Characteristics and prognostic impact of plasma fibrin monomer (soluble fibrin) in patients with coronary artery disease.

We measured fibrin monomer (FM), soluble fibrin, as a marker of thrombin activity in plasma samples obtained in parallel with the first two routine samples for cardiac markers in 165 patients with acute chest pain admitted consecutively to our hospital. A reference limit of FM in a healthy population was set at 3.0 mg/l. Elevated plasma FM was observed in 48.8% of patients with acute coronary syndromes, in 42.3% of patients with specific non-coronary disease, in 31.5% of those with stable angina pectoris and in 18.2% of patients with non-specific chest pain. No significant difference was observed between sample 1 and sample 2 in patients not receiving thrombolytic treatment during the sampling period (P = 0.46). In patients with coronary artery disease, FM was significantly related to the level of cardiac troponin T (P = 0.001), but no correlation was observed between the individual plasma FM and cardiac troponin T values. Outcome analysis during the following 30 months after the index event in patients with acute coronary syndromes revealed higher FM levels in those with coronary re-events or death than in patients without new events (P = 0.001). This observation indicates a prognostic potential of FM in risk evaluation of patients with coronary artery disease.

Assessment of haematuria: automated urine flowmetry vs microscopy.

BACKGROUND: Microscopy of the urine sediment may be a useful method in the distinction between a glomerular and a non-glomerular source of urinary bleeding. However, microscopic techniques are time consuming and hampered by inter-observer variations. In the present study we have therefore compared bright-field microscopy with automated urine flowmetry (Sysmex UF-100), examining their ability to differentiate between glomerular and non-glomerular haematuria. METHODS: Fresh urine samples were obtained from 112 patients with a well-defined, single cause of a positive dipstick test. Their urine specimens were examined within 4 h in a blinded manner. Of them, 79 specimens had a positive dipstick for blood and thus could be evaluated for haematuria. RESULTS: The Sysmex UF-100 had a sensitivity and specificity of 0.83 and 0.94 respectively in detecting non-glomerular bleeding. The positive and negative predictive values were 0.95 and 0.78 respectively. The corresponding values of microscopy were 0.79 and 0.90 respectively, and 0.93 and 0.74 respectively. CONCLUSIONS: Automated flowmetry can be used in the distinction between glomerular and non-glomerular haematuria.

Plasma total homocysteine response to oral doses of folic acid and pyridoxine hydrochloride (vitamin B6) in healthy individuals. Oral doses of vitamin B6 reduce concentrations of serum folate.

Plasma total homocysteine response was compared in four groups of healthy individuals given orally divided doses of vitamin supplementations for a duration of 5 weeks. The vitamin supplements; A, 0.3 mg folic acid; B, 120 mg vitamin B6; C, combination of 0.3 mg folic acid and 120 mg vitamin B6 or D, 0.6 mg folic acid reduced the concentrations of plasma total homocysteine 20, 17, 32 and 24%, respectively. However, the intergroup comparisons did not show a significant difference in the effects of vitamin supplements. Multivariate analysis with correction for differences in pre-supplement values indicated a significant effect of vitamin B6 supplementation on plasma total homocysteine and serum folate. Our data show that plasma total homocysteine concentrations are reduced with low to medium divided doses of folic acid alone or in combination with vitamin B6.

Atherothrombogenic risk modulation by n-3 fatty acids was not associated with changes in homocysteine in subjects with combined hyperlipidaemia.

Favourable effects of n-3 fatty acids on the atherogenic risk profile were recently demonstrated in subjects with combined (type IIb) hyperlipidaemia, not responding to a therapeutic diet. Re-examination of a previous patient material was performed to assess the influence of n-3 fatty acids on homocysteine and several coagulation factors. Subjects were randomly allocated to receive either a concentrated compound of 85% eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) (n = 28), or corn oil (n = 29), in a daily dose of 4g for 12 weeks. The intervention was double-blind. Homocysteine remained unchanged in both groups after 12-week treatment. N-3 fatty acids supplementation did not affect the levels of fibrinogen, coagulation factor VII or tissue factor pathway inhibitor (TFPI), while plasminogen activator inhibitor (PAI) increased significantly (Student's t-test; p <0.05). Total blood platelets were significantly reduced in subjects receiving n-3 fatty acids (Student's t-test; p <0.05), whereas bleeding times increased non-significantly.

Cardiac troponins I and T in patients with suspected acute coronary syndrome: a comparative study in a routine setting.

We compared cardiac troponin I (cTnI), using Access, Sanofi Pasteur, and cardiac troponin T (cTnT), using Elecsys, Boehringer Mannheim, in the first two routine blood samplings in a routine panel of cardiac markers for the biochemical diagnostic evaluation of patients with symptoms of acute myocardial infarction (AMI). No significant differences in the overall clinical performances of cTnI and cTnT were observed for the diagnosis of AMI (n = 68), but cTnI demonstrated lower initial sensitivity and higher specificity compared with cTnT. cTnT was increased to higher relative values than cTnI (P = 0.023). Discordances were found between cTnI and cTnT in sample I but not in sample II; positive cTnT/negative cTnI was more common than the opposite discordance (P = 0.027). cTnT was more frequently increased in patients with unstable angina pectoris (UAP) than cTnI (P = 0.038), with no significant differences between sample I and sample II; discordant results with respect to cTnI and cTnT appeared in 6 (33%) of these patients, all of which were positive for cTnT and negative for cTnI. Four patients with UAP (22%) developed AMI within 4 months; three were associated with increased cTnI and cTnT at the time of initial testing, and one was discordant (positive cTnT). In patients classified with no acute coronary syndrome (n = 84), five concordant positives for cTnI and cTnT were observed, indicating the existence of a myocardial injury of recent origin in these patients. AMI evolved in one of these patients 5 months later. We conclude that cTnT and cTnI detect acute myocardial injury with equal clinical performance in AMI patients classified by WHO criteria. cTnT was more frequently increased in patients with UAP than cTnI, but the clinical significance of this discordance could not be determined from this study.

Systemic thrombin generation and activity resistant to low molecular weight heparin administered prior to streptokinase in patients with acute myocardial infarction.

One hundred patients were included in a randomized open trial to assess the systemic factor Xa (FXa) and thrombin inhibitory effect as well as the safety profile of low molecular weight heparin (LMWH) given subcutaneously in conjunction with streptokinase (SK) in patients with acute myocardial infarction (MI). The treatment was initiated prior to SK, followed by repeated injections every 12 h for 7 days, using a dose of 150 anti-Xa units per kg body weight. The control group received unfractionated heparin (UFH) 12,500 i.u. subcutaneously every 12 h for 7 days, initiated 4 h after start of SK infusion. All patients received acetylsalicylic acid (ASA) initiated prior to SK. Serial blood samples were collected prior to and during the first 24 h after initiation of SK infusion for determination of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III (TAT) complexes, fibrinopeptide A (FPA) and cardiac enzymes. Bleeding complications and adverse events were carefully accounted for. Infarct characteristics, as judged by creatine kinase MB isoenzyme (CK-MB) and cardiac troponin T (cTnT), were similar in both groups of patients. A comparable transient increase in F1 + 2, TAT and FPA was noted irrespective of heparin regimen. Increased anti-Xa activity in patients given LMWH prior to thrombolytic treatment had no impact on indices of systemic thrombin activation. The incidence of major bleedings was significantly higher in patients receiving LMWH as compared to patients receiving UFH. However, the occurrence of bleedings was modified after reduction of the initial LMWH dose to 100 anti-Xa units per kg body weight. In conclusion, systemic FXa- and thrombin activity following SK-infusion in patients with acute MI was uninfluenced by conjunctive LMWH treatment.

Cardiac markers in the early hours of acute myocardial infarction: clinical performance of creatine kinase, creatine kinase MB isoenzyme (activity and mass concentration), creatine kinase MM and MB subform ratios, myoglobin and cardiac troponin T.

We compared early markers of acute myocardial infarction (AMI) in the first 6 h from the onset of symptoms in 133 non-traumatized patients arriving at the emergency department with chest pain suggestive of AMI. Clinical performance parameters were calculated on the basis of 45 patients with AMI and 88 patients with a non-AMI diagnosis. At admission and in the first 0-3 h after the onset of chest pain the creatine kinase-MB (CK-MB) subform ratio was the most sensitive test at a comparable specificity level of 0.95. In the time interval of 3-5 h, myoglobin, the CK-MB mass concentration and the CK-MB subform ratio were associated with the greatest areas under receiver operating characteristic (ROC) curves, but differences between these tests were small and non-significant. At 6 h from the onset of pain, differences in clinical performance between the same three tests were even smaller whether or not samples drawn after the start of thrombolytic treatment were included in the test comparison. For confirmation of AMI at 6 h after onset of pain, CK-MB (activity and mass concentration) demonstrated the highest positive likelihood ratio, and for exclusion of AMI at 6 h the CK-MB subform ratio was associated with the highest negative likelihood ratio. However, differences between the CK-MB subform ratio, CK-MB mass concentration and myoglobin were not significant as estimated by the substantial overlap between the confidence intervals of the likelihood ratios and the ROC areas at 6 h. Cardiac troponin T (cTnT) demonstrated an ROC area equal to the CK-MB isoform ratio and myoglobin at 6 h. However, the likelihood ratio for ruling out AMI was lower, mostly due to the elevated cTnT in unstable coronary disease not defined as AMI. We conclude that the CK-MB subform ratio, CK-MB mass concentration and myoglobin do not demonstrate any significant differences in clinical performance for ruling in or ruling out acute myocardial infarction at 6 h after the onset of chest pain.

Cardiac troponin T immunoassay on biotin--streptavidin-coated microplates: preliminary performance in acute myocardial infarction.

We have adapted the cardiac troponin T (TnT) immunoassay system (ELISA troponin T, Boehringer Mannheim, Germany), which is based upon streptavidin-biotin immunoassay technology, to a sensitive microplate system. A coating of microplates with biotinylated bovine serum albumin (biotin-LC-BSA) remained stable for months. A secondary streptavidin coating was prepared as the first step of the assay. By using o-phenylenediamine (o-PD) as a substrate for the peroxidase-anti-TnT conjugate, the system allowed rapid kinetic measurement of TnT levels. The upper limit of a reference population (97.5th percentile) was found to be 0.04 mu g l-1. Intra-assay imprecision at 0.08 mu g l-1 was 8%, and 3-4% between 0.28 and 4 mu g l-1. Between-assay imprecision was 6.2% at 0.28 mu g l-1. Studies of TnT and CK-MB mass concentration in acute myocardial infarction patients, treated with streptokinase, demonstrated a clinical sensitivity of the TnT microplate system similar to that of the CK-MB mass concentration test, during the first 8 h after initiation of thrombolytic therapy, at discriminator levels of 0.1 mu g l-1 (TnT) and 8 mu g l-1 (CK-MB mass concentration). The early CK-MB/TnT ratio was lower in patients with signs of successful reperfusion (early peak CK-MB) than in the remaining patients (p<0.001). Serum samples from two patients with renal failure and one patient with rhabdomyolysis demonstrated strong non-linear behaviour with dilution, indicating the presence of an interfering factor. Kinetic measurement compared favourably with end-point analysis with respect to sensitivity and total analysis time. The system described greatly reduces the costs of TnT measurements compared to the ES systems. The total assay time is 70-90 min.

Improvement of serum lipids and blood pressure during intervention with n-3 fatty acids was not associated with changes in insulin levels in subjects with combined hyperlipidaemia.

OBJECTIVES: To investigate the effect of an omega-3 fatty acid concentrate K85 on serum lipids, lipoproteins, insulin metabolism and blood pressure in subjects with combined hyperlipidaemia. DESIGN: After a run-in dietary period of 10 weeks, subjects were randomly allocated to receive either encapsulated K85 (n = 28) or corn oil (n = 29). The intervention was double-blind. SETTING: An outpatient centre in Stavanger, Norway. SUBJECTS: Fifty-seven of 141 individuals, who, after the run-in period continued to meet the inclusion criteria: serum triglycerides of > or = 2.0 mmol L-1 and total serum cholesterol of > or = 6.0 mmol L-1. INTERVENTION: Encapsulated K85, containing 85% eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA), or corn oil, both administered in a daily dose of 4 g for 12 weeks. MAIN OUTCOME MEASURES: Change in metabolic and haemodynamic parameters related to intervention. RESULTS: Serum EPA and DHA increased significantly in the K85 group during the treatment period. The body-mass index remained unchanged. A 28% reduction in serum triglycerides was noted in the K85 group from the first 4 weeks. Total serum cholesterol was significantly reduced with both regimens. Serum high-density lipoprotein cholesterol increased significantly during the first 8 weeks in the K85 group. Significant reductions in systolic and diastolic blood pressures were noted in subjects on K85. The treatment did not affect serum glucose, plasma insulin and proinsulin levels. Insulin:glucose and proinsulin:glucose ratios remained unchanged. CONCLUSIONS: The atherogenic risk profile was improved with K85 in subjects with combined hyperlipidaemia, but n-3 fatty acids supplementation did not affect glucose/insulin homeostasis.

Procoagulant (thromboplastin) activity in human bronchoalveolar lavage fluids is derived from alveolar macrophages.

Fibrin deposition in the alveolar space and the lung interstitium is a prominent feature of many types of inflammatory pulmonary diseases. Cells of the monocyte/macrophage line are the primary cells supplying procoagulant activity in inflammatory lesions. In the present study we found that both lung alveolar macrophages (LAM) and bronchoalveolar lavage fluids (BALF) from humans contained procoagulant activities. The procoagulant in BALF was associated with membrane vesicles which sedimented at 100,000 g for 1 h. By electron microscopy the BALF ultrasediment was seen to consist almost exclusively of membrane material and this was confirmed by monitoring the content of different marker enzymes for specific subcellular structures. Using macrophage membrane markers, at least part of the BALF-ultrasediment was shown to be derived from LAM. On the basis of phospholipase C sensitivity, antibody neutralization and the site of action of the procoagulant in the sequential activation of coagulation factors, both the LAM-associated and the BALF-associated procoagulant activity was identified as thromboplastin (tissue factor) or thromboplastin-factor VII complexes. This suggests that alveolar macrophages and the LAM-derived thromboplastin-containing microvesicles may contribute to intraalveolar and interstitial fibrin deposition in vivo and probably also have consequences for the development of pulmonary fibrosis.

Effects of phospholipase C, a tissue thromboplastin inhibitor, on pulmonary microembolism after missile injury of the limb.

Tissue thromboplastin probably plays an important role in the development of post-traumatic pulmonary microembolism. Infusion of purified human tissue thromboplastin in animals resulted in an intravascular coagulation and respiratory insufficiency. This could be inhibited by previous infusion of phospholipase C (PLC) from Bacillus cereus. We have studied the effects of PLC infusion on the course of post-traumatic pulmonary microembolism, induced by a high-energy (c. 700 J) missile trauma to the hind legs of pigs. The trauma resulted in a major muscular injury and an indirect femoral fracture. Untreated pigs developed intrapulmonary microemboli. The degree of microembolism in the lungs was measured quantitatively by external detection over the right lung of radiolabeled platelets and fibrin. Infusion of 80 micrograms PLC/kg/hour resulted in an accumulation of blood PLC associated with toxic reaction leading to increasing tachycardia and circulatory collapse after 10 hours. PLC infusion of 20 micrograms/kg/hour did not inhibit the pulmonary microembolism. A PLC-dose in between, viz. 40-50 micrograms/kg/hour, proved to efficiently inhibit most of the microembolism during the infusion period. Cessation of PLC infusion after 24 hours was accompanied by a later increase in pulmonary trapping of platelets and fibrin and decreases in paO2. Concomitantly there were opacities seen on chest X-rays. The results show that tissue thromboplastin is an important etiologic factor in post-traumatic pulmonary microembolism and that inhibition with phospholipase C can be of value in the prophylaxis of the syndrome.

Thromboplastin (tissue factor) in plasma membranes of human monocytes.

The synthesis of thromboplastin, a potent trigger of blood coagulation, can be induced in human peripheral blood monocytes. Indirect evidence suggests that newly synthesized thromboplastin becomes in part available on the cell surface. We have attempted to study the localization and availability of thromboplastin more directly by isolating plasma membranes from isolated human peripheral blood monocytes. The specific activities of the plasma membrane markers increased 16-22-fold in these preparations with a recovery of about 15%. The contamination by mitochondria, lysosomes, nuclei and endoplasmic reticulum was low as estimated by marker enzymes and electron microscopy. In both unstimulated and stimulated monocytes thromboplastin was largely recovered in this plasma membrane fraction, providing direct evidence for its membrane localization. Phospholipase C (E.C. 3.1.4.3) is a potent inactivator of thromboplastin through its hydrolysis of the phospholipids necessary for thromboplastin activity [Otnaess, Prydz, Bjørklid & Berre (1972) Eur. J. Biochem. 27, 238-243]. About 70% of the total membrane thromboplastin activity was inactivated when whole cells were treated with phospholipase C and the membranes subsequently isolated. Following stimulation to induce thromboplastin synthesis, the plasma membranes showed a shift in their relative content of phosphatidylcholine and phosphatidylethanolamine consistent with a transmethylation process.

Inhibitory effect of 3-deazaadenosine on the thromboplastin response of stimulated human monocytes.

Immune complexes (IC), 12-O-tetradecanoylphorbol-13-acetate (TPA), endotoxin (LPS) and phytohaemagglutinin (PHA) induce thromboplastin activity in human peripheral blood monocytes. In the presence of transmethylation inhibitors 3-deazaadenosine (DZA) and homocysteine a dose-dependent inhibition of the thromboplastin response reaching about 60 per cent was observed, when IC, LPS or PHA was used as the stimulant. TPA-induced thromboplastin synthesis was more resistant (maximum 20 per cent inhibition).

The effect of phospholipase C in sheep.

Based on earlier studies in rats, phospholipase C (PLC) seemed to be a very promising prophylactic agent for certain types of thrombo-embolic disease. Recent studies in rabbits have, however, demonstrated that phospholipase C is more toxic than expected from the previous data. To gain more knowledge about its toxicity in larger animals we have studied its effect in sheep. Estimated LD50 for the enzyme in sheep was between 0.4 and 0.2 mg PLC/kg given as a 23 min infusion and below 0.2 mg/kg given as a bolus. Cellular necrosis was a common feature in several tissues of sheep dying from PLC. This explained the pulmonary oedema, decreased oxygen tension and renal failure with haematuria, proteinuria and glucosuria which occurred. PLC was probably filtered out in the glomeruli and totally reabsorbed in the tubuli until they were destroyed by PLC. An increase in different plasma enzymes suggested that PLC exerted a toxic effect on both muscle cells and hepatocytes. The blood glucose level remained about 20% lower in the PLC-treated animals than in the controls for more than 2 weeks. Pulmonary oedema and renal failure were the probable causes of death.

Comparison of protein and lipid composition of the human platelet alpha-granule membranes and glycerol lysis membranes.

Platelet glycerol lysis membranes and alpha-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to beta 2-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the alpha-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of 125I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000-135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the alpha-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.

Monocytes and platelets share the glycoproteins IIb and IIIa that are absent from both cells in Glanzmann's thrombasthenia type I.

By means of an antiserum specific to the complex of the platelet membrane glycoproteins IIb and IIIa we demonstrate here that monocytes and purified monocyte membranes share these glycoproteins with platelets. The monocyte glycoprotein IIb-IIIa complex showed complete immunological identity with the platelet counterpart and, furthermore, dissociated after EDTA treatment exactly as did the platelet complex. In Glanzmann's thrombasthenia type I, monocytes as well as platelets lack this antigen completely.

Intravenous injections of tissue thromboplastin and phospholipase C in rabbits.

Previous experiments (Gollub et al. [10]. Giercksky et al.) have shown that phospholipase C (PLC) has a protective effect on rats and rabbits receiving thromboplastin infusions. As a step towards the possible use of PLC in humans, we have investigated further the use of PLC in rabbits. Infusions of human or rabbit thromboplastin caused a rapid fall in factors V and VIII. LD50 for our standard human thromboplastin preparation was estimated to be 1.5--1.8 ml/kg. Highly purified PLC was given as an intravenous bolus immediately before thromboplastin. No improvement of survival and only a slight difference in factors V and VIII were observed, in contrast to earlier observations in the rat. This may in part be due to differences in the susceptibility to PLC of the thromboplastins used. PLC was also more toxic in rabbits than in rats.