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Fibrin clots with reduced permeability, increased clot stiffness and reduced fibrinolysis susceptibility may predispose to cardiovascular disease (CVD). Little is known, however, about the structure of fibrin clots in patients with end-stage renal disease (ESRD). These patients suffer from a high risk of CVD in addition to their chronic low-grade inflammation. Using permeability, compaction and turbidity studies in 22 ESRD patients and 24 healthy controls, fibrin clots made from patient plasma were found to be less permeable (p < 0.001), less compactable (p < 0.001), and less susceptible to fibrinolysis (p < 0.001) than clots from controls. The maximum rate of turbidity increase was also higher for the patients than controls (p < 0.001), and scanning electron microscopy revealed higher clot density of fibrin fibers in clots from patients than clots from controls (p < 0.001). Patients had higher plasma concentrations of fibrinogen, C-reactive protein and interleukin 6 than controls. These plasma markers of inflammation correlated significantly with most of the fibrin structure characteristics observed in the patients. In contrast, plasma markers of azothemia showed no such correlations. The results suggest that in ESRD patients fibrin clots are significantly different from healthy controls, and that the fibrin structure characteristics in the patients are associated primarily with the inflammatory plasma milieu rather than with level of azothemia.

Genetic analyses are increasingly integrated in the clinical laboratory, and internal quality control programmes are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) by ammonium acetate precipitation and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i. e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 microg/ml blood, A260/A280 ratio >1.75, negative DNAsis tests), and the reagent blank was contaminated in <1% of the results. Occasionally, results were re-analysed because of positive reagent blanks (< 1%) or because of problems with the controls (<5%). On confirmation, we observed 4 genotyping discrepancies. Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses.

Do extremely old persons have a genetically favourable profile which has protected them from cardiovascular death? We have tried to answer this question by measuring DNA polymorphisms of selected cardiovascular risk indicators [factor VII, FVII (R/Q353, intron 7 (37bp)n, and -323ins10), beta fibrinogen (-455G/A), plasminogen activator inhibitor type 1, PAI-1 (-675(4G/5G)), tissue plasminogen activator, t-PA (intron 8 ins311), platelet receptor glycoprotein IIb/IIIa, GPIIb/IIIa (L/P33), prothrombin (20210G/A), methylene tetrahydrofolate reductase, MTHFR (A/V114), angiotensin converting enzyme, ACE (intron 16 ins287), and angiotensinogen (M/T235)]. Blood was collected from 187 unselected Danish centenarians, and 201 healthy Danish blood donors, aged 20-64 years (mean age 42 years). Genomic DNA was amplified using PCR and the genotype was determined by RFLP methods or allele-specific amplification followed by agarose gel electrophoresis. The frequencies of the high-risk alleles in centenarians were: for FVII R/Q353 0.91; for FVII intron 7 (37bp)n 0.67; for FVII-323 ins10 0.90; for fibrinogen 0.16; for PAI-1 0.52; for t-PA 0.59; for GPIIb/IIIa 0.16; for prothrombin 0.008; for MTHFR 0.33; for ACE 0.52; and for angiotensinogen 0.36. Comparable frequencies were observed in the blood donors. Subgroup analysis of men and women separately gave similar results. The genotype frequencies in the centenarians and the blood donors were similar for all polymorphisms, and this study suggests that common variations in genes associated with cardiovascular risk do not contribute significantly to longevity.

Greenland Inuit are a population with a low risk of cardiovascular disease. Recently, we stated that frequencies of potentially high risk alleles of the apolipoproteins, fibrinogen, factor V, glycoprotein IIIa and factor VII (FVII) genes have different allele frequencies in the Inuit when compared with Caucasian populations. We have extended this study and evaluated whether or not this was also true for the genetic polymorphisms of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), angiotensin-converting enzyme (ACE) and angiotensinogen in a group of 133 Greenland Inuit, aged 30-34 gamma. In addition, we compared the plasma levels of these factors and those of C-reactive protein (CRP) and D-Dimer in Inuit and in Danes, comparable for age and gender. Frequencies (f) were assessed of the alleles that are known as the potential high risk alleles in Caucasians. In the Inuit, the f(insertion allele) of the t-PA intron8ins311 polymorphism was 0.37 (CI 0.32-0.43), the f(4G allele) of the PAI-1 promoter polymorphism was 0.88 (CI 0.83-0.91), the f(deletion allele) of the ACE intron16ins287 polymorphism was 0.40 (CI 0.33-0.47) and the f(M-allele) of the angiotensinogen M/T353 polymorphism was 0.30(CI 0.25-0.38). As for fibrinogen and FVII polymorphisms, these frequencies are all significantly different from what is reported for Caucasian populations. In the Inuit, plasma levels of fibrinogen and D-Dimer were higher than in the Danes, the PAI-1 levels were lower and FVII, t-PA and CRP levels were comparable. The observed allele frequencies of the polymorphisms of t-PA, fibrinogen, FVII, ACE, angiotensinogen and the plasma levels of PAI-1 and D-Dimer were in accordance with the low CVD risk in the Inuit, considering the observed associations between these measures and CVD risk in Caucasian populations, but for other measures this was not the case (allele frequencies of the PAI-1 polymorphism, and plasma levels of fibrinogen, FVII and t-PA). In conclusion there are clear differences in genetic background and plasma levels of risk factors in Greenland Inuit compared with Caucasian populations, and these differences were sometimes, but not always, in accordance with the observed low cardiovascular disease risk of the Inuit population.

This study was conducted in order to examine possible anticoagulant properties of the lungs during tissue thromboplastin-induced intravascular coagulation. Rabbit brain tissue thromboplastin (n = 17) or saline (n = 6 + 3) was infused above the right atrium (n = 11 + 3) of the heart or in the arcus aorta (n = 6) for a period of 120 min in non-pregnant New Zealand rabbits. Rabbits infused with tissue thromboplastin responded with significantly (p < 0.05) more excessive changes in a number of haemodynamic variables (heart rate, PaO2,PaCO2, blood pH etc.) compared with rabbits infused with saline. Similarly, the prothrombin time (p < 0.05) and the activated partial thromboplastin time (p < 0.05) were significantly more prolonged in rabbits receiving tissue thromboplastin compared with control animals. Also the concentration of blood platelets (p < 0.05), plasma fibrinogen (p < 0.05), antithrombin (p < 0.05), and protein C (p < 0.05) decreased significantly in thromboplastin-treated animals compared with control animals. In all these haemostatic variables there was a common trend that animals infused with tissue thromboplastin in the arcus aorta responded more excessively than animals infused in the right atrium of the heart, and these deviations were statistically significant for fibrinogen (p < 0.05) and prothrombin time (p < 0.05). Similarly, animals infused with tissue thromboplastin in the arcus aorta had an increased number of microthrombi in the lungs and kidneys compared with animals receiving tissue thromboplastin above the right atrium. As the lungs are the first pass organ when you infuse above the right atrium the results from this study suggest that the lungs play a key role in protecting the organism against excessive tissue thromboplastin-induced activation of coagulation.

Many reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25 young patients with a previous instance of spontaneous deep vein thrombosis documented by phlebography and in 50 healthy controls. The two populations were comparable with respect to a number of base-line variables (age, height, weight, etc.), while the patients had significantly lower fibrinolytic activity (p < 0.02), and significantly higher protein concentrations of t-PA (p < 0.0001) and PAI-1 (p < 0.0006). We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5.2 ng/ml and for PAI-1 18 ng/ml. The sensitivity and specificity for these cut-off points were for t-PA 73% (95% confidence interval 63%-84%) and for PAI-1 67% (confidence interval 55%-77%). The negative predictive value with a cut-off point t-PA concentration of 5.2 ng/ml was 85% (95% confidence interval 70%-94%). We observed a significantly negative association between concentration of t-PA and fibrinolytic activity (rs = -0.47; p < 0.005) and also between PAI-1 and fibrinolytic activity (rs = -0.78; p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the present study was to find out whether plasminogen activator inhibitor type-1 (PAI-1) controls the formation of plasmin in patients with ischaemic heart disease. We examined PAI activity, PAI-1 antigen, tissue type plasminogen activator (t-PA) activity, t-PA antigen, plasmin-alpha2-antiplasmin complex (PAP-complex) and fibrin degradation products D-dimer in 62 patients before (unstimulated) and after infusion of 1-desamino-8-D-arginine vasopressin (DDAVP; stimulated). DDAVP was used in a standardized dose to trigger the release of t-PA from the vascular endothelium. We observed that under basal conditions (unstimulated) median plasma t-PA activity for the whole group of patients was 86.5 mIU/ml (0-900), and after stimulation 2550 mIU/ml (0-6800), P < 0.0001; median plasma concentration of t-PA antigen was 14.7 ng/ml (7.0-115.5) under basal conditions, and after stimulation 34.1 ng/ml (15.8-58.6), P < 0.0001; median plasma PAI activity was 16.9 IU/ml (1.5-144.8) under basal conditions, and after stimulation 3.1 IU/ml (0-118.5), P < 0.0001; median plasma concentration of PAI-1 antigen was 21.5 ng/ml (8.1-132.2) under basal conditions, and after stimulation 14.9 ng/ml (4.8-149.0), P < 0.0001; the median plasma concentration of PAP-complex was 469.5 ng/ml (185.0-1802.0) under basal conditions, and after stimulation 695.5 (243.0-2292.0), P < 0.0001; median plasma concentration of D-dimer was 298.0 ng/ml (103.0-948.0) under basal conditions, and after stimulation 296.5 ng/ml (97.0-917.0), P < 0.0008.(ABSTRACT TRUNCATED AT 250 WORDS)

The tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery. The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours. Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracorporeal circulation (p less than 0.01), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors. Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p less than 0.01) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p less than 0.01). Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p less than 0.05). We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery.

In a longitudinal study the plasma levels of antithrombin-III, alpha 2-macroglobulin, alpha 2-antiplasmin, histidine-rich glycoprotein, and protein C were followed in two groups of patients with acute myocardial infarction (AMI), one with and one without deep vein thrombosis (DVT). None of the sequentially studied periods revealed significant differences between the two groups of patients. However, small but consistently higher levels of histidine-rich glycoprotein in patients with DVT suggested the existence among patients submitted for myocardial infarction of a subgroup with increased thrombophilic potential. It was concluded that the inhibitors studied are of little value as possible indicators of the presence of DVT at early stages of the disease when clinical signs are absent and when antithrombotic prophylaxis should preferably be initiated.

In a prospective study of deep vein thrombosis (DVT), detected by the Tc-plasmin test, in 34 patients with acute myocardial infarction sequential determinations were made in plasma by immunologic methods of histidine-rich glycoprotein (HRG) and total plasminogen and the concentrations of free plasminogen calculated. Mean plasma HRG concentrations were consistently higher in the group of patients, in which Tc-plasmin scanning had revealed the existence of DVT. The effect of HRG caused the level of free plasminogen to be only 50-60% of the level of total plasminogen. Fluctuations of HRG caused only minor changes in free plasminogen concentrations. Our data suggest that HRG acts as a weak, negative acute phase reactant.

Among 29 patients recovering from a first-time acute myocardial infarction (AMI) 9 patients suffered reinfarction during a four year follow-up period. The 9 reinfarction patients were found to belong to a subgroup of 17 patients with a selective depression to near zero of euglobulin t-PA activity. None of the group of 12 patients with marked t-PA activity suffered a relapse. Euglobulin fibrinolytic activity as usually assayed did not distinguish between groups with or without relapse.

An overestimation of the plasminogen concentration occurs in patients with elevated levels of fibrin degradation products (fdp) and/or fibrinogen, when using assays based on the activation of plasminogen by streptokinase (Sk) followed by the hydrolysis of a synthetic chromogenic substrate. This source of error could be overcome by addition of fibrinogen in excess to the plasminogen assay thereby obtaining maximum stimulation of the Sk-plasminogen complex. The late degradation products of fibrinogen and fibrin, Dcate and Ddimer, respectively, had no potentiating effect on the Sk-plasminogen complex. The modified assay was designed as a manual method or for a centrifugal analyzer. The excellent reproducibility is illustrated by the coefficient of variation (CV) within series being 1.3% (n = 24) and from day to day being 1.9% (n = 20).

We determined during the acute stage of myocardial infarction selected fibrinolysis variables (tissue-type plasminogen activator, intrinsic plasminogen activators, tissue-type plasminogen activator inhibition, C1-inactivator) and related the observed changes to changes in two acute phase reactants (C-reactive protein, fibrinogen). Acute myocardial injury induce significant increases in blood of tissue-type plasminogen activator inhibition (day one, p less than 0.05), C-reactive protein (day three, p less than 0.01), fibrinogen (day six, p less than 0.01), and C1-inactivator (day eight, p less than 0.01). Tissue-type plasminogen activator activity measured as C1-inactivator resistant fibrinolytic activity showed a minimum day two after the acute attack (p less than 0.01), whereas plasminogen activator activities arising from the intrinsic system of fibrinolysis remained constant. The observed changes did not parallel the occurrence of deep vein thrombosis indicated by a positive Tc-plasmin test (41% of the patients).