Early changes in serum creatinine level and estimated glomerular filtration rate predict pancreatic necrosis and mortality in acute pancreatitis: Creatinine and eGFR in acute pancreatitis.

AIM: The aim of the study was to evaluate the significance of serum creatinine level (SCL) and estimated glomerular filtration rate (eGFR) measured in an early phase of acute pancreatitis (AP) for prediction of pancreatic necrosis (PNec) and mortality. METHODS: One hundred and forty-seven patients with AP were retrospectively reviewed in the study. Serum creatinine level and estimated glomerular filtration rate (calculated using the abbreviated Modification of Diet in Renal Disease equation) on admission and 48 h thereafter were analyzed for each patient. These parameters were compared with contrast-enhanced computed tomography images performed within 96 h from admission (n = 103). Usefulness of SCL and eGFR for prediction of PNec and fatal outcome of AP was evaluated using a receiver operator characteristic curve analysis and comparison of average parameter values. RESULTS: We confirmed pancreatic necrosis in 41 (39.8%) of 103 patients using computed tomography examination. Both creatinine and estimated glomerular filtration rate measured on admission (p < 0.001, p < 0.001 respectively) and 48 h later (p = 0.001, p < 0.001 respectively) were significantly associated with the presence of pancreatic necrosis. Moreover, serum creatinine level and eGFR measured on the 1st day proved to be a good predictor of fatal outcome. Both, mortality and presence of pancreatic necrosis were significantly higher in the group with elevated serum creatinine level and low eGFR values. CONCLUSIONS: SCL and eGFR on admission are useful indicators of PNec and mortality.

Magnification chromoendoscopy in comparison to standard chromoendoscopy for detection of intestinal metaplasia in renal transplant recipients.

PURPOSE: Renal transplantation is associated with frequent gastrointestinal complications. Intestinal metaplasia is a feature of atrophic gastritis whereas the diagnosis of Barrett's esophagus is based on histological demonstration of specialized metaplasia. Both conditions are associated with increased risk of adenocarcinoma. The aim of the present study was to assess whether magnification endoscopy improves the diagnostic accuracy of intestinal metaplasia in stomach and in esophagus. MATERIAL AND METHODS: In this non-randomized, feasibility study thirty one (12 women and 19 men) renal transplant recipients, with a mean age of 44.0 years were evaluated for the presence of intestinal metaplasia. Standard esophagogastroscopy with methylene blue staining was followed by magnification endoscopy. The presence of gastritis and intestinal metaplasia was classified according to modified updated Sydney classification. RESULTS: Of 31 patients, 16 patients had endoscopic and histopathological evidence of gastric intestinal metaplasia, and standard endoscopy with methylene blue staining was sufficient for diagnosis (15 from 16). Magnification endoscopy allowed identification of 6 patients with specialized intestinal metaplasia in Barrett's esophagus, which would be otherwise missed. CONCLUSIONS: In this study diagnostic accuracy of standard endoscopy for identification of intestinal metaplasia in the stomach was not improved by the use of magnification endoscopy, but the latter was an accurate method of predicting specialized intestinal metaplasia in Barrett's esophagus. The use of magnification endoscopy in the clinical setting of renal transplantation needs further studies.

Gastric juice ammonia and urea concentrations and their relation to gastric mucosa injury in patients maintained on chronic hemodialysis.

PURPOSE: This study was undertaken to test the hypothesis that high concentrations of urea in gastric juice would have an influence on Helicobacter pylori infection in patients maintained on chronic hemodialysis (HD). MATERIAL AND METHODS: We investigated 30 patients (17 males, 13 females; mean age 50.8 +/- 2.9 years) with end-stage renal disease (ESRD) undergoing hemodialysis treatment (HD) for at least 6 months, who were compared to 31 patients (16 males, 15 females; mean age 61.3 +/- 2.2 years) with dyspeptic symptoms. Biopsies from the gastric antrum and body were taken for histological investigation. Urea and ammonia were measured in gastric juice, and the severity of gastritis was evaluated according to Sydney criteria. RESULTS: H. pylori infection was found in 19 (63%) HD patients and in 22 (71%) control subjects. Gastric juice urea concentration was significantly higher in HD patients than in controls and H. pylori infection caused a significant decrease in urea concentration in both groups. There was an inverse correlation between urea and ammonia concentration in gastric juice in both groups. Ammonia concentration in both groups was higher in H. pylori infected patients. In H. pylori negative subjects ammonia/urea ratio was lower in HD patients in comparison to controls. Ammonia/urea ratio was raised by H. pylori infection in both groups, and the difference between HD and control groups persisted. H. pylori infection was associated with polymorphonuclear infiltration of gastric mucosa. There was a significant correlation between gastric ammonia and mucosal polymorphonuclear leukocytes infiltration and gastritis score. CONCLUSIONS: Higher urea levels in the gastric juice of chronically hemodialyzed patients do not seem to be a risk factor for infection with Helicobacter pylori.

The nephrologist's role in the oncology--haematology ward.

Cancer patients require care by a multidisciplinary medical team. Although nephrology usually is not the "core" specialty in such a multidisciplinary team, nonetheless it may substantially improve the quality of care. This paper reviews nephrologist's role in the management of the cancer patient.

Long-term effects of erythropoietin on platelet serotonin storage and platelet aggregation in hemodialysis patients with reference to ketanserin treatment.

Correction of uremic platelet serotonin (5-HT) storage pool deficiency is one of the very early hemostatic effects of erythropoietin (Epo) therapy. In this work, platelet 5-HT with relation to primary hemostasis was studied in 15 hemodialysis patients treated with Epo for 8 months. Moreover, effects of ketanserin, a blocker of platelet and vascular smooth muscle cell 5-HT2A receptors, in these patients were followed. The parameters studied were compared with relevant values in healthy controls and in hemodialysis patients not treated with Epo, and remeasured in the long-term Epo patients after a 14-day oral ketanserin trial. Platelet 5-HT content in the eighth month of Epo therapy was not different from the one in untreated patients. Ristocetin- and collagen-induced platelet aggregation were enhanced in comparison with both control groups, as opposed to unaltered response to ADP and arachidonic acid. Fibrinogen concentration was lower than in the untreated group. An inverse correlation between ADP-induced platelet aggregation and the skin bleeding time (r=-0.536, p<0.05) and a positive one between the former and platelet 5-HT (r=0.644, p<0.01) were found. Platelet count correlated positively with both platelet 5-HT (r=0.823, p<0.0002) and ADP-induced platelet aggregation (r=0.596, p<0.02). Ketanserin produced a decrease in ristocetin-induced platelet aggregation, fibrinogen, and prolongation of the bleeding time. The first two of the changes correlated positively with their pre-ketanserin values (r=0.923, p<0.00001 and r=0.839, p< 0.0001, respectively). Post-ketanserin, positive correlations between depressed ristocetin- and arachidonic acid-induced platelet aggregation (r=0.760, p<0.005), and between collagen- and corresponding values of arachidonic acid- (r=0.622, p<0.02), ADP-induced platelet aggregation (r=0.396, p<0.01), and platelet 5-HT (r=0.654, p<0.05) were found. Efficient hemostasis in hemodialysis patients on protracted Epo therapy is, in part, dependent on enhanced platelet aggregability. Correction of platelet 5-HT storage pool deficiency is not evident in this stage but 5-HT still influences complex mechanisms of primary hemostasis. Ketanserin is of anticoagulant value in these patients but its effects must be weighted against possible exacerbation of the anemia.

Modulation of hematopoiesis by ketanserin in erythropoietin-treated uremic patients: evidence from platelet studies.

Recombinant human erythropoietin (EPO) not only ameliorates the anemia of renal failure but also modulates platelet function and corrects uremic platelet serotonin (5-hydroxytryptamine, 5-HT) storage pool deficiency. We studied if ketanserin, a blocker of platelet and vascular smooth muscle receptors for 5-HT, could reverse any EPO-induced changes in hemostasis. A complete blood count, immunoreactive serum EPO concentration, skin bleeding time (BT) and whole blood platelet aggregation (electric impedance method) induced by ristocetin and ADP, and intraplatelet and whole blood 5-HT, were determined in seven chronic hemodialysis (HD) patients before and after 1, 2, 4 and 8 weeks of EPO therapy, and repeated after a 4-week co-treatment with oral ketanserin. Stimulation of erythropoiesis was accompanied by a rise in the platelet count ( P < 0.05), shortening of the BT ( P < 0.02), an increase in platelet aggregability, and by replenished intraplatelet 5-HT store. Ketanserin co-treatment produced an unexpected 33% fall in serum EPO level ( P < 0.02), a decrease in the platelet count ( P < 0.05), prolongation of the BT ( P < 0.05) and depressed platelet aggregation in response to both agonists. There was no change in the amount of intraplatelet 5-HT while whole blood 5-HT concentration decreased significantly ( P < 0.02). Strong positive correlations between the decrease in whole blood 5-HT and the prolongation of the BT ( r = 0.786, P < 0.05), and between the former parameter and the fall in the platelet count ( r = 0.820, P < 0.05) were found. In conclusion, we report dual erythro- and thrombocytopoietic effects of EPO combined with correction of a platelet defect in the storage of 5-HT and enhanced platelet aggregability. The ketanserin-induced falls in serum EPO concentration and the platelet count provide new evidence of the dependency of thrombocytopoiesis on EPO in the initial weeks of the therapy. The 'antiplatelet' effects of ketanserin observed in this study seem to be due to reduction in circulating thrombocyte number rather than from any inhibitory effect on their aggregation.

The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S.

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.

[Altered circadian rhythm of platelet aggregation in patients on longterm hemodialysis]. / Zaburzony dobowy rytm agregacji plytek krwi u pacjentów przewlekle hemodializowanych.

Chronic uremia is associated with a bleeding tendency, and paradoxically with propensity for thrombotic complications. Several physiological systems are subject to circadian rhythm, including among others hemostasis, platelet aggregability and fibrinolysis. Alterations in these rhythms were suggested to be involved in the pathogenesis of sudden cardiac and cardiovascular complications of diabetes mellitus. As cardiovascular events are the most frequent cause of death in dialyzed patients, we studied circadian rhythm of platelet function in chronically hemodialyzed patients in relation to blood and plasma serotonin. We investigated 16 patients (mean age 49.7 +/- 12.2 years, 10 females, 6 males) who had been maintained on chronic hemodialysis. Control group consisted of 8 age matched healthy volunteers. Blood was collected after 15 min. rest at 8:00, 11:00, 17:30, and 23:00 from antecubital veins. Platelet aggregation was measured according to the method of Born. Following concentrations of aggregating agents were used: ADP 5 microM; collagen, 2 micrograms/ml; arachidonic acid 0.75 microM, serotonin 1 microM; and ristocetin 1.5 micrograms/ml. Serotonin was measured in whole blood and plasma by HPLC method. In PRP from healthy subjects aggregatory responses to ADP and arachidonic acid were significantly higher at 17:30 than at 8:00. In uremic patients aggregatory response to ADP and ristocetin was more intensive at 11:30 and 23:00 in comparison to 8:00. Whole blood 5-HT did not change during the day, while plasma 5-HT concentration increased significantly in uremics at 11:30 in comparison to initial value. In conclusion, our study demonstrates that in chronically dialyzed patients circadian changes in platelet aggregation are different from normal persons.

Diurnal variation in serum remnant-like lipoproteins, platelet aggregation and fibrinolysis in healthy volunteers.

Postprandial triglyceridemia and 'remnantemia' may better reflect the atherosclerotic risk than triglyceride (TG) levels in the fasting state. Recently, a new method was developed based on a monoclonal antibody recognizing an epitope distal to the carboxyl end of apo B48 which allows easy measurement of remnant-like lipoproteins (RLP). This study was performed in order to investigate RLP response to a standardized fat meal and establish a normal diurnal pattern of RLP in blood and compare it to platelet aggregation and fibrinolysis in healthy young men. We investigated 7 male volunteers (age range 18-23 years) who received a standardized fat meal (Othsuka Pharmaceutical Company, Japan) containing 32.9% lipids, 2.5% protein, 2.5% carbohydrate, 0.3% calcium and 0.1% phospholipids, and 74 mg/100 g cholesterol (C) at 7:30. The energetic value of this cocktail was 341 kcal/100 g. Area under curve (AUC) responses in TG, RLP-TG and RLP-C after the meal were as follows: for TG 28.66 +/- 8.94; for RLP-TG 17.54 +/- 5.55; for RLP-C 1.27 +/- 0.42 mg x dl-1 x h-1. These responses were correlated to each other. Surprisingly, collagen-induced platelet aggregation in whole blood was negatively related to RLP-C AUC. Fluctuation patterns of TG, RLP-TG and RLP-C concentrations during the day were remarkably similar, peaking in this particular group of subjects at 10:00-12:00 and at about 23:00, whereas cholesterol was decreasing late in the night and very early in the morning. This pattern was different from those of platelet aggregation and fibrinolysis parameters.

Inverse relationships between haemoglobin and ristocetin-induced platelet aggregation in haemodialysis patients under erythropoietin therapy.

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.

The effect of a 5HT2 receptor antagonist sarpogrelate (MCI-9042) treatment on platelet function in Buerger's disease.

The effect of a new, specific 5-HT2 receptor antagonist sarpogrelate (MCI-9042) treatment on platelet function and serotonin levels in both plasma and whole blood in Buerger's disease, was assessed in a pilot study. We investigated 10 patients suffering from Buerger's disease. Sarpogrelate in a dose of 3 x 100 mg a day was given p.o. for a period of 8 weeks. It was well tolerated and no major side effects were noted. It was judged to be effective in some patients as assessed by its effect on both subjective complaints and objective evaluation of ankle pressure index (API). Sarpogrelate induced a significant decrease in plasma serotonin (5-HT) concentration starting after the 4th week which lasted through to the 8th week of the study, whereas plasma tryptophan concentration increased significantly after 2 and 4 weeks. There were no changes in plasma 5-HIAA concentration. On the other hand whole blood 5-HT concentration increased significantly after 2 weeks, and there was also a tendency to increase in whole blood tryptophan concentration (p = 0.052). Platelet aggregation induced by ADP and collagen did not show any statistically significant changes. Surprisingly, platelet aggregation induced by serotonin increased significantly after 2 weeks and even more so after 4 weeks of treatment, and then it returned to baseline values after 8 weeks. There was no effect on platelet count, APTT, TT and fibrinogen concentration.

Comprehensive study on platelet function, hemostasis, fibrinolysis, peripheral serotonergic system and serum lipids in nephrotic syndrome.

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.

Construction, expression and preliminary characterization of glycosylation mutants of human protein S.

Human protein S (HPS) is a vitamin K dependent plasma glycoprotein involved in the regulation of activated protein C and possibly fibrinolysis. Its c-DNA sequence shows three N-glycosylation consensus sequences (Asn-X-Ser/Thr). In order to study influence of N-linked glycosylation on HPS function, set of mutants of HPS was constructed. Mutants were generated, starting from an SV40/Adeno derived pD5HPS2 expression vector, using PCR enabled, site specific methodology. They included single amino acid substitutions at each of three N-glycosylation consensus sequences: Asn458-->Gln, Ser460-->Gly, Asn468-->Gln, Thr470-->Gly, Asn489-->Gln, Thr491-->Gly. Variant HPS were expressed in stable 293 human kidney cell lines in the presence of vitamin K1 (we did not succeed in expressing variant Asn489-->Gln) and purified from conditioned media using pseudoaffinity chromatography on QAE-Sepharose. Variant Asn468-->Gln showed decreased gamma-carboxyglutamate content. All of the mutants were active in a clotting type assay based on factor Va inactivation, and they were compared to wt-HPS and plasma HPS. In conclusion, we have constructed, expressed and purified set of HPS mutants useful in studying the role of N-glycosylation in HPS function.

Ketanserin: a new perspective in posttransplant erythrocytosis?

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.

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.

[Evaluation of selected parameters of hemostasis in patients with nephrotic syndrome]. / Ocena wybranych parametrów ukladu hemostazy u chorych z zespolem nerczycowym.

Thromboembolic complication are often seen in patients with nephrotic syndrome (NS). A comprehensive study of haemostasis and fibrinolysis has been performed in a group of 39 patients with nephrotic syndrome. We have found increased level of fibrinogen, fibronectin, fibrin monomers, alfa 2 macroglobulin, protein C and C 1 esterase inhibitor, whereas level of alfa 2 antiplasmin, alfa 1 antitrypsin were decreased. Patients with thromboembolic complications had particularly low levels of antithrombin III and histidine rich glycoprotein.

The effector roles of kringle 1 and kringle 2 in the enzymatic properties of recombinant tissue-type plasminogen activator as revealed by generation of recombinant molecules containing each kringle linked to the protease domain.

Recombinant DNA technology has been employed to construct and express in human kidney 293 cells cDNAs encoding deletion-mutant recombinant (r) tissue-type plasminogen activators (tPA) retaining only the kringle 1 ([K1tPA]) and serine protease (P) functional domains (r-[K1tPA]P), and the kringle 2 ([K2tPA]) and P domains (r-[K2tPA]P), along with a variant of r-tPA containing a W253S mutation (r-tPA/W253S). Of these mutants, only r-[K2tPA]P retained its ability to interact with omega-amino acid effector molecules. The Km for single-chain wild-type (wt) r-tPA toward the synthetic substrate H-D-Ile-L-Pro-L-Arg-p-nitroanilide (S2288) was decreased by approximately 3-fold in the presence of a saturating concentration of human fibrinogen (Fg), along with a small (1.14-fold) increase in the kcat for this same reaction. The kinetic activation (dissociation) constant (KA) for Fg was 2.4 microM. Fg did not influence the steady-state amidolytic properties of two-chain wtr-tPA. Similar effects of Fg on the Km for hydrolysis of S2288 were displayed for single-chain forms of r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, with additional small effects of Fg on the kcat of this reaction. The KA for Fg toward these proteins ranged from 2.4 microM for wtr-tPA to 5.2 microM for r-[K1tPA]P. The amidolytic properties of the two-chain forms of these variants were also unaffected by Fg. The activation rate of [Glu1]-plasminogen ([Glu1]Pg) by wtr-tPA was stimulated approximately 7-fold by Fg and approximately 139-fold by the same concentration (in Fg equivalents) of human fibrin (Fn) (Fn/Fg stimulatory ratio = 19.9). The Fn/Fg ratio was 10.6, 20.9, and 18.0 for r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S, respectively. Quantitative [Glu1]Pg-enriched clot lysis assays revealed that r-[K1tPA]P, r-[K2tPA]P, and r-tPA/W253S were approximately 18, 72, and 54%, respectively, as effective as wtr-tPA in catalyzing the plasminogen activation event leading to lysis. The antifibrinolytic agent epsilon-aminocaproic acid, inhibited clot lysis with approximately equal effectiveness in [Glu1]Pg-enriched clots when wtr-tPA, r-[K1tPA]P, r-[K2tPA]P, or r-tPA/W253S were employed as the activators. These studies demonstrated that Fg- and Fn-based stimulatory effects on the enzymatic properties of r-tPA and its variants were generally not proportional to the macroscopic binding abilities of these proteins with omega-amino acids or with Fg and Fn.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue plasminogen activator, plasminogen activator inhibitor, and other parameters of fibrinolysis in the early stages of taurocholate acute pancreatitis in rats.

It is well known that fibrinolytic activity in the early stages of acute experimental pancreatitis (AEP) as assessed by euglobulin lysis time (ELT) is depressed. The aim of this study was to evaluate changes in the fibrinolytic system in the early stages of taurocholate AEP in rats. Tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor 1 (PAI-1) activity, plasminogen, alpha 1 proteinase inhibitor (alpha 1 PI), alpha 2 antiplasmin (alpha 2 AP), antithrombin III (AT III), fibrinogen, and ELT were measured 0.5, 1, 3, and 6 h after the induction of taurocholate AEP in rats, as well as in sham-operated animals and the control group, which was not submitted to any operation. T-PA activity decreased significantly after 3 and 6 h of AEP; PAI activity had a time course reverse to t-PA and was parallel to alpha 1 PI activity. ELT was slightly prolonged after 0.5, 1, and 3 h, whereas alpha 2 AP activity and plasminogen levels increased significantly; AT III activity was increased after 1 h in comparison to control group. Sham operation caused nonsignificant changes in fibrinolysis. Increase of PAI activity and decrease of t-PA could be a reasonable explanation for inhibited plasma euglobulin fibrinolytic activity noted in the early period of AEP.

Plasminogen activator inhibitor activity as a possible indicator of disease activity in rest angina with angiographically insignificant coronary artery stenosis.

To assess the role of fibrinolytic system, 19 patients with rest angina and insignificant coronary artery stenosis and 23 controls performed symptom-limited multistage exercise. Vasospasm was angiographically demonstrated in 12 patients. Pre- and peak exercise blood samples from each patient were assayed to determine the fibrinolytic components. The patients displayed significantly increased PAI activity both under the basal conditions (p less than 0.01) and at peak exercise (p less than 0.01) as compared with the controls. However, the values of other fibrinolytic components, such as t-PA antigen, t-PA/PAI-1 complex and free PAI-1 antigen, in the controls and patients were similar. Nineteen patients were divided into two groups according to PAI activity levels under basal conditions. Nine patients displayed high PAI activity (more than the mean + 1 SD of the control value) under the basal conditions. When compared to the remaining 10 patients, the high PAI activity group had both a significantly short time interval from the last attack to the time of getting the blood sample (p less than 0.05), and a worse short-term prognosis (p less than 0.05). Thus, the level of PAI activity under basal conditions reflected the extent of disease activity, suggesting that PAI activity may be a useful clinical indicator of the severity of rest angina in patients without significant coronary stenosis.