Search

Background: Increased plasma soluble von Willebrand factor antigen (vWF : Ag) level, a marker of vascular endothelial cell dysfunction, is a strong predictor of atherosclerotic cardiovascular disease (CVD) in the general population. We studied cross-sectional associations between vWF : Ag level, prevalence of CVD, and related factors including pre-dialysis arterial blood pressure (BP) and some markers of inflammation in maintenance haemodialysis (HD) patients. Methods and results. Plasma vWF : Ag level measured by an enzyme-linked immunosorbent assay (ELISA) was higher in 110 HD patients than in 20 controls. On bivariate regression analysis, vWF : Ag level was directly associated with the presence of CVD, age, fibrinogen and the use of enoxaparin (vs unfractionated heparin) during HD procedures, and inversely with albumin and pre-dialysis BP. The patients with prevalent CVD were older, had higher vWF : Ag, white blood cell and platelet counts, fibrinogen and triglycerides, lower albumin levels, and were less frequently on combination antihypertensive therapy. Multivariable analyses identified low pre-dialysis BP, hypoalbuminaemia and hyperfibrinogenaemia (in descending order of significance) as independent predictors of high vWF : Ag level. There were no associations between vWF : Ag levels and gender, ABO blood type, smoking, body mass index, renal failure cause, duration of HD therapy, K(t)/V, normalized protein catabolic rate, dialysate buffers, dialysers, viral hepatitis, erythropoietin treatment, specific antihypertensive drugs, haemoglobin, white blood cell and platelet counts, liver enzymes, phosphorous, total cholesterol, and triglycerides., Conclusion: Elevated plasma levels of endothelial dysfunction marker vWF : Ag in maintenance HD patients are associated with established cardiovascular mortality risk factors such as low pre-dialysis blood pressure and the activated acute phase response.

Background: The level of soluble thrombomodulin (sTM), a traditional marker of endothelial injury, is also dependent on renal excretory function. We studied serum sTM in chronic haemodialysis (HD) patients to determine which factors are predictive of its levels in this population., Methods and Results: sTM levels of 10.7 (5.72-30.7) ng/ml in 100 HD patients were higher than in 30 controls (P<0.0001). In a bivariate regression analysis, immunoreactive sTM was positively associated with the presence of hepatitis B virus surface antigen and/or anti-hepatitis C virus antibodies measured by third generation ELISAs (P<0.0001), and was related to certain markers of liver injury and biosynthetic dysfunction. sTM was also directly associated with time on dialysis (P=0.001), or use of unfractionated heparin (UFH) (vs enoxaparin) (P=0.0007), erythropoietin (P=0.008), ACE-inhibitors (P=0.034), acetate-buffered dialysate (vs bicarbonate) (P=0.040), pre-dialysis systolic (P=0.012), and diastolic blood pressure (P=0.043). It was negatively associated with lipoprotein(a) (P=0.029). sTM was not related to age, sex, smoking, cause of renal failure, prevalence of cardiovascular disease, amount of HD delivered, preserved residual renal function, ferritin, C-reactive protein, and other vasoactive medications used. In a multivariable analysis, a positive hepatitis marker (P=0.0002), the use of UFH (P=0.030) and erythropoietin (P=0.019), and raised pre-dialysis blood pressure (P=0.024) were positive independent predictors of high sTM level., Conclusion: These data indicate that, in addition to endothelial activation, elevated sTM levels in HD patients may be related to viral infection and/or liver dysfunction, and influenced by modifiable factors such as increased blood pressure, and the type of heparin and erythropoietin treatment used.

In chronic renal failure disturbances of hemostasis are predominantly due to the defective platelet function and platelet/vessel wall interactions. Erythropoietin, used for treatment of renal anemia, affect hemostasis in dialyzed patients. The aim of the work was to assess platelet aggregation and P-selectin concentration in patients on continuous ambulatory peritoneal dialysis (CAPD) during erythropoietin therapy. The studied were performed on 12 CAPD patients, administered with subcutaneous erythropoietin in a dose of 2000 U three times a week for 3 months. Hemoglobin, hematocrit and erythrocyte count increased significantly after 1 month of the treatment. Platelet aggregation in platelet-rich plasma induced by collagen, ristocetin and serotonin increased significantly after 3 months of erythropoietin therapy when compared to the baseline values. Whole blood platelet aggregation and P-selectin concentration did not change significantly during 3 months of erythropoietin therapy. Erythropoietin treatment in CAPD patients did not affect significantly platelet aggregation and activation.

In patients that developed a chronic renal failure the augmentation in tryptophan degradation is reflected in the increase in plasma metabolites of kynurenine pathway. Hemodialysis is one of therapeutic approaches that significantly reduce all plasma kynurenine metabolites in uremic patients. In spite of haemodialysis, plasma concentration of kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, xanthurenic acid and quinolinic acid were still elevated in uremic patients in comparison with healthy volunteers. These data shows significant disturbances in kynurenine metabolism in uremic patients. Accumulation of these substances in uremic blood is capable to account for certain uremic symptoms.

Von Willebrand factor (vWF) abnormalities are involved in the hemostatic disturbances of uremia. Studies of vWF in both anemic and recombinant erythropoietin (EPO)-treated maintenance hemodialysis (HD) patients have given inconsistent results that could be partially dependent on the methodology. Therefore, the reciprocal relationships between four different vWF assays were studied in HD patients in relation to EPO therapy. Plasma vWF activity measured by ELISA using monoclonal antibodies against platelet glycoprotein (GP) Ibalpha (vWF:Act), plasma vWF antigen concentration by ELISA employing polyclonal antihuman vWF antibodies (vWF:Ag), and functional vWF activity by ristocetin-induced platelet aggregation (RIPA) in both platelet-rich plasma (PRP) and whole blood were studied in 70 HD patients. In the whole group, no correlations between these assays were found. In the non-EPO patients (n = 32), vWF:Act was correlated with vWF:Ag (r = 0.504, p = 0.003) but not with vWF activity by RIPA in PRP. In the EPO-treated patients (n = 38), vWF:Act was higher than in the untreated ones (p = 0.001), and vWF:Ag was related to the whole-blood RIPA (r = 0.386, p = 0.016). In conclusion, the platelet GP Ibalpha-binding epitope of the vWF molecule assessed by vWF:Act ELISA is intact in HD patients. However, this assay does not reflect the functional vWF activity as measured by RIPA in PRP. In EPO-treated patients, the GP Ibalpha-binding domains of vWF are more available but the plasmatic vWF monomers are neither more numerous by vWF:Ag ELISA nor more hemostatically active by RIPA PRP. The whole blood RIPA test has some potential for the quantification of vWF monomers in EPO-treated HD patients., (Copyright 2001 S. Karger AG, Basel)

So far it is not clear how erythropoietin affects the anticoagulant properties of vascular endothelium in uremia. Since serotonin is also thought to play a role in the pathogenesis of thrombosis, the aim of the study was to evaluate major components of extrinsic coagulation pathway, markers of endothelial cell injury, lipoprotein (a) and peripheral serotonergic mechanisms during rHuEPO therapy in hemodialyzed patients. The study was performed on chronically hemodialyzed patients divided into two groups: with rHuEPO treatment and without rHuEPO therapy in relation to the control group. In uremic patients, thrombomodulin and von Willebrand factor, activity of factor VII, tissue factor pathway inhibitor (TFPI) activity, TFPI and tissue factor (TF) concentrations, lipoprotein (a) level were significantly higher when compared to healthy volunteers. Treatment with rHuEPO resulted in a further significant rise in markers of endothelial cell injury: thrombomodulin and von Willebrand factor and TFPI concentration. Extrinsic coagulation factors: activities of factor VII and X, TFPI activity and TF activity and concentration, lipoprotein (a) and vitronectin remained unchanged during rHuEPO therapy. Platelet serotonin content and whole blood serotonin were significantly lower in uremic patients relative to healthy volunteers and during rHuEPO treatment they increased significantly. Whole blood serotonin reached normal values. Plasma serotonin, significantly elevated in uremia, did not change during rHuEPO therapy. Serotonin uptake by uremic platelets was significantly impaired and remained unaltered during rHuEPO administration. Serotonin release by uremic platelets was also significantly depressed but a significant improvement was observed in rHuEPO-treated patients. Our data suggest that endothelial injury, TF pathway components and peripheral serotonergic system disturbances may predispose to thromboembolic complications and play a role in the pathogenesis of atherosclerosis in uremic patients, particularly treated with rHuEPO. Increase in TFPI may compensate the increase in TF in these patients., (Copyright 2000 S. Karger AG, Basel)

The aim of the study was to follow effects of multiple dialyzer reuse on some coagulation parameters. In a random group of 10 patients undergoing intermittent hemodialysis treatment, platelet coagulant, fibrinogen, BTG and PF4 concentrations were determined after 1st dialyzer usage and after 2 consecutive dialysis sessions after which the dialyzer was reused by manual method with 1% Dialyne solution. An increase in fibrinogen concentration after 240 min of HD procedure was shown when dialyzer was used for 1st and 3rd time. BTG activity increased after 240 min of HI procedure when the dialyzer was used for 3rd time. There was a fall in platelet count after 30 min of HD during the 1st and the 3rd dialyzer use. The fall in platelet count lasted all dialysis session when dialyser was used for 3rd time. The PF4 activity was decreased after 30 min of HD session after the 1st and the 2nd dialyzer use.

Coagulation parameters--platelet aggregation, beta-thromboglobulin, fibrinopeptide A were analyzes in patients with final chronic renal insufficiency during haemodialysis after a low molecular weight heparin application. In the study was documented that standard heparin is able to induce platelet aggregation. A low molecular weight heparin is suggested as sefex, because no influence on platelet aggregation was determined.

Dermatan sulphates have been shown to inhibit thrombus formation and thrombus growth in different experimental model of venous thrombosis. At variance with heparins, they show a remarkably low haemorrhagic potential. On the other hand, very few data are available on the effect of dermatan sulphates on arterial thrombus formation. We evaluated the effects of a low molecular weight (LMW)-dermatan sulphate, a high molecular weight (HMW)-dermatan sulphate and sulodexide (a mixture of fast-moving heparin fraction and dermatan sulphate) in comparison with LMW- and HMW-heparin, in a model of arterial thrombosis in rats. The insertion of an artificial prosthesis into the abdominal aorta of the animals induced the formation of an occluding thrombus within 2-5 days. The time in which 50% of the loops became occluded (OT50) was also calculated and used to compare the efficacy of the different drug treatments. This was 2.84 days for control animals and 4.25 and 5.80 days for HMW- and LMW-dermatan sulphate, respectively. Neither drug changed the "template" bleeding time, even at higher doses. In contrast, HMW-heparin at doses (8 mg/kg, sc, twice a day) inducing an antithrombotic activity comparable to that of dermatan sulphates, dramatically prolonged the bleeding time. LMW-heparin at the same doses was ineffective. Sulodexide (10 mg/Kg, sc, twice a day) prolonged the occlusion time to the same extent as HMW-heparin (OT50 5.10 vs. 4.14 days), with less an effect on the bleeding time (144 +/- 6 s vs. > 300 s, respectively). Histological examination confirms that the prolongation of occlusion time induced by the drugs is really related to thrombus formation inhibition at the site of arterial wall injury. Acetyl salicylic acid (ASA) (100 mg/kg/day in drinking water as lysine acetylsalicylate) did not modify the effect of Desmin 370 and Sulodexide on both occlusion and bleeding time. However, while it did not increase the antithrombotic activity of HMW-heparin, it significantly prolonged its haemorrhagic effect. In conclusion, dermatan sulphates are effective inhibitors of arterial thrombosis in rats, without inducing bleeding complications.

Background: Amelioration of the anaemia of chronic renal failure and subsequent improved haemorheology result in correction of bleeding diathesis as evidenced by shortening of the skin bleeding time (BT). However, the relationship between the haematocrit and platelet-vessel wall interactions in haemodialysis (HD) patients under recombinant human erythropoietin (rHuEpo) therapy, assessed by platelet aggregation in response to ristocetin is more complex and somewhat inconsistent., Methods: We investigated the relationship between haemoglobin (Hb) levels and whole blood ristocetin-induced platelet aggregation (electric impedance method) in 28 HD patients treated with rHuEpo, and with normal BT. The measurements were repeated in 16 subjects after having reduced platelet aggregability with orally administered ketanserin., Results: Ristocetin-induced platelet aggregation in the whole group was comparable to those found in 21 age-matched healthy subjects (normals) and in 25 HD patients not treated with rHuEpo (uraemics). Interestingly, a significant inverse correlation between this aggregation and Hb concentration was found (r = -0.392, P < 0.05). In the group of 16 patients, the pre-ketanserin aggregation was more intensive than in the normals and uraemics (P < 0.05). Ketanserin produced a fall in ristocetin-induced platelet aggregation (P < 0.02), prolongation of the BT (P < 0.02) and, unexpectedly, a decrease in serum Epo concentration (P < 0.0002) and the Hb level (P < 0.001). Again, an inverse correlation between depressed ristocetin-induced platelet aggregation and lowered Hb concentration was found (r = -0.590, P < 0.02). Moreover, a strong positive correlation between the extent of preketanserin platelet aggregation and the decrease in the intensity of this process that followed the trial was observed (r = 0.919, P < 0.000005). There were no changes in other haematological parameters or arterial blood pressure., Conclusions: Considering the role of von Willebrand factor and fibrinogen in mediating ristocetin-induced platelet aggregation, and enhanced synthesis and/or release of these macromolecules in response to uraemia or inflammation, we suggest that exaggerated whole-blood platelet aggregability to ristocetin points to blunted erythropoiesis in HD patients on rHuEpo therapy.

Since thromboembolic complications in transplanted patients are generally attributed to combined abnormalities in platelets and coagulo-lytic system, some hemostatic parameters tPA (tissue plasmogin activator):Ag and activity, its inhibitor-PAIAg and activity, tPA/PAI, thrombin-antithrombin (TAT) and plasmin-antiplasmin complexes (PAP), urokinase-uPA, euglobulin clot lysis time-ECLT, fibrinogen, plasminogen, protein C activity, D-dimer, prothrombin fragments1+2 (F1+2), fibrin monomers, fibronectin, lipoprotein-a, and von Willebrand factor(vWF), were evaluated using commercially available kits. The studies were performed on kidney transplant recipients treated with CsA, azathioprine and prednisone (n=21), and healthy volunteers (n=21). ECLT was significantly prolonged in kidney transplant recipients together with a rise in F1+2,lipoprotein-a, fibrinogen, fibronectin, and vWF when compared with controls. The TPA level was lower, whereas the PAI level was higher in kidney transplant recipients when compared with controls. In conclusion, CsA-treated kidney transplant recipients show evidence of pronounced impairment in fibrinolysis and endothelial damage in comparison with healthy volunteers.

A pathogenetic role for fibrin deposition and platelet activation in the kidney is thought to play a role in the pathogenesis of acute renal failure (ARF). Thus, some fibrinolytic parameters and platelet function have been studied in 17 patients with ARF and compared to healthy volunteers and subjects with chronic renal failure (CRF). Since serotonin may participate in pathological processes resulting from platelet/vessel wall interactions, its level in the whole blood and plasma was also assayed. In ARF and CRF platelet aggregatory responses in both whole blood and in platelet rich plasma upon stimulation with various agonists (collagen, arachidonic acid, ADP, ristocetin) were lower than those obtained in healthy volunteers. Increased levels of lipoprotein (a), von Willebrand factor (vWF) and fibronectin were found in ARF relative to controls. Protein C activity was significantly lower in patients with ARF. Euglobulin clot lysis time was prolonged in ARF and CRF, reflecting a decreased overall fibrinolytic activity. Activity of tissue plasminogen activator (tPA) inhibitor (PAI) and PAI:Ag were higher in ARF, whereas tPA:Ag, urokinase, tPA/PAI complexes, thrombin-antithrombin complexes (TAT), plasmin-antiplasmin (PAP) complexes, fibrinogen, and F1+2 did not differ between ARF and controls. In CRF elevated levels of TAT, PAP, fibrinogen and prothrombin fragments F1+2 were found, whereas concentration of fibronectin was lowered when compared to controls. In both groups of renal failure patients increased levels of fibrin monomers and d-dimer were found relative to healthy volunteers. Whole blood serotonin was significantly lower, whereas plasma serotonin was significantly higher in patients with ARF and CRF relative to controls. Serotonin uptake and its release from platelets were markedly diminished in patients with ARF and CRF. Chronic renal failure exhibit a slightly different pattern of coagulopathies that acute renal failure.

Several data suggest that platelet-derived serotonin (5-HT), previously classified as a trivial modulator of blood-borne cardiovascular disease (1), may play a decisive role in various pathological processes, resulting from abnormal platelet-vessel wall interactions (2). Following platelet activation upon a contact with a damaged vessel wall, 5-HT, released from platelets upon the activation of 5-HT2 receptors, may amplify the action of other agents on vascular smooth muscle cells and platelets (3,4). Platelets play a unique role in 5-HT metabolism: they take it up, store in their dense granules and release upon stimulation. Platelet dysfunction, alteration in platelet count, their consumption and turnover are common in renal diseases (5). Storage pool deficiency regarding 5-HT and ADP was reported in renal failure (6). Previously we reported that 5HT2 receptor blockers may serve as potent antiplatelet drugs in uremic patients, which are prone to thrombotic complications (7,8). Up to date there have been a few reports dealing predominantly with 5-HT levels in renal patients. Thus, we focused on peripheral serotonergic mechanisms including 5-HT uptake and release and kinetics of its uptake in uremic patients.

A comprehensive study on platelet aggregation, hemostasis, fibrinolysis and serum lipids in relation to peripheral serotonergic system has been performed on 41 nephrotic patients. Enhanced platelet aggregatory responses in both whole blood and in platelet rich plasma (PRP) were found upon stimulation with different agonists when compared to healthy volunteers. Increased levels of fibrinogen, fibrin monomers, and protein C activity were observed in nephrotic patients. Euglobulin clot lysis time was significantly prolonged in nephrotic patients. Activity of tissue plasminogen activator (tPA) inhibitor was higher in nephrotic syndrome, whereas tPA activity was significantly lower in these patients when compared to controls. Urokinase concentration, lipoprotein (a), cholesterol, LDL and VLDL levels were significantly higher in nephrotic patients over controls. Whole blood serotonin was significantly lower, whereas plasma serotonin was significantly higher in nephrotic patients relative to controls. Serotonin uptake and its release from platelets were markedly diminished in patients with nephrotic syndrome. Disequilibrium in the coagulolytic system, platelet hyperactivity, hyperfibrinogenemia, disturbances in peripheral serotonergic system together with lipid abnormalities may contribute to the progression and development of atherosclerosis and an enhanced risk of thromboembolic complications in nephrotic syndrome.

Platelet aggregation was studied in the whole blood and platelet rich plasma (PRP) upon stimulation with various agonists (collagen- 0.5 microgram/ml in the whole blood and PRP, ADP 10 microM in the whole blood and 2 microM in PRP, ristocetin 0.75 mg/ml in the whole blood and 1.25 mg/ml in PRP and arachidonic acid (AA) 0.5 mM in the whole blood and PRP) following incubation with ET-1 at final concentrations of 0.1 microM and 0.2 microM for 1 and 5 min. Collagen- and ADP-induced platelet aggregation in PRP was found to be diminished following incubation with ET-1 for 1 and 5 min at the concentrations of 0.1 microM and 0.2 microM. Aggregation of platelets upon stimulation with AA and ristocetin remained unaltered following incubation with ET-1. In the whole blood ristocetin-induced platelet aggregation was significantly diminished following incubation with ET-1 for 5 min, whereas AA-induced platelet aggregation was not significantly changed following preincubation with ET-1. Our results suggest that platelets may possess ET-1 receptor(s) and that ET-1 may also interact with other blood cells.

We have recently shown that ketanserin, an antagonist of peripheral serotonin 5-HT2 receptors lowers blood erythropoietin (Epo) levels in some chronic hemodialysis patients. Based on this finding, a preliminary study was undertaken to investigate the effect of a 3-week oral ketanserin administration on serum Epo concentration and relevant hematological parameters in 4 renal allograft recipients with posttransplant erythrocytosis (PTE). We found a marked decrease in Epo concentrations following ketanserin administration, from 48% to 76% of the abnormally elevated pretreatment values with subsequent increases at 3 weeks after discontinuation of the drug in all patients studied. In 3 of them a corresponding decrease or no rise in the erythrocyte count were noted. During the 6-week study period, the need for monthly phlebotomies was eliminated in these patients. It is hypothesized that ketanserin diminishes erythropoietin synthesis and may become a new drug in the treatment of posttransplant erythrocytosis.

Disturbances in serum lipids, hemostasis and platelet functions are frequent features in some kidney diseases and may contribute to the progression of atherosclerosis with its complications. Recently, an attention has been paid on beneficial effects of fish oil on serum lipids and hemostasis, and proteinuria. The purpose of this work was to assess platelet functions, some hemostatic parameters and serum lipids in patients with chronic glomerulonephritis treated with Trienyl. The study was performed on 7 patients with glomerulonephritis, before, 3 and 6 months following fish oil treatment. A small and nonsignificant rise in cholesterol, HDL and LDL was found, whereas triglycerides level fell significantly following 3 and 6 months of therapy Fibrinogen concentration was lowered significantly 6 months following fish oil administration. Platelet aggregation in platelet-rich plasma remained unaltered during therapy, whereas platelet responses to ADP and arachidonic acid in the whole blood were inhibited after 6 months of the therapy. Unsaturated omega 3 fatty acids in Trienyl alter lipid metabolism, platelet/vessel wall interactions and proteinuria and therefore might be beneficial in therapy of glomerulonephritis, particularly in combination treatment.

Erythropoietin treatment is known to correct anemia and to improve hemostasis. Since platelets may contribute to thromboembolic complications, we assessed platelet aggregation in whole blood and platelet-rich plasma from chronically hemodialyzed patients treated with erythropoietin and evaluated in vitro effects of this drug on aggregatory responses of uremic and normal platelets. Recombinant human erythropoietin was given to uremic patients at a dose of 2,000 IU subcutaneously three times a week. Platelet aggregation in whole blood and platelet-rich plasma was induced by collagen, ADP, arachidonic acid, and ristocetin. In uremic patients, erythropoietin therapy resulted in an enhancement of platelet sensitivity to various agonists, particularly in platelet-rich plasma, reaching values comparable to those of healthy volunteers. In vitro studies we were unable to show any direct effect of erythropoietin, used at concentrations that occurred post intravenous administration, on platelet aggregation both in whole blood and in platelet-rich plasma.

Ketanserin, an antagonist of peripheral serotonin receptors when given to hemodialyzed patients treated with recombinant human erythropoietin (rHuEpo) corrects some changes in hemostasis but also apparently delays an increase in hematocrit. We wished to elucidate the effects of oral administration of ketanserin on serum Epo, some hematological and biochemical blood parameters, arterial blood pressure (BP), and bleeding time in hemodialyzed patients receiving rHuEpo therapy. We noted a 33% decrease in Epo concentration (p < 0.05) after a 4-week ketanserin trial in patients in the initial stage of rHuEpo therapy. Although a concomitant decrease in erythrocyte count and hemoglobin did not reach statistical significance, these changes correlated positively with decreasing Epo level (r = 0.749 and 0.787, respectively). Ketanserin administered for 14 days to patients between 32 and 34 weeks of rHuEpo therapy also produced a decrease of 26% in Epo concentration (p < 0.005). This decrease correlated (r = 0.629) with a decrement in the red blood cell (RBC) count (p < 0.005). Hemoglobin concentration followed the same pattern (p < 0.005). However, the decreases in the reticulocyte count did not reach statistical significance. The decrease in hormone concentration resulted in a concomitant thrombocyte decrease (p < 0.05) only in patients who received ketanserin in the interval between 8 and 12 weeks of rHuEpo therapy. The previously normal bleeding time was significantly prolonged (p < 0.05) in both groups of patients. There were no changes in leukocyte count, iron status parameters, or calcium, phosphorus, or bilirubin concentration. Administration of ketanserin even for 4 weeks did not influence BP in the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The contant of dimethyl-L-arginine in the ultrafiltrate obtained during dialysis session from 30 patients with end-stage renal failure maintaned on chronic hemodialyses was studies. 14 patients were normotensive and 16 were hypertensive. In patients with hypertension dimethyl-L-arginine level was significantly elevated (mean value 7.67 mumol/l), when compared to patients with normal blood pressure (mean value 2.37 mumol/l). Prolongation of the dialysis session is postulated in hypertensive patients.

Serotonergic mechanisms are partially responsible for changes in haemostasis and blood pressure rise in haemodialyzed patients treated with recombinant human erythropoietin (rHuEPO). Ketanserin--an antagonist of platelet and blood vessel serotonin receptors, given concomitantly to patients on rHuEPO therapy, prevents certain changes in haemostasis. Preliminary observations suggest that ketanserin also inhibits an increase in haemoglobin and haematocrit. So far there have been no reports on ketanserin affecting erythropoiesis. We studied an influence of the 2-week oral ketanserin administration on some haematologic parameters, serum erythropoietin concentration, blood pressure and relevant biochemical blood indexes in 15 haemodialyzed patients treated with rHuEPO for 32 weeks. We found a significant fall in serum EPO concentration (p < 0.005) measured with the ELISA technique that correlated positively (r = 0.629) with a decrease in erythrocyte count (p < 0.005). Haemoglobin concentration followed the same pattern (p < 0.005). The previously normal bleeding time prolonged significantly (p < 0.02) after ketanserin therapy. There were no changes in plasma iron and ferritin concentration, total iron binding capacity, transferrin saturation index, calcium, phosphorus and bilirubin concentration nor reticulocytosis. The decrement in EPO concentration did not affect peripheral blood platelet or leukocyte counts. The 14-day treatment with ketanserin did not influence arterial blood pressure in the patients. The study shows that ketanserin administered to haemodialyzed patients on long-term rHuEPO therapy induces a decrease in erythropoietin concentration and inhibits erythropoiesis. This phenomenon seems to result from diminished endogenous hormone synthesis caused by ketanserin.

Thrombotic events sometimes complicate erythropoietin therapy. Fibrinolytic system may play a role in their pathogenesis. The studies were performed on 22 chronically hemodialyzed patients with end-stage renal failure treated with recombinant human erythropoietin (rHuEPO, Eprex, Cilag) for 12 weeks. Alpha 2 antiplasmin (alpha 2AP), antithrombin III (AT III), C1 esterase inhibitor (C1 INH), plasminogen activator inhibitor (PAI) activities, alpha 2macroglobulin (alpha 2M), fibrinogen, fibrin monomers concentration and euglobulin clot lysis time (ECLT) were measured before and after 1, 2, 4, 8 and 12 weeks of rHEPO therapy. A fall in PAI activity (p < 0.05) was found after 1 week, while alpha 2AP and C1 INH activities decreased after 12 weeks of the therapy (p < 0.001 and p < 0.05, respectively). The activity of AT III, the main inhibitor of the coagulation cascade fell after 4 weeks of the treatment with rHuEPO (p < 0.001). Fibrin monomers concentration was found to be decreased after 12 weeks of rHuEPO administration. Fibrinogen and alpha 2M concentration showed no statistically significant changes during rHuEPO therapy. A decrease in plasma fibrinolytic inhibitor activities may be considered as a protective mechanism against thrombotic tendency observed during rHuEPO therapy.

Recombinant human erythropoietin was administered to 12 patients with end-stage renal failure on long-term hemodialysis. They responded to the therapy with a shortening of the prolonged bleeding time, starting from the 1st week of therapy, before a significant increase in hemoglobin concentration was achieved. We also observed an increase in the activity of tissue plasminogen activator and a decrease in the activity of its inhibitor. There were no changes in platelet count but a significant increase in blood and platelet serotonin concentration was found. The shortening of the prolonged bleeding time before the correction of the anemia correlated with the rise in blood and platelet serotonin concentration during erythropoietin therapy. We suggest the possible involvement of an serotonergic mechanism in the hemostatic action of recombinant human erythropoietin.

Patients with uraemia have a defect haemostasis caused by severe anaemia and disturbances of platelet/vessel wall interactions. Recombinant human erythropoietin (rHuEPO) treatment not only corrects anaemia, but also shortens the bleeding time. There are few reports dealing with changes of haemostasis during the first month of rHuEPO treatment. We studied platelet function after 1, 2, 4, 8, and 12 weeks of rHuEPO treatment. Erythropoietin was given to 19 dialysed patients with chronic uraemia in a dose of 2000 u subcutaneously 3 times a week. Bleeding time showed a significant fall as early as after the first week of rHuEPO treatment (p < 0.05). After the first month the bleeding time became normal in most of the patients. A significant rise in ristocetin-induced platelet aggregation was observed from the first week of therapy. It showed a strong correlation with the shortening of the bleeding time. Collagen-induced aggregation followed the same pattern but the changes were not striking. There was not significant difference in platelet adhesion, platelet aggregation in the whole blood and those induced by ADP and arachidonic acid. Platelet serotonin concentration was also showed to increase during rHuEPO therapy. We conclude that rHuEPO improves haemostasis by influencing platelet aggregation possibly involving a serotoninergic mechanism but on the other hand may increase a tendency to thrombosis.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and dGTP, resulting in feedback inhibition of ribonucleotide reductase (RR; EC 1.17.4.1) and hence DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of dGTP in G1-phase, but not in S-phase cells, because of the utilization of dGTP for DNA synthesis and limited capacity to synthesize dGTP from dG. These results support the hypothesis that dG/dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is RNA synthesis, which was inhibited at an early stage in dGuo-L cells.

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.

To determine whether the toxic effects and changes in many cell functions caused by antitumor 1-nitroacridines are related to their enzymatically mediated covalent interstrand DNA cross-linking (J. Konopa, J. W. Pawlak, and K. Pawlak. Chem.-Biol. Interact., 43: 175-197, 1983), the cross-linking potency of the derivatives with structural modifications in position 9 of the acridine nucleus was estimated as their in vitro threshold concentrations (0.3 to 4.5 microM), beyond which the first interstrand DNA cross-links could be detected in DNA of cultured HeLa S3 cells with a polyethylene glycol 6000-Dextran T500 assay. Statistically significant (p less than 0.05) correlations exist between the cross-linking potency of 1-nitroacridines and their in vivo antitumor activity and toxicity against mice with Sarcoma 180 tumors in solid form (3 to 1065 mumol/kg of body weight), as well as their in vitro cytotoxicity against cultured HeLa or HeLa S3 cells (0.0005 to 7.2 microM), indicating that the interstrand DNA cross-linking potency might be one of primary determinants of in vivo and in vitro biological activity of 1-nitroacridine antineoplastic drugs. Susceptibility of the parent agents to reduction does not appear to be a rate-limiting factor of DNA cross-linking potency of 1-nitroacridines and their metabolic transformations (J. W. Pawlak, and J. Konopa. Biochem. Pharmacol., 28: 3391-3402, 1979), because no significant differences were observed among the agents with respect to their polarographic half-wave potentials estimated under anaerobic conditions.

The mechanism of the cellular toxicity of four inosinate dehydrogenase (IMP-DH) inhibitors with different antitumor and antiviral pharmacological profiles was investigated in mouse lymphoma (S-49) cell culture. Drug effects on cell growth, nucleotide pools, and DNA and RNA synthesis were measured in the presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, while they also demonstrated some growth-inhibitory effects when added alone in high concentrations. All four drugs reduced cellular guanosine triphosphate levels and caused secondary changes of the uridine, cytidine, and adenosine triphosphate pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed except with mycophenolic acid which may represent a pure IMP-DH inhibitor. Both tiazofurin and selenazofurin interfered with the uptake and/or metabolism of uridine and thymidine tracers; however, this effect appeared not to contribute to their cellular toxicity in vitro. Moreover, selenazofurin and tiazofurin impaired the utilization of exogenous guanine salvage supplies for DNA and RNA synthesis, and guanine was particularly ineffective in reversing the toxic effects of tiazofurin on cell growth. This finding is important in view of the available guanine salvage supplies in vivo. Since tiazofurin, selenazofurin, and their known metabolites failed to inhibit hypoxanthine-guanine-phosphoribosyl transferase, guanosine monophosphate kinase, and guanosine diphosphate kinase in cell extracts or permeabilized cells, these drugs may interfere with salvage transport across cellular membranes. The toxic effects of mycophenolic acid and ribavirin were similarly reversed by salvage supplies of up to 200 microM guanine, which suggests that ribavirin primarily acts as an IMP-DH inhibitor under these conditions. This result could explain the rather low antitumor efficacy of both mycophenolic acid and ribavirin in vivo. However, increasing the guanine salvage supply in the medium above 200 microM further reversed the toxic effects of mycophenolic acid to maximum rescue, while it increased the toxicity of ribavirin (300 microM). This finding suggests the presence of a toxic mechanism of ribavirin at higher concentrations that is dependent upon the presence of guanine supplies sufficient to fully overcome the IMP-DH inhibition. This study documents that each antimetabolite displays a unique spectrum of activities with multiple toxic targets.