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Role of cytosolic calcium and actin polymerization on agonist-induced secretion by the platelets of liver cirrhosis patients/Karaciger sirozlu hastalarin trombositlerinde aktin polimerizasyonu ve sitozolik kalsiyumunun etkisi.


Cirrhosis of the liver is anatomically defined to be an end-stage liver disease characterized by fibrosis and structurally abnormal nodules [1]. Cirrhosis leads to multiple complications like ascites, gastrointestinal and esophageal variceal bleeding as a result of portal hypertension and platelet dysfunction [2]. Thrombocytopenia is a significant feature of chronic liver diseases including cirrhosis and attributed to passive platelet sequestration in the spleen. The platelet dysfunction in cirrhosis is probably both intrinsic to the platelets and secondary to soluble plasma factors. Witters et al have implicated the necessity of platelet research in liver diseases to provide new pathophysiological insights [3].

Blood hemostasis is affected by platelets by adhering to the sites of vascular injury, releasing compounds from their granules, aggregating together to form hemostatic platelet plug and providing procoagulant surface on their phospholipid membrane to arrest bleeding [4]. Defect in any one of the above events lead to platelet dysfunction and defective hemostasis.

Human platelets are secretory cells, and exocytosis is the mode of secretion which results in the release of a-granules, lysosomes and dense granules. The nature of a-granules and lysosomes has been reported in many (patho) physiological conditions [5,6]. Normally, the secretory organelles are randomly dispersed in the cytoplasm of resting platelets. Upon activation, platelets lose their discoid shape, develop multiple pseudopods, and concentrate the cell organelles in the centre of the cell. This alteration in the physical structure is closely associated with the polymerization of globular or monomeric actin (G-actin) to filamentous actin (F-actin) that leads to secretion of substances into the surrounding medium [7].

About 3-8 dense granules are present per platelet [8,9], which contains serotonin, adenosine tri phosphate (ATP), adenosine di phosphate (ADP), calcium ([Ca.sup.2+]) and pyrophosphate (PPi) [10,11]. The contents, especially serotonin and ADP amplify the platelet responses induced by strong platelet agonists and stabilize platelet aggregates [12,13]. The procoagulant actions of these components are proved experimentally [14]. Many studies have shown that ATP has a complex role in the regulation of platelet aggregation [15,16]. [Ca.sup.2+] is essential for both platelet aggregation [17] and the release reaction [18]. Addition of [Ca.sup.2+] ionophore to platelets resulted in aggregation and release of stored contents from platelets [18].

We have previously reported that defective [Ca.sup.2+] release from the internal stores may account for inhibition of platelet aggregation in cirrhosis [19]. Though many scientists have reported that platelet aggregation and adhesion are abnormal in liver cirrhosis of different etiology [20, 21], the study regarding the secretory functions of platelets in liver cirrhosis is limited. Hence, the present investigation was focused on evaluating the level of secretion of hemostatic elements simultaneously with changes in the level of cytosolic [Ca.sup.2+] and actin polymerization in platelets stimulated by the agonist collagen in vitro. The study is also aimed to assess the relationship between bleeding time (BT) and the secretory capacity of platelets in liver cirrhosis.

Subjects and Methods


The study comprised of fifty patients registered in the Department of Surgical Gastroenterology and Proctology, Stanley Medical College and Hospital, and the Department of Digestive Health Diseases, Government Peripheral Hospital, Chennai. Cirrhosis was confirmed by ultrasound and Doppler use-ultrasound. Variceal bleeding was confirmed by endoscopy. Patients screened for other platelet disorders not associated with liver complications and patients under any prescribed medication were excluded from the study. Patients were grouped as bleeders (n=27) and non-bleeders (n=23) as per Child-Pugh classification. The age range of the patients was 32 to 54 years with a mean of 46[+ or -]6.38 years. Age and sex-matched healthy volunteers (n=50) between 34 to 55 years with a mean of 48 [+ or -] 6.56 years with normal liver biochemistry were used as control subjects. The clinical characteristics of the study groups are presented in Table I.

The study protocol was ethically approved and the blood sample was collected with the consent of each patient.

Isolation of platelets

Platelets were isolated by the method of Aster and Jandl [22]. Ten millilitres of fasting blood was collected by venous arm puncture and mixed with 1.6 ml of acid-citrate-dextrose solution. The blood was centrifuged at 275g for 10 min at room temperature to obtain platelet rich plasma (PRP). The PRP was again centrifuged at 400g for 5 min to remove any contaminating red blood cell (RBC). Then the PRP was centrifuged at 1000g to pellet out the platelets and washed in platelet buffer I (0.12 M NaCl, 0.0129 M trisodium citrate and 0.03 M glucose) followed by washing in buffer II (0.154 M NaCl, 0.01 M Tris and 0.001 M EDTA, pH 7.4). The washing procedure was continued until the pellet was red cell- free. The platelet pellet was suspended in platelet storage buffer containing 0.109 M NaCl, 4.3 mM [K.sub.2]HP[O.sub.4], 16 mM [Na.sub.2]HP[O.sub.4], 8.3 mM Na[H.sub.2]P[O.sub.4] and 5.5 mM glucose, pH 7.5 and stored at 4[degrees]C. All the estimations were performed under sterile conditions within 5 h of sample collection.


Fura-2AM, serotonin, DNA, DNaseI, guanidine hydrochloride, digitonin and EGTA were purchased from Sigma-Aldrich Chemicals, Bangalore, India. ADP, ATP, HEPES and phenyl methyl sulfonyl fluoride (PMSF) were purchased from Sisco Research Laboratories, Mumbai, India. All other chemicals and reagents used were of analytical grade.

Assay of platelet cytosolic [Ca.sup.2+]

Platelet cytosolic [Ca.sup.2+] was measured with the fluorescent indicator Fura-2 by the method described by Pollock et al. [23]. 2[micro]M Fura-2 AM (Sigma-Aldrich Chemicals, Bangalore) was added to PRP and incubated for 45 min at 37[degrees]C and then centrifuged at 700 g for 20 min. The supernatant plasma was discarded and the platelet pellet was resuspended in HEPES buffer (140 mM NaCl, 5 mM KCl, 5mM K[H.sub.2]P[O.sub.2],1 mM Mg[Cl.sub.2], 5 mM glucose and 10 mM HEPES, pH 6.5) at 37[degrees]C. The platelet concentrate was repeatedly washed twice and centrifuged at 640 g for 20 min and finally the pellet was resuspended in HEPES buffer, pH 7.4 at a concentration of 1x[10.sup.4] cells/ml. Fluorescence intensities at 510nm emission were measured at 37[degrees]C using an ELGCO SL174 spectrofluorometer with the excitation wavelength of 340 and 380 nm. The cells were lysed with 50[micro]M digitonin followed by addition of 1mM Ca[Cl.sub.2] to obtain maximal fluorescence, [F.sub.max]. Then, the lysed cells were added with 10mM EGTA in 20mM Tris base and the pH of the lysed cells was adjusted to 8.5 to yield the [Ca.sup.2+] independent fluorescence of Fura-2, [F.sub.min]. [Ca.sup.2+] was then calculated according to the formula,

[Ca.sup.2+] = kd x (F - [F.sub.min])/ ([F.sub.max] - F)

Where F is the Fura-2 fluorescence of resting or stimulated platelets. [k.sub.d] was taken to be 224nM [24].

Assay of serotonin

Serotonin was determined by the spectrofluorometric method [25]. Briefly, the isolated platelets were suspended in 5ml of ice-cold saline to wash out the adhering blood proteins and centrifuged at 1000g for 20 min. The washing procedure was repeated twice and the pure platelet pellet obtained was finally suspended in 3.5ml of 0.02N HCl and agitated gently to lyse the platelets. One ml of the lysate was diluted with 2ml of water and the proteins were precipitated by the addition of 0.2ml of 10% ZnS[O.sub.4] and 0.1ml of 1N NaOH. To the supernatant, 0.3ml of 12N HCl was added and the fluorescence was measured in an ELGCO SL174 spectrofluorometer at 490 nm after excitation at 385 nm. The amount of serotonin was determined from the calibration curve obtained by using known concentrations of serotonin (Sigma-Aldrich Chemicals, Bangalore) as the standard.

Assay of adenine nucleotides

Adenine nucleotide content was measured by HPLC. The chromatographic separation was performed on a 250x4.60 mm column packed with 10-pm silica particles to which octadecyl groups has been bonded. The column was eluted with 50mM potassium phosphate buffer, pH 7.0. Samples were analyzed with isocratic elution at a flow rate of 1.2 ml/min. Twenty microliters of sample or a standard solution was manually injected using Hamilton syringes. Chromatographic peaks in experimental samples were detected by absorbance at 254 nm and identified by comparison to known standards.

Assay of PPi and inorganic phosphate (Pi)

The enzymatic method of determination of PPi content was based on the oxidation of NADH to NAD per mole of PPi consumed which was measured spectrophotometrically at 340nm [26]. The Pi content was measured spectrophotometrically at 620nm [27].

Preparation of platelets for secretion studies The platelet count in each group was adjusted to 2.5x[10.sup.9] cells/[micro]l. The platelets were activated with 2[micro]M collagen and the reaction was terminated at different time intervals (0, 3, 6, 9 min) by mixing the sample with one third volume of ice-cold 0.633M formaldehyde in 0.05M EDTA for serotonin and one-third volume of 0.15M NaCl for adenine nucleotides and 10% TCA for PPi and Pi determinations. The contents were kept on ice for up to 60 min and then centrifuged at 12,000g for 2 min at room temperature [28]. The extracellular fluid of the platelet suspension was used for the estimation of serotonin, adenine nucleotides, PPi and Pi as described previously.

Isolation of platelet cytoskeleton

Platelet cytoskeleton was obtained from the isolated platelets [29] with slight modifications. Briefly, washed platelets (1:1 in platelet storage buffer) was added with 10 vol of Triton X-100 solubilization buffer (1% Triton X-100, 40mM KCl, 10mM imidazole-chloride, 10mM EGTA, 2mM Na[N.sub.3], pH 7.0) and mixed by inversion. The flocculent precipitate was cooled on ice for 12 min and collected by centrifugation at 3000g for 2 min. The translucent platelet membrane pellet was carefully collected from the microfuge tube and used for the estimation of G- and total actin content. The isolated platelets after activation with 2[micro]M collagen were lysed with Triton X-100 solubilization buffer at different time intervals and processed similarly for the estimation of G-, F- and total actin content.

Assay of actin content

Measurement of unpolymerized (G- actin) and total actin was done by DNase inhibition assay [30] with slight modifications [7] to stabilize the inhibitory activity of the platelet lysate. Briefly, platelet suspension was lysed with equal volume of ice-cold Triton X-100 lysis buffer (2% Triton X-100, 10mM EGTA and 100mM Tris-HCl, pH 7.4). About 50-[micro]l aliquot was immediately removed, mixed with equal volume of dissociating buffer (1.5 M guanidine-HCl, 20 mM Tris-HC1, 1 M sodium acetate, 6 mM Ca[Cl.sub.2], 1 mM Na-ATP, pH 8.4), and incubated on ice for 15 min. The contents were then added with 20 [micro]l of DNase G (beef pancreas, DN 100: Sigma-Aldrich Chemicals, Bangalore) at a concentration of 0.2 mg/ml in 50 mM Tris-HC1, pH 7.5, 0.1 mM Ca[Cl.sub.2], 10 [micro]M PMSF. Sixty [micro]l of this mixture was removed and added to 3 ml of DNA (calf thymus, type I: Sigma-Aldrich Chemicals, Bangalore) at a concentration of 40 [micro]g/ ml in 100 mM Tris-HC1, 4 mM MgS[O.sub.4], 1.8 mM Ca[Cl.sub.2], pH 7.5. Total actin concentration was determined after treating the samples at 0[degrees]C with an equal volume of 1.5 M guanidine-HCl in 1.0 M sodium acetate/l mM Ca[Cl.sub.2]/1 mM ATP/20 mM Tris HCl, pH 7.5. The change in absorbance at 260 nm was recorded for 5 min.

Standard curves were generated in exactly the same way using purified rabbit skeletal muscle actin. The DNase inhibitor activity measured in the absence of guanidine- HCl represents the amount of G- actin in a sample and the activity after treating the sample with guanidine-HCl measures total actin. From the difference between the total actin and the G- actin content, F- actin content was determined. G- actin and F- actin content were expressed as % of total actin content.

Statistical analyses

The Data was analyzed by using commercially available statistic software package (SPSS for Windows V.12.0). ANOVA test was applied for statistical analysis. Spearman's rank correlation analysis was conducted for the correlation of paired values. Results were presented as mean [+ or -] SD. Statistical p-value <0.05 was used to establish significance.


Concentrations of serotonin, adenine nucleotides, cytosolic [Ca.sup.2+], PPi, Pi and G- actin content in unstimulated platelets. The levels of serotonin, adenine nucleotides, ATP and ADP, PPi, Pi, cytosolic [Ca.sup.2+] and cytoskeletal G-actin content of unstimulated whole platelets are presented in Table 2. There was no significant difference in the levels of adenine nucleotides, PPi and Pi between non-bleeders and normal subjects but a significant decrease in the levels of adenine nucleotides was observed in bleeders when compared with normal subjects. There was a significant decrease in the concentrations of serotonin and [Ca.sup.2+] between patients with bleeding complication and those without bleeding complication. G- actin content determined by DNAse I inhibition assay showed no significant changes in the level of actin contents in cirrhotic patients when compared to that of normal subjects.

Secretory response of platelets to collagen

To measure the secretory response of the platelets obtained from experimental subjects, the platelets were activated with 2[micro]M collagen and the level of secretion was measured in terms of release of serotonin, adenine nucleotides, PPi, and Pi (Figure 1). There was a significant decrease in the levels of serotonin, ADP, PPi and Pi secreted by platelets of both categories of cirrhosis patients when compared to those of normal subjects. ATP/ ADP ratio showed a significant elevation in cirrhosis patients when compared to normal subjects. The elevation was more significant in bleeders when compared to non-bleeders. The level of polymerized actin (F-actin) was found to be significantly low in cirrhotic bleeders when compared to that of normal subjects and non-bleeders (Figure 2).

Time-dependent secretory response of platelets to collagen

The response of platelets to agonist was measured in terms of serotonin and adenine nucleotides, ATP and ADP at different time intervals (Figure 3). The time-dependent secretory response of cirrhotic platelets to collagen showed that maximum secretion of serotonin and ADP was reached in normal platelets earlier than that of cirrhotic platelets. ATP secretion was found to be delayed in normal platelets whereas it showed a steady rise in platelets isolated from cirrhosis platelets (Figure 3). In platelets of cirrhosis patients, though there was a time-dependent raise in ADP and serotonin levels, the net secretion was lesser than those of normal subjects. But the amount of ATP secretion was higher than that of normal subjects.


Time-dependent changes in the level of cytosolic [Ca.sup.2+] and polymerization of actin during secretion

The changes in the cytosolic [Ca.sup.2+] were monitored at regular time intervals during the course of secretion (Figure 4). Gn normal platelets, there was a proportionate increase in the level of cytosolic [Ca.sup.2+] as the procoagulants release proceeded. There was a time dependent, but, low and delayed response shown by cirrhotic platelets. The level of increase was not significant in cirrhotic bleeders (Figure 4). Cirrhotic patients without bleeding complications showed elevation in the level of cytosolic [Ca.sup.2+] during the secretion process.

During collagen-induced secretion, the time course of actin polymerization was also monitored simultaneously (Figure 5). In cirrhotic cells the level of actin polymerization was significantly lower than that of normal platelets and reflected in the level of F-actin, the polymerized form.

Spearman correlation analyses showed that there was a positive correlation between BT-ATP/ADP ratio, [Ca.sup.2+]-serotonin and [Ca.sup.2+]-F-actin. A significant negative correlation was observed between the paired values of BT-[Ca.sup.2+] and BT--serotonin (Table 3).


Platelets play pivotal roles in the hemostatic process including detection of vascular lesions, adherance at sites of injury, recruitment of additional platelets and consolidation into a hemostatic plug [31]. Most of the cirrhosis patients considered in the study had low platelet count and altered bleeding time. Thrombocytopenia is generally associated with deficient platelet function and thereby the related abnormality [3]. In our study the defective secretory response observed in all the cirrhosis patients and more significantly in patients with bleeding complication could be correlated with the platelet count.



ADP, secreted from dense granules induces other platelets to degranulate and potentiate coagulation. Furthermore, ADP in conjunction with thromboxane [A.sub.2] and thrombin cause platelet contraction and the formation of a secondary hemostatic plug to bind fibrinogen which acts as a crosslink between the platelets allowing aggregation. Other molecules within platelet dense granules include ATP and ionized [Ca.sup.2+] which are necessary for several steps of the coagulation cascade. Serotonin plays an essential role as vasoconstrictor to minimize the loss of blood. Studies in patients with dense granule abnormality indicate that deficiency in serotonin, ATP, ADP and [Ca.sup.2+] are highly correlative and the alterations are interrelated [10].



We could find a significant alteration in the levels of serotonin and adenine nucleotides in resting platelets of cirrhotic bleeders. When the platelets were activated with collagen there was a significant change in the levels of secretion of serotonin, ADP and ATP in liver cirrhotic patients with bleeding complications when compared to those of non-bleeders and normal subjects. Our results show that both serotonin and nucleotide secretion is low in cirrhotic bleeders which can be correlated with hemostatic disturbances.

Serotonin is not synthesized in platelets but is actively taken up from the plasma and accumulates in dense granules where it is likely complexed with ATP and potentially with [Ca.sup.2+] [32]. The serotonin released by exocytosis is relatively stable and functions as a weak agonist on serotonin receptors (-5H[T.sub.2]-receptors) [33]. Dense granule derived serotonin, therefore acts to activate additional platelets and thus recruits them into aggregates [31]. The positive feedback effect of serotonin may be of secondary importance to its vasoconstrictive action which reduces the blood flow at the site of injury and thereby limits blood loss [32].

In normal platelets, the level of ADP secretion is significantly greater than that of ATP. In cirrhotic subjects, ATP secretion was found to be elevated when compared to that of ADP. ATP competitively inhibits ADP action [34] and thus regulates platelet activation. Elevated ATP secretion has been reported in various inflammatory and shock conditions, primarily as a consequence of nucleotide release from platelets, endothelium and blood vessel wall [35-37]. In this investigation we have shown that in cirrhotic condition, platelets secrete elevated level of ATP, which antagonize the action of ADP, and this might contribute to the platelet activation defect in liver cirrhosis. Our observation also showed that the secretory response of platelets to agonists is delayed in liver cirrhosis. It can be correlated with similar activation of platelets in vivo by the endogenous collagen exposed on the ruptured vascular walls.

Our results show that the activation of platelets by agonists was associated with parallel elevation in the level of [Ca.sup.2+] in normal platelets. The level of elevation was not significant in the platelets of cirrhosis patients. So, the altered secretory capacity of cirrhotic platelets may be accounted by the low availability of [Ca.sup.2+] which functions in a diverse manner to regulate the secretory process. In our previous study, we have reported that the enzyme activity is significantly low in cirrhotic platelets that cannot affect [Ca.sup.2+] efflux required for activation [38].

The role of cytosolic [Ca.sup.2+] in platelets has been studied in various pathophysiological conditions. It has been found that micromolar levels of [Ca.sup.2+] ions added to the extracellular medium elicit secretion of serotonin in the presence of millimolar levels of Mg-ATP. [Ca.sup.2+] transport inhibitors were shown to interrupt with the secretion of serotonin by platelets [39]. The state of assembly of the platelet cytoskeleton appears to be under the control of intracellular concentration of free [Ca.sup.2+]. The ability of [Ca.sup.2+] to control the assembly and disassembly of the platelet cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation [40]. Secretory granules bound to the end of an actin filament can move to the plasma membrane as a result of the contraction of acto-myosin that is activated by [Ca.sup.2+] ions [41]. In this investigation we have studied the variation in the level of calcium release between bleeders and non-bleeders. When compared to non-bleeders, bleeders have shown a slow release of [Ca.sup.2+] from the subcellular organelles to cytoplasm that might affect secretory action of platelets.

It is necessary for the platelet to possess an actin cytoskeleton that can be rapidly restructured upon activation [42]. It has also been reported that an actin-binding protein, inhibited from interacting with actin by a rise in [Ca.sup.2+] may be a factor that permits the rapid organization of the platelet actin cytoskeleton. Few investigators have suggested that actomyosin-dependent granule centralization and membrane fusion act synergistically to facilitate granule secretion [43-45]. It has been demonstrated that agonist induced activation results in conversion of unpolymerised, monomeric, globular G- actin to filamentous F- actin [46]. The finding of the present study reveal that normal unstimulated platelets exhibit 80% of their total actin in the monomeric state but the cirrhotic platelets showed a non-significant decrease in the total and G- actin contents. When the cirrhotic platelets were stimulated, F- actin formation was significantly lower than that of normal platelets. The low level of formation of polymerized F- actin in the platelets of liver cirrhotic patients as observed in our investigation may be associated with the decreased granule secretion in cirrhotic bleeders. However, the exact role of actin polymerization in platelet granule secretion remains to be elucidated in liver cirrhosis. A study on the structural and morphological changes of cirrhotic platelets on activation has been undertaken in our laboratory to give supportive evidence for the present work.

The statistical correlation between bleeding time and the various parameters analysed reveal that bleeding time, a reliable indicator of platelet functions is negatively correlated with the secretory response evidenced by the levels of nucleotide and serotonin secretion. BT was also found to have significant positive correlation with the level of ATP, an inhibitor of platelet secretion. Cytosolic [Ca.sup.2+] level is found to have a parallel correlation with the levels of serotonin secretion and F- actin formation. This study reveals that actin polymerization is also defective in the platelets of patients with cirrhosis and this may be responsible for the low secretory response to agonists.


It is concluded that the secretion of hemostatic elements by the platelets in response to the agonist collagen is defective in liver cirrhosis. Low [Ca.sup.2+] release from the internal store and impaired actin polymerization seem to alter the normal secretory capacity of platelets in liver cirrhosis associated with bleeding complication.


The authors (SA and AG) thank University Grants Commission, New Delhi, India for the financial assistance.

Received: September 3, 2008 Accepted: March 30, 2009

Gelis tarihi: 03 Eylul 2008 Kabul tarihi: 30 Mart 2009


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Sam Annie-Jeyachristy (1), Arumugam Geetha (1), Rajagopal Surendran (2), Subburayan Jeevan Kumar (3), Sarangapani Arulprakash (3)

(1) Department of Biochemistry, Bharathi Women's College (Affliated to University of Madras), Chennai, Tamil Nadu, India

(2) Department of Surgical Gastroenterology and Proctology, Stanley Medical College and Hospital, Chennai, Tamil Nadu, India

(3) Department of Digestive Health Diseases, Government Peripheral Hospital, Chennai, Tamil Nadu, India

Address for Correspondence: Arumugam Geetha, Department of Biochemistry, Bharathi Women's College (Affliated to University of Madras), Chennai, 600 108, Tamil Nadu, India Phone: 044 25286411 E-mail:
Table 1. Clinical characteristics of subjects included in this study

Clinical findings No. of cirrhotic No. of normal
 patients subjects

Total number 50 50
Male / female ratio 30/20 35/15
Age in years (range) 32-54 34-55
Grade (according to Child - Pugh class)
 A 13 -
 B 12 -
 C 25 -
Ascitic 35 -
Non-ascitic 15
Hepatic encephalophathy 6 -
Platelet count
 <100x[10.sup.9] cells/L 27 -
 >100x[10.sup.9] cells/L 12 -
 >250x[10.sup.9] cells/L 11 50
Prothrombin time
 <4 seconds 12 50
 4-6 seconds 11 -
 >6 seconds 27 -
Bleeding time
 Normal (60-90 s) 11 50
 Abnormal (90-125 s) 39 -

Table 2. Adenine nucleotides, serotonin, calcium, pyrophosphate,
inorganic phosphate and membrane G -actin content if the unstimulated
platelets of cirrhotic patients and in normal healthy volunteers.
(mean [+ or -] SD)

 Cirrhotic patients

Parameters Normal subjects Non-bleeders
 (n=50) (n=23)

Cytosolic [Ca.sup.2+] 28 [+ or -] 3.0 24 [+ or -] 2.8 *
 (nM/[10.sup.4] cells)
Serotonin (nM/mg protein) 2.0 [+ or -] 0.41 1.8 [+ or -] 0.21 *
ATP (nM/mg protein) 20.5 [+ or -] 3.1 19.5 [+ or -] 2.0NS
ADP (nM/mg protein) 12.5 [+ or -] 1.9 11.5 [+ or -] 1.8NS
ATP/ADP ratio 1.64 [+ or -] 0.18 1.70 [+ or -] 0.19NS
PPi (nM/mg protein) 6.0 [+ or -] 0.08 5.7 [+ or -] 0.08NS
Pi (nM/mg protein) 12.6 [+ or -] 2.1 12.0 [+ or -] 2.0NS
G- actin content 80 [+ or -] 8.2 75 [+ or -] 7.7NS
 (% Total actin)

 Cirrhotic patients (n=50)

Parameters Bleeders (n=27)

Cytosolic [Ca.sup.2+] 17 [+ or -] 2.0 **#
 (nM/[10.sup.4] cells)
Serotonin (nM/mg protein) 1.7 [+ or -] 0.25 *#
ATP (nM/mg protein) 17.6 [+ or -] 1.9 *#
ADP (nM/mg protein) 10.0 [+ or -] 1.6 *#
ATP/ADP ratio 1.76 [+ or -] 0.19NS #
PPi (nM/mg protein) 5.6 [+ or -] 0.08NS
Pi (nM/mg protein) 11.7 [+ or -] 2.0NS
G- actin content 70 [+ or -] 7.3NS
 (% Total actin)

** p < 0.001, * p < 0.05, NS - non-significant, when compared with
normal subjects

## p < 0.001, # p < 0.05, NS-non-significant, when compared with

[Ca.sup.2+], calcium; ATP, adenosine triphosphate; ADP, adenosine di
phosphate; PPi, pyrophosphate; Pi, inorganic phosphate;
G-actin, Globular actin;

Table 3. Spearman rank correlation coefficient between
BT serotonin, BT - ATP/ADP ratio, BT - [Ca.sup.2+], [Ca.sup.2+]
- serotonin and [Ca.sup.2+] - F-actin

Variables [r.sub.s]

BT - serotonin -0.50
BT - ATP/ADP ratio 0.52
BT - [Ca.sup.2+] -0.55
[Ca.sup.2+] - serotonin 0.48
[Ca.sup.2+] - F- actin 0.57

All correlation coefficient (Spearman rho-rs) values were significant
at P <0.05

BT, bleeding time; ATP, adenosine tri phosphate; ADP, adenosine di
phosphate; [Ca.sup.2+], calcium; F-actin, filamentous actin
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Article Details
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Title Annotation:Research Article
Author:Annie-Jeyachristy, Sam; Geetha, Arumugam; Surendran, Rajagopal; Kumar, Subburayan Jeevan; Arulprakas
Publication:Turkish Journal of Hematology
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2009
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