Andrzej Rydzewski

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.

Changes in Plasminogen Activator Inhibitor 1 and Tissue-Type Plasminogen Activator during Exercise in Patients with Coronary Artery Disease

As depressed fibrinolysis is implicated in the pathogenesis of coronary artery disease, we have studied the activation of fibrinolysis during maximal, symptom-limited exercise in a group of 68 men. After exercise they were divided, according to their coronary angiography and exercise 201Tl emission computed tomography results, into three groups. Group 1: persons with normal exercise 201Tl emission computed tomography results and no underlying diseases who served as controls; group 2: patients with coronary artery disease without exercise-induced myocardial ischemia, and group 3: patients with coronary artery disease with transient, exercise-induced myocardial ischemia. Before and at peak exercise we measured the plasminogen activator activity (PAA) in the euglobulin fraction of plasma by an amidolytic method and the concentrations of tissue plasminogen activator (t-PA), activator-inhibitor complex - plasminogen activator inhibitor 1 (PAI-1) complexed with t-PA - and total PAI-1 by enzyme immunoassay. The concentration of free PAI-1 in plasma was calculated by subtraction of the concentration of activator-inhibitor complex from that of total PAI-1. Under basal conditions, group 3 had significantly higher free and total PAI-1 levels than group 1. There were no statistically significant differences between the three groups in PAA, t-PA, and activator-inhibitor complex levels. At peak exercise, group 1 showed the highest release of t-PA accompanied with highest increases in PAA as well as in activator-inhibitor complex, the proportion of released t-PA antigen not bound to PAI-1 being highest in group 1. Free PAI-1 decreased significantly, but there were no differences between individual groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical significance of plasminogen activator inhibitor activity in patients with exercise-induced ischemia

To assess the fibrinolytic system in patients with exercise-induced ischemia and its relation to ischemia and severity of coronary artery disease (CAD), 47 patients with CAD confirmed by results of coronary angiography underwent symptom-limited multistage exercise thallium-201 emission computed tomography. All patients with CAD had exercise-induced ischemia as assessed from thallium-201 images. Pre- and peak exercise blood samples from each patient and preexercise blood samples from control subjects were assayed for several fibrinolytic components and were also assayed for plasma adrenaline. The extent of ischemia was defined as delta visual uptake score (total visual uptake score in delayed images minus total visual uptake score in initial images) and the severity of CAD as the number of diseased vessels. In the basal condition, plasminogen activator inhibitor (PAI) activity was significantly higher in patients with exercise-induced ischemia as compared to control subjects (p less than 0.01), although there were no significant differences in other fibrinolytic variables between the two groups. Moreover, PAI activity in the basal condition displayed a significantly positive correlation with the extent of ischemia (r = 0.47, p less than 0.01). Patients with exercise-induced ischemia were divided into two groups (24 with single-vessel disease and 23 with multivessel disease). There were no significant differences in coronary risk factors, hemodynamics, or plasma adrenaline levels during exercise between single-vessel and multivessel disease except that delta visual uptake score was significantly higher in multivessel disease (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Fluctuations of euglobulin lysis time, tissue plasminogen activator, and free and total plasminogen activator inhibitor levels in plasma in daytime

Blood was taken from healthy 15 males and 10 females at 9:30 h, 10:00 h and 12:30 h. The euglobulin fraction was prepared and clotted by the addition of human thrombin. The clot lysis time shortened significantly from 9:30 h to 10:00 h (p less than 0.01) and further to 12:30 h (p less than 0.01). Plasma levels of tissue plasminogen activator (t-PA) antigens did not change from 9:30 h to 10:00 h, but slightly and significantly to 12:30 h (p less than 0.01). Plasma levels of free plasminogen activator inhibitor-1 (PAI-1) and complex of t-PA-PAI-1 decreased from 9:30 h to 10:00 h (p less than 0.02) and to 12:30 h (p less than 0.01). Plasma levels of total PAI-1 (free plus complex) decreased from 9:30 h to 10:00 h (p less than 0.01) and to 12:30 h (p less than 0.01). These results suggest that a major factor contributing to the enhanced fibrinolytic activity of the euglobulin fraction may be a level of PAI-1 (free and total).

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 Buergers disease, was assessed in a pilot study. We investigated 10 patients suffering from Buergers 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.

Determination of plasminogen activator inhibitor-1 (PAI-1) in plasma using two different anticoagulants and methods.

Platelets contain plasminogen activator inhibitor (PAI-1) in mainly inactive form which may be released during platelet activation. We wanted to compare effects of anticoagulant and time of blood storage on PAI-1 determination by two different methods

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.

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.

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.

Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma

Effects of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, in vitro were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1, 1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma in vitro.