in HCV-induced liver cirrhosis. Further studies assessing the levels of vWF, being a member of primary hemostasis and an acute-phase inflammatory reactant reflecting an active HCV infection, together with assessing HCV RNA viremia and studying their relationship with platelet dysfunction in HCV-induced liver cirrhosis are worthwhile. The study of platelet functional changes in relation to various etiologies of chronic liver disease remains an intriguing area, demanding further research.[30]

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