Jacek Borawski

A STUDY OF PLATELET FUNCTIONS, SOME HEMOSTATIC AND FIBRINOLYTIC PARAMETERS IN RELATION TO SEROTONIN IN HEMODIALYZED PATIENTS UNDER ERYTHROPOIETIN THERAPY

Erythropoietin corrects anemia and improves hemostasis, but on the other hand bears a risk of thrombotic complications. Therefore in the present study an attempt has been made to evaluate bleeding time, platelet functions and some hemostatic and fibrinolytic parameters in relation to blood and platelet serotonin before and after 1, 2, 4, 8 and 12 weeks of treatment. 22 chronically hemodialyzed patients were administered with human recombinant erythropoietin (rHuEPO) in a dose of 2000 IU s.c. 3 times a week. Bleeding time was shortened significantly as early as after 1 week of the therapy, whereas hematocrit and hemoglobin increased after 2 weeks. These changes lasted throughout the study. Only a transient rise in platelet count, collagen-induced platelet aggregation, beta-thromboglobulin and VIII:C activity were observed during therapy relative to baseline values. ADP- and arachidonic acid-induced platelet aggregation seemed to be unaffected by rHuEPO treatment, whereas a gradual and progressive enhancement in platelet aggregation in response to ristocetin was found, starting from the 2nd week of the therapy. It lasted throughout the study and correlated inversely with the bleeding time and positively with a rise in both blood and platelet serotonin. rHuEPO did not alter plasminogen, fibrinogen, platelet factor 4, alpha 2 macroglobulin levels, protein C activity and euglobulin clot lysis time. A decline in protein C and S concentrations and antithrombin III activity observed during the therapy were counterbalanced by a fall in the activity of alpha 2 antiplasmin, C1 esterase inhibitor and plasminogen activator inhibitor. It is concluded that rHuEPO may improve platelet/vessel wall interactions possibly by means of serotonergic mechanisms. A lowered activity of inhibitors of fibrinolysis may be regarded as a protection against a general tendency to thrombosis during rHuEPO therapy.

The Hematocrit-Corrected Erythrocyte Sedimentation Rate Can Be Useful in Diagnosing Inflammation in Hemodialysis Patients

Background/Aims: We aimed to determine predictors of erythrocyte sedimentation rate (ESR), and the ESR level pointing to the presence of inflammation in 60 chronic hemodialysis (HD) patients. Methods/Results: On bivariate analysis, increased Westergren ESR of 62 (4–160) mm/h correlated inversely with hematocrit (Hct) and serum albumin, and positively with age, plasma fibrinogen, serum C-reactive protein (CRP), immunoglobulins A and G, α1-acid-glycoprotein and α1-antitrypsin. On multivariable analysis, independent predictors of the ESR were raised CRP (p < 0.0001), low Hct (p < 0.0001), increased fibrinogen (p < 0.0001) and immunoglobulin A (p = 0.009), and older age (p = 0.015). The Hct-corrected ESR level [ESR × (Hct/45)] of 38 (4–91) mm/h was independently predicted by CRP (p < 0.0001), fibrinogen (p < 0.0001), and age (p = 0.001). In the patients with normal CRP and albumin, the Hct-corrected ESR value was normal (23 mm/h) and lower than that of 59 mm/h in the subjects with elevated CRP and hypoalbuminemia. Using these cut-off points, the positive and negative predictive values of the Hct-corrected ESR on the presence of inflammation were 1.0, and its sensitivity and specificity were 100%. Conclusion: Increased Westergren ESR in HD patients is associated with activated acute-phase response, anemia, and aging. The Hct-corrected ESR values of 23 and 59 mm/h precisely select the HD patients with severe inflammation from those without.

Relationship between oxidative stress and extrinsic coagulation pathway in haemodialyzed patients

Enhanced oxidative stress (SOX), endothelial dysfunction and haemostatic abnormalities are common in end-stage renal failure patients undergoing maintenance haemodialysis (HD). We studied associations among circulating immunoreactive total lipid peroxides as a marker of short-time SOX, autoantibodies against oxidized LDL as a surrogate of prolonged SOX, copper/zinc superoxide dismutase (Cu/Zn SOD) as a major antioxidant enzyme, tissue factor (TF) as a principal initiator of extrinsic coagulation pathway counteracted by its inhibitor (TFPI), and prothrombin fragment 1+2 (F 1+2) as a surrogate of activated haemostasis.Pre-dialysis blood levels of all the markers studied were higher in 24 clinically stable HD patients compared to 11 healthy controls. Spearmans correlations among the three SOX markers were positive but nonsignificant in both HD patients and controls. In HD subjects, increased Cu/Zn SOD levels directly correlated with those of TF (rho=0.551, p=0.005) and TFPI (rho=0.501, p=0.001); the coagulation markers were also positively associated with each other (rho=0.663, p=0.0004). In healthy subjects, the relations between Cu/Zn SOD, TF and TFPI levels were inverse but not significant, and the direct association between TF and TFPI was nonsignificant either. In conclusion, increased plasma levels of Cu/Zn SOD, the antioxidant enzyme with emerging endothelial cell-protective and antithrombotic properties, may be a novel part of the system counteracting activated extrinsic coagulation system in maintenance HD patients.

Tissue Factor and Thrombomodulin in Hemodialysis Patients: Associations with Endothelial Injury, Liver Disease, and Erythropoietin Therapy

Patients receiving maintenance hemodialysis (HD) present with hemostatic abnormalities, which may be aggravated by comorbid conditions, especially liver disease. The factors that influence plasma levels of thrombomodulin (TM), an initiator of the anticoagulant protein C pathway, and those of tissue factor (TF), which triggers the extrinsic coagulation pathway, were assessed. In 63 HD patients, TM and TF levels were higher than those in healthy controls. In bivariate analysis, TF positively correlated with TM, and both were directly associated with the presence of viral hepatitis B or C marker, serum liver enzymes, use of erythropoietin therapy, hemoglobin levels, and duration of HD therapy, and inversely correlated with body mass index. TF was also positively associated with plasma von Willebrand factor (vWF) antigen, and inversely associated with activated partial thromboplastin time. In multivariate analysis, increased vWF, alanine aminotransferase, and use of erythropoietin independently predicted both TF and TM levels. HD patients with vWF and ALT levels lower than middle, and not treated with erythropoietin had normal TF but increased TM concentrations compared with levels in healthy controls. Increased plasma levels of TM and TF in patients on maintenance HD are surrogates of vascular endothelial injury. Liver disease and use of erythropoietin treatment are also important determinants of these markers, and should be considered in further studies.

Inflammatory markers and platelet aggregation tests as predictors of hemoglobin and endogenous erythropoietin levels in hemodialysis patients.

Background/Aims: Chronic inflammation is a common cause of severe anemia and hyporesponsiveness to recombinant erythropoietin (EPO) therapy in maintenance hemodialysis (HD) patients. We compared various acute-phase markers and ex vivo platelet aggregation tests in relation to clinical conditions in order to find factors predictive of hemoglobin (Hb) and endogenous EPO levels in a cross-section of clinically stable HD patients. Methods: In 100 subjects, pre-HD blood levels of C-reactive protein (CRP), α1-acid-glycoprotein (AGP), α1-antitrypsin (AT), immunoglobulin (Ig) M and G (by nephelometry), antigens of endothelial von Willebrand factor (vWF), type 1 plasminogen activator inhibitor and thrombomodulin, interleukin-6, lipoprotein(a) [Lp(a)] and EPO (by ELISA), and albumin, fibrinogen, iron metabolism indices, thyroid-stimulating hormone, phosphorus, parathormone, total cholesterol, triglycerides, viral hepatitis B/C markers, liver enzyme, and aluminium were determined. Platelet aggregations in response to ristocetin (RIPA), adenosine diphosphate, and collagen were measured in whole blood (electric impedance method) and platelet-rich plasma (optical aggregometry). Results: Hb levels inversely correlated with IgM, Lp(a), soluble vWF antigen, phosphorus, and all platelet aggregations in whole blood, but not in platelet-rich plasma. HD duration and triglycerides were positive correlates of anemia. In a multivariable analysis, increased IgM, short HD duration, increased Lp(a) and enhanced whole blood RIPA (in descending order of significance) were independent predictors of low Hb levels. In 51 patients not treated with recombinant EPO, serum levels of this hormone inversely correlated with whole blood RIPA, AT, age, vWF antigen, AGP, and positively with viral hepatitis marker. Anemia and EPO levels were not affected by gender, body mass index, cause of renal failure, residual renal function, HD dose, protein catabolic rate, use of different heparins or dialysate buffers, ACE inhibitor therapy, and parathyroid or thyroid function. In additional 10 patients, single HD session resulted in an increase in IgM levels associated with a fall in total lymphocyte counts. Conclusion: Subclinical inflammation is an important determinant of anemia in maintenance HD patients. Increased serum IgM reflecting a microinflammatory effect of HD procedures, enhanced whole blood RIPA as a surrogate of vascular endothelial damage, and Lp(a) as its promoter could be markers of such impaired erythropoiesis.

Effects of Recombinant Erythropoietin Therapy on Circulating Endothelial Markers in Hemodialysis Patients

Effects of a 4-week course of recombinant human erythropoietin (rHuEpo) therapy on four circulating endothelium-derived cardiovascular risk markers were studied in 20 patients receiving maintenance hemodialysis in relation to surrogates of chronic inflammation, liver function, and arterial blood pressure. Soluble intercellular adhesion molecule-1 (sICAM-1), antigens of plasminogen activator inhibitor-i (PAI-i:Ag) and von Willebrand factor (vWF:Ag), and soluble thrombomodulin (sTM) were determined by immunoenzymatic assays. C-reactive protein; (xi acid-glycoprotein; ax-antitrypsin; immunoglobulin M, A, and G; interleukin-6; lipoprotein(a); fibrinogen; total protein; albumin; total cholesterol; hepatitis B and C markers; liver enzymes; prothrombin time; and phosphorus were measured by routine methods. The rHuEpo treatment resulted in a 25% increase in sICAM-1 (Wilcoxons p = 0.001), a 50% increase in PAI-i:Ag (p = 0.004), a 15% increase in sTM (p = 0.002), and did not change vWF:Ag levels. The increase in sICAM-1 concentration directly correlated with that of PAI-i:Ag (Spearmans rho = 0.483, p = 0.031). The rHuEpo-induced increases in hemoglobin, platelets, and pre-dialysis diastolic blood pressure levels did not correlate with the increments in the endothelial markers studied. In conclusion, short-term rHuEpo therapy activates vascular endothelium in patients receiving maintenance hemodialysis. This specific effect may influence cardiovascular risk.

Chronic viral hepatitis C, oxidative stress and the coagulation/fibrinolysis system in haemodialysis patients

INTRODUCTION The aim of the present study was to establish whether the presence of chronic viral hepatitis (PVH) could be implicated in the elevation of oxidative stress (SOX) and haemostasis system in haemodialysis (HD) patients. MATERIALS AND METHODS In HD patients with and without PVH and in controls we compared the markers of: coagulation pathway- tissue factor (TF) and its inhibitor (TFPI), prothrombin fragment F (1+2) (F (1+2)); fibrinolysis: tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its soluble receptor (suPAR), plasminogen activator inhibitor 1 (PAI-1), plasmin/antiplasmin complexes (PAP); and a marker of SOX-Cu/Zn superoxide dismutase (Cu/Zn SOD) levels. RESULTS Patients, particularly those with PVH, showed a significant increase in the markers of the coagulation, fibrinolysis and oxidative status as compared to controls. All parameters of coagulation/fibrinolysis system were directly associated with the PVH and Cu/Zn SOD levels, and there was a relationship between the PVH and Cu/Zn SOD levels. Multivariable analysis showed that PVH and increased SOX were identified as independent variables significantly associated with the disturbances of coagulation/fibrinolysis system in these patients. CONCLUSIONS We concluded that PVH is a novel determinant of the increased oxidative stress as well as the disturbances of coagulation/fibrinolysis system in haemodialysis patients.

Serum α1-Antitrypsin But Not Complement C3 and C4 Predicts Chronic Inflammation in Hemodialysis Patients

Background. We studied whether predialysis serum levels of positive acute phase markers such as α1-antitrypsin (AT), and complement components C3 and C4 could identify the presence of chronic inflammation in maintenance hemodialysis (HD) patients. Methods/Results. In 103 stable HD patients, AT directly correlated with C-reactive protein (CRP) (P<0.005), α1 acid-glycoprotein (P<0.005), fibrinogen (P<0.05), lipoprotein (a) (P<0.01) and von Willebrand factor antigen (P<0.05), while C3 and C4 were not related to any of these inflammatory markers. In the patients with elevated CRP and hypoalbuminemia, the mean AT value of 1.74 ± 0.50 g/L was higher (P = 0.008) than that of 1.38 ± 0.27 g/L in the subjects with normal CRP and albumin. Using the above cut-off levels, the positive and negative predictive values of AT on the presence of severe inflammation were 0.86 and 0.62, respectively, and the sensitivity and specificity were 86% and 73%, respectively. Conclusion. Serum AT levels above 1.74 g/L and below 1.38 g/L may select the HD patients with severe inflammation from those without. Measurements of C3 and C4 are not helpful in this situation.

RELATIONS BETWEEN OXIDATIVE STRESS, HEPATOCYTE GROWTH FACTOR, AND LIVER DISEASE IN HEMODIALYSIS PATIENTS

Background: Hepatocyte growth factor (HGF) and copper/zinc superoxide dismutase (Cu/Zn SOD) protect against tissue injury, including that due to oxidative stress (SOX). We studied whether they could be associated with each other, SOX markers, prevalence of viral hepatitis, and cardiovascular disease (CVD) and their laboratory surrogates in maintenance hemodialysis (HD) patients. Methods: In 24 patients, pre-dialysis serum HGF, plasma Cu/Zn SOD, total lipid peroxides, and serum autoantibodies against oxidized LDL were measured by ELISAs. Viral hepatitis B and C markers were determined by third generation microparticle ELISAs, and CVD was identified on a clinical basis. Results: In HD patients, circulating HGF, Cu/Zn SOD, and the other SOX markers were higher than in healthy controls, and HGF directly correlated with Cu/Zn SOD levels (P = 0.0006). Both HGF (P = 0.021) and Cu/Zn SOD (P = 0.017) were positively associated with prevalence of viral hepatitis and serum alanine aminotransferase activity (P = 0.021 and P = 0.040, respectively). Presence of CVD directly correlated with HGF (P = 0.001) but not with Cu/Zn SOD levels (P = 0.087). Circulating HGF positively related to serum C-reactive protein (P = 0.043). In patients without viral hepatitis and CVD, both HGF and Cu/Zn SOD were lower than in those with, and higher than in healthy controls. CVD (P = 0.003) and viral hepatitis (P = 0.024) were independent predictors of increased HGF, while positive viral hepatitis marker predicted increased Cu/Zn SOD levels (P = 0.019) in HD patients. There were no associations between HGF and the SOX markers in controls. Conclusions: In maintenance HD patients, circulating Cu/Zn SOD and HGF levels are increased, likely as a part of the reparatory reaction against liver damage. Viral hepatitis status and liver function should be considered in further studies of Cu/Zn SOD in these subjects.

Effect of Hemodialysis on Plasma Levels of Vascular Endothelial Markers

Vascular endothelial cell dysfunction is linked to hemostatic abnormalities and accelerated atherosclerosis in patients receiving maintenance hemodialysis (HD). The relationships between pre-dialysis plasma levels of immunoreactive thrombomodulin, von Willebrand factor, tissue factor and its inhibitor were studied, and the effects of HD procedure on these endothelial markers were observed. All the markers were higher in 39 HD patients than in 15 healthy controls (p<0.0001). HD treatment resulted in a 50% increase in tissue factor pathway inhibitor (p<0.0001), but did not influence the other markers. This increment directly correlated with the post-dialysis decrease in diastolic (p=0.011) and mean arterial blood pressure (p=0.039), and the surface area of the dialysis membrane (p=0.007). There were no associations between the increase in tissue factor pathway inhibitor and the amount of fluid removed, dose of enoxaparin, or other HD-specific factors. In conclusion, HD is responsible for an increase in plasma tissue pathway factor inhibitor level. The release of tissue factor pathway inhibitor during HD is not only due to heparin injection but also to the contact between blood and artificial dialysis membrane, and to HD-activated hemodynamic forces.