Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects

Although protein C (PC) and activated protein C (APC) have been postulated to be useful for treating patients with thrombosis, their critical effect remains to be studied in human subjects. To examine whether purified PC or APC are useful for treating patients with thrombosis without showing any adverse effect, we studied effects on coagulation and fibrinolysis in normal human subjects. When highly purified human PC was administered intravenously to healthy subjects, plasma levels of immunoreactive PC decreased with a half-life of 10.9 h. Intravenously administered APC decreased with a half-life of 23 min as measured by prolongation of activated partial thromboplastin time (APTT). However, 1.7 h was obtained for the plasma half-life of APC when it was measured immunologically. These findings suggested that a significant fraction of the administered APC was rapidly inhibited by plasma inhibitor. Upon administration of APC, APTT was prolonged and plasma levels of clotting factor VIII (F-VIII) decreased transiently as measured by clotting assay. However, when determined by a chromogenic assay method in which 120-fold diluted plasma samples were used, plasma levels of F-VIII remained unchanged. Plasma levels of F-V did not decrease after APC administration. These findings suggested that prolongation of APTT and apparent decrease in plasma F-VIII clotting activity might be due to the in vitro-effect of APC present in plasma samples used. Diurnal fluctuation of plasminogen activator inhibitor in normal subject was not affected by administration of APC. Thus, PC or APC seems to function selectively at the site of thrombin-formation without lowering plasma levels of coagulation factors.     

Effects of oral and transdermal estrogen replacement therapy on markers of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in postmenopausal women

We compared the effects of oral estradiol (2 mg), transdermal estradiol (50 microg), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in 27 postmenopausal women at baseline and after 2 and 12 weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the prothrombin activation marker prothrombin fragment 1+2 (F1+2). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-1 (PAI-1) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-1 activity, and increased D-dimer concentrations, suggesting increased fibrinolysis. The concentration of soluble E-selectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (LDL) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (HDL) cholesterol, apolipoprotein AI and apolipoprotein All concentrations increased significantly. LDL particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.     

Plasma levels of coagulation and fibrinolysis markers in acute ischemic stroke patients with lone atrial fibrillation

Atrial fibrillation (AF) is a well-defined risk factor for ischemic stroke. Patients with lone AF represent a subgroup of AF patients with the lowest lifelong stroke risk. Nonvalvular atrial fibrillation (NVAF) confers a hypercoagulable state resulting in an increased risk of thromboembolism. This study was performed to determine the contributory role of alteration in the hemostatic markers of thrombin generation and fibrinolysis in patients with lone AF during acute ischemic stroke episode. We studied thrombin-antithrombin complexes (TAT), prothrombin fragments 1+2 (F1+2), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) concentrations in patients with acute middle cerebral artery ischemic stroke due to atherosclerotic large artery disease (n=50), lone AF (n=24) and cardioembolism (n=21). The values were compared with those of age-matched control subjects with lone AF and sinus rhythm (n=21 and 15, respectively). The mean F1+2 concentration was higher in the control subjects with lone AF in comparison with those without AF (p=0.014). Patients with stroke due to possible cardioembolism, from lone AF or other causes, had higher TAT (and marginally higher F1+2) concentrations than those with atherosclerotic stroke (p<0.001). tPA concentrations were not different among groups (p=0.89). PAI-1 levels were marginally higher in stroke patients with lone AF and atherothrombotic large artery disease compared to the controls without AF (p=0.05). These results suggest that in the acute period of ischemic stroke secondary to lone AF, enhancement of the coagulatory activity occurs as a result of increased thrombin generation, similar to other possible sources of cardioembolism. Observed hemostatic alterations in acute ischemic stroke associated with lone AF may indicate some therapeutic and prognostic implications. Received: 3 April 2000 / Accepted in revised form: 20 September 2000     

Hemodynamic changes and systemic activation of coagulation and fibrinolysis during controlled endotoxemia in pigs

In this study, we have established a pig model that can combine extensive hemodynamic monitoring with simultaneous repetitive (serial) blood sampling for the study of multiple variables related to the hemostatic system. Sixteen healthy young pigs were studied to evaluate the influence of continuous endotoxin infusion on hemodynamic patterns and activation of coagulation and fibrinolysis. The chief aim of the study was to investigate the applicability of analytical methods primarily developed for use with human plasma samples in quantification of factors and reaction products of the porcine coagulation and fibrinolytic systems, and further, to use these methods to study the longitudinal changes in the plasma levels of these hemostatic variables as a consequence of endotoxin infusion. We found that acute, controlled endotoxemia induced a hemodynamic state of shock and reduced pulmonary gas exchange. Simultaneously, a gradual increase in peripheral blood mononuclear cell tissue factor activity was demonstrated, and increased maximally 5.5-fold 4 hours after onset of endotoxin infusion. Thrombin-antithrombin complexes increased in plasma to maximum levels after 3 hours, accompanied by an ethanol gelation test that was regularly positive after 1 to 2 hours, and fibrin monomer levels that gradually increased maximally 3.8-fold after 6 hours. These changes were followed by gradual decreases of both fibrinogen and factor VII levels, mainly due to consumption. Plasma levels of tissue type plasminogen activator activity peaked at 1.5 hours (11.3-fold increase), whereas the peak of plasminogen activator inhibitor-1 activity (14-fold increase at 4.5 hours) was delayed compared to tissue plasminogen activator and completely extinguished plasma tissue plasminogen activator activity. The sequential activation of coagulation and fibrinolysis established a procoagulant state favoring disseminated intravascular coagulation and microthrombus formation, potentially leading to multiple organ dysfunction.     

Activation of coagulation and fibrinolysis during surgery, analyzed by molecular markers

Activation of hemostasis during surgery was investigated in 30 elective cases, who underwent either gastric (group G) or hepatic (group H) resection by a serial determination of various molecular markers such as fibrinopeptide A (FPA), fibrinopeptide B beta 15-42 (B beta 15-42) D-dimer, thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC). In both groups, the values of FPA and TAT were significantly elevated intraoperatively, indicating an occurrence of hypercoagulable state. The degree of the elevation was more marked in group H, probably due to greater tissue damage during hepatic resection. Also in both groups, the values B beta 15-42 and PIC were significantly increased during surgery, while the amount of D-dimer was within normal range in most cases, indicating the occurrence of the primary fibrinolysis. These findings are compatible with our previous observations on the postoperative changes in hemostasis. There were statistically significant but variable correlations between the values of fibrinopeptides and the enzyme-inhibitor complexes. The absolute values of the molecular markers of fibrinolysis were always higher than those of coagulation, suggesting that a considerable amount of plasmin, rather than thrombin, is released by surgical tissue damages.     

Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples.

We sought to assess the longitudinal stability of risk factors for atherosclerosis and thrombosis. including several coagulation. fibrinolysis, and inflammation factors, in frozen plasma samples stored at -70 degrees C for months or years. We reviewed data collected on 29 different control pools over periods ranging from 7 to 59 months for two functional assays (factor VII and fibrinogen) and seven antigen measurements (C-reactive protein. D-dimer, plasmin-alpha2-antiplasmin complex, plasminogen activator inhibitor-1, protein C, protein S, and tissue plasminogen activator), totaling more than 15,000 data points. Screening of the data using least squares regression revealed only sporadic associations between monthly means and time, with no consistent trends. Analysis by repeated measures and summary measure methods revealed no evidence of sample degradation over time for the factors studied. Our finding of longitudinal stability in the biochemical properties of frozen plasma strengthens the presumption of sample stability on which molecular epidemiologic studies are based.     

The effect of ALG on coagulation and fibrinolysis

Rapid intravenous injection of ALG is known to induce an anaphylactoid reaction and platelet aggregation. It is also claimed to activate the coagulation system. In the present study, rapid intravenous injection of ALG in splenectomized dogs resulted in thrombocytopenia and decrease of fibrinogen. It was also found to increase the disappearance rate of ¹²⁵I-labelled fibrinogen. At the same time successively increasing levels of FDP were observed. The changes induced by ALG alone were significantly reduced by pretreatment of the dogs with Heparin, Trasylol or Aspirin. Protection was also given by thrombocytopenia. On the other hand, inhibition of the fibrinolytic system with AMCA resulted in formation of fibrin thrombi in the lung. It is suggested that ALG activates the coagulation system by trapping of platelets and leukocytes resulting in a vessel wall injury as well as an activation of the complement system.     

Marathon run I: effects on blood coagulation and fibrinolysis

Blood coagulation and fibrinolysis were assessed in 13 Finnish amateur runners aged 31 to 48, and one 65-year old taking part in a non-competitive marathon (42.2 km). After the run the mean values of partial thromboplastin time showed a very significant shortening, whereas the mean values of the prothrombin time and of plasma fibrinogen were not significantly altered. The mean values of euglobulin lysis time were significantly shorter and the mean values of fibrin degradation products increased highly significantly. After the run, protamine sulphate was positive or strongly positive in all subjects, whereas the ethanol gelation test was negative in all runners; no cryofibrinogen was detected in any participant. Thus, running a marathon race affects the haemostatic balance and activates the fibrinolytic mechanism. The effects of training and physical fitness on the above parameters are discussed.     

Protein C activity levels in endotoxin-induced disseminated intravascular coagulation in a dog model

The levels of protein C (PC) and other coagulation factors were monitored during endotoxin-induced disseminated intravascular coagulation (DIC) in the dog. Initial evaluation of the effectiveness of intradermal administration of bolus endotoxin quantities into the dog, demonstrated induction of DIC in the canine, without the severe side effects associated with bacterial sepsis. Quantitative determination of canine plasma protein C levels were performed using a multiple step amidolytic assay, that included a specific precipitation of the vitamin K-dependent proteins from citrated plasma, followed by thrombin activation (and neutralization) and subsequent measurement of the activated protein C (APC) by chromogen hydrolysis. This investigation demonstrated, that over a twenty-four hour interval, intradermal administration of endotoxin produces a gradual decrease in the PC activity levels, concomitant with a significant reduction in the Factor V, Factor VIII and fibrinogen levels and platelet count, and a prolongation of the Prothrombin Time and Partial Thromboplastin Time. During the first 6 hours, protein C levels fell below the pre-levels and remained significantly lower in the surviving dogs. Thus, this endotoxin-induced DIC animal model permits evaluation of various hemostatic parameters, yet diminishes the severe clinical findings associated with DIC.     

Activated coagulation/fibrinolysis system and platelet function in acute thrombotic stroke patients with increased C-reactive protein levels

The relationship between systemic infection or inflammation and an increased risk of thrombotic diseases has recently raised renewed interest. In order to determine the mechanisms underlying this relationship, we determined plasma levels of coagulation/fibrinolysis markers and platelet function in patients with acute thrombotic stroke (<24 h after onset) prior to treatment, and compared the results between cases with elevated and normal C-reactive protein (CRP) levels and controls. Plasma levels of thrombin-antithrombin complex (TAT), plasmin-antiplasmin complex, and D-dimer were significantly higher in patients with elevated CRP levels than in those with normal CRP levels and controls (P<0.005). Platelet aggregation induced by 1 and 10 microM ADP was significantly higher in patients with elevated CRP levels than those with normal CRP levels (P<0.05). These findings suggest that activation of the coagulation/fibrinolysis system and platelet function may be in part related to stroke onset in patients with increased CRP levels.     

The effects of gynaecological surgery on coagulation activation, fibrinolysis and fibrinolytic inhibitor in patients with and without ketorolac infusion

The effects of gynaecological surgery on the fibrinolytic and inhibitor mechanisms were followed up for 24h post-operatively in patients receiving a single dose of ketorolac infusion (n = 18) as compared with those not receiving ketorolac infusion (n = 11). A pre-operative state of lower mean t-PA activity and higher PAI-1 levels with increased platelet activation than that reported in normal subjects were observed in both groups of patients. Increased t-PA activity upon anaesthetic induction together with a decreased level at 24h post-operation was seen in both groups. However, fibrinolytic ‘shut-down’ was not evident as significant increase in D-dimer levels was observed post-operatively, suggesting an enhanced lytic state concurrent with an enhanced activation of coagulation and diminished platelet activation although β-TG remained above the normal level; plasmin from this enhanced lytic state affects platelet adhesion and cleaves platelet glycoprotein Ib thus inhibit release reaction. Ketorolac infusion elicited a significant response in PAI-1 activity within 24h post-operation and this was not seen in the non-ketorolac group in spite of the rising trend by 24h post-operation which did not achieve statistical significance. There were no statistical signifcant differences in blood loss and duration of surgery between the two groups of patients. Overall, both groups of patients showed similar haemostatic changes post-operatively for 24h, a longer duration of post-operative study would have revealed any subtle changes in the molecular markers of thrombosis which was not the objective of this study.     

Activation markers of coagulation and fibrinolysis: alterations and predictive value in acute coronary syndromes

Several alterations of the coagulation, of the fibrinolysis and of inflammation are known in patients with acute coronary syndromes. To extent current knowledge of the pathophysiology and to optimize therapeutical strategies, the new molecular markers can be used in clinical studies. Furthermore, several studies were undertaken to assess the prognostic value of activation markers of these systems for patients with unstable angina pectoris and acute myocardial infarction with or without thrombolytic therapy. The majority of studies focussed on markers of thrombin activation, fibrinogen, fibrin degradation products and t-PA and its main inhibitor PAI-1. While there are stimulating results from larger studies, the value for prognosis for the individual patient still is limited by the overlap of patients with good versus a poor outcome.     

Serial changes in fibrinolysis and coagulation activation markers in acute and convalescent phase of ischemic stroke

BACKGROUND: The changes in the activity of a number of plasma markers of coagulation and fibrinolysis have previously been studied in patients with ischemic stroke, with conflicting results. We aimed to find out the changes in the activities of a wide array of markers of the coagulation and the fibrinolytic system of mildly or moderately affected first-ever ischemic stroke patients. METHODS: In a prospective, longitudinal, case-control study, we studied plasma plasminogen activator inhibitor type-1 (PAI-1) activity, tissue-type plasminogen activator antigen (t-PA:Ag), d-dimer, prothrombin fragment 1+2 (F 1+2), and thrombin-antithrombin III complex (TAT) levels in 55 consecutive patients on admission, 1 week, 1 month, and 3 months after an ischemic stroke. Sex- and age-matched controls were studied once. All patients underwent blood sampling at each study time point; comprehensive stroke risk factors were recorded, and the etiology of the ischemic stroke was determined. All patients were contacted 3 years later for possible recurrent ischemic events. RESULTS: PAI-1 activity was increased in the acute phase and at 3 months, D-dimer levels were significantly higher at 1 week and 1 month after stroke, whereas t-PA:Ag, TAT and F 1+2 levels remained stable during the whole study period. CONCLUSIONS: The changes of the fibrinolytic and coagulation system activity in the patients with mild or moderate ischemic stroke appeared minor compared with the results of previous studies, which included more severely ill patients.     

The effects of angiotensin metabolites on the regulation of coagulation and fibrinolysis in cultured rat aortic endothelial cells

Not only angiotensin II (Ang II) but also other angiotensin metabolites such as angiotensin I (Ang I), angiotensin III (Ang III), angiotensin IV, or angiotensin 1-7 have recently been reported to have various activities. Few data, however, are available on the regulation of thrombus formation. In this study, we investigated the effects of angiotensin metabolites on the mRNA expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue type plasminogen activator (TPA) in cultured rat aortic endothelial cells. None of the used angiotensin metabolites altered TFPI or TPA mRNA expression levels. Ang I, Ang II, and Ang III made TF and PAI-1 mRNA inductions which were inhibited by an selective antagonist of angiotensin II type 1 receptors. These metabolites made TF predominant to TFPI or PAI-1 to TPA, and could render endothelial cells thrombogenic.     

The effect of dihydroergotamine on platelets, coagulation and fibrinolysis, studied in healthy humans and in dogs

The effect of dihydroergotamine (DHE) on platelets, coagulation and fibrinolysis was studied in healthy volunteers ex vivo before and after intravenous administration (0.5 mg). In addition, the effect of DHE in different concentrations on platelet aggregation was evaluated in vitro. Haematocrit was found to increase, as did factor VIII:C and antithrombin III - though the latter increases were not significant when correction was made for the altered haematocrit. Though platelet function ex vivo was unchanged, inhibition of adrenaline induced platelet aggregation was noted in vitro when the plasma concentrations of added DHE were higher than those obtained with clinically relevant doses. No effect on fibrinolysis was noted.     

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

Introduction

The aim of the present study was to establish whether the presence of chronic viral hepatitis (PVH) could be implicated in the elevation of oxidative stress (SOX) and haemostasis system in haemodialysis (HD) patients.

Matherials and methods

In HD patients with and without PVH and in controls we compared the markers of: coagulation pathway- tissue factor (TF) and its inhibitor (TFPI), prothrombin fragment F _1 + 2 (F _1 + 2); fibrinolysis: tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its soluble receptor (suPAR), plasminogen activator inhibitor 1 (PAI-1), plasmin/antiplasmin complexes (PAP); and a marker of SOX - Cu/Zn superoxide dismutase (Cu/Zn SOD) levels.

Results

Patients, particularly those with PVH, showed a significant increase in the markers of the coagulation, fibrinolysis and oxidative status as compared to controls. All parameters of coagulation/fibrinolysis system were directly associated with the PVH and Cu/Zn SOD levels, and there was a relationship between the PVH and Cu/Zn SOD levels. Multivariable analysis showed that PVH and increased SOX were identified as independent variables significantly associated with the disturbances of coagulation/fibrinolysis system in these patients.

Conclusions

We concluded that PVH is a novel determinant of the increased oxidative stress as well as the disturbances of coagulation/fibrinolysis system in haemodialysis patients.