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The species specificity of thromboplastin (tissue factor) is an important fact to take into consideration when clotting assays or experiments are planned. We have (re)investigated all possible combinations of thromboplastin and plasma from several of the most commonly used species of experimental animals.
Substantial thrombomodulin activity could be detected in tissue thromboplastin preparations from placenta or from lung but not from brain. When the amount of these preparations was adjusted to contain 1 unit of tissue factor activity, up to 0.85 units of thrombomodulin activity could be measured, corresponding to the generation of 17 pmol/ml/min of activated protein C when 1.5 microM human protein C was activated by 20 nM human alpha-thrombin in the presence of 5 mM CaCl2. After treatment by phospholipase C, thrombomodulin activity was reduced in these samples. Addition of mixed brain procoagulant phospholipids partially restored thrombomodulin activity in the phospholipase C-treated samples. These results emphasize the role of phospholipids in the expression of optimal thrombomodulin activity in tissue thromboplastin preparations from placenta or from lung.
Thromboplastin activity in amniotic fluid from 39 patients pregnant in weeks 14-40 has been studied. Amniotic fluid was obtained by amniocentesis. All fluids were fractionated and the activity studied in both supernatant and sediment in a highly specific and sensitive test system. All samples tested showed a very high thromboplastin activity. No correlation to gestational age was found. Amniotic samples from 19 of the patients were incubated with endotoxin. Endotoxin did not further increase the procoagulant activity.
Mouse placental cells have been isolated and grown in cultures. These cells produce a procoagulant which is identical to thromboplastin (factor III) by two criteria. The procoagulant activity increases with time, culminating on the 5th day of culture. The increase is inhibited by cycloheximide, alpha-amanitin, and actinomycin D, showing that de novo synthesis of protein and RNA is necessary. About 90-95% of the total cellular thromboplastin activity in whole cells is available for inactivation by particle-bound trypsin and thus present on the cell surface. Endotoxin and phytohaemagglutinin did not further increase the procoagulant activity.
A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca(3)(PO(4))(2) adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185,000) and was specifically inhibited by an antiserum directed against activated PTA. These data suggested that PTA can be converted into its active form by trypsin. PTA was not activated by thrombin, chymotrypsin, papain, ficin, plasmin, plasma kallikrein, tissue thromboplastin, or C. Trypsin converted PTA to its active form enzymatically. Whether trypsin serves to activate PTA in vivo is not yet clear.
To determine the sensitivity for detection of coagulation factor deficiencies by commercial reagents for canine plasma, 5 commercial activated partial thromboplastin time (APTT) reagents with different types of contact activator and phospholipid of various origin were examined. Thirty canine plasma samples with minor or moderate deficiencies of coagualition factors that influence the APTT were examined. Significant differences were found for the sensitivity of various reagents, but no correlation was found with the type of contact activator. Following the test instructions provided by the manufacturers, the number of APTT results that were prolonged beyond the reference range varied between 20 and 30 (sensitivity = 0.67-1.00); the number of corresponding results using a standardized test protocol varied between 19 and 28 (sensitivity: 0.63-0.93). The most sensitive reagent contained kaolin as a contact activator and a human placental thromboplastin. The results of this study indicate that the APTT test optimized for human plasma is also a sensitive screening test of the intrinsic system of canine plasma, provided that a suitable reagent is used.
Circulating anticoagulants are endogenously produced substances that interfere with in vitro tests of coagulation like activated partial thromboplastin time (APTT), and cause prolongation of the clotting times. Evaluation of the abnormal APTT involving various factor assays and mixing studies may provide inconclusive and ambiguous results. Tissue thromboplastin inhibition test (TTIT) is one of the screening assays for detection of circulating anticoagulants. However, this test is influenced by the presence of unfractionated heparin (UFH) from concentrations 0.2 U/mL and higher. Since low-molecular-weight heparins (LMWHs) are increasingly used for the prevention of thrombotic disorders and may replace UFH in the future, in this study the authors studied the influence of LMWHs on the performance of TTIT and compared the results with UFH. UFH and LMWHs showed a prolongation of TTIT in the concentration range of 0.25-1.0 U/mL. The marked prolongation of the TTIT with UFH and different LMWHs is in decreasing order of UFH > ardeparin > tinzaparin > dalteparin > enoxaparin. Patients with circulating anticoagulants who are given LMWHs may have false-positive results of TTIT and this influence should be kept in mind during patient management.
Reference intervals for two well-recognized tests for the lupus anticoagulant were determined using 98 healthy subjects. The purpose of the study was to compare the reference intervals for the dilute tissue thromboplastin inhibition test on this group of healthy subjects calculated by parametric and nonparametric statistical methods, and to compare these results with results obtained on subsets of 20 and 40 randomly selected individuals from the group of 98. The same procedures were followed for the dilute Russell's viper venom test. Results were recorded in seconds of clotting times and in ratios (subject/mean of that set or subset). Statistical analysis revealed Gaussian distribution of the results in the large group as well as in each subset for both tests. The results showed more variation between sets of the dilute tissue thromboplastin inhibition test than of the dilute Russell's viper venom test. Nonparametrically calculated reference intervals were wider than those determined by the parametric method, especially if confidence intervals are provided for both reference limits in a group of 94 controls or in a subset of 40 subjects. The nonparametric technique uses all data for the calculation of reference interval of such sample sizes no matter how widely spread the results are. There was no significant difference between the reference intervals of subsets and the whole group (p > 0.05) calculated by both statistical techniques. Very few outliers were observed among these subjects in both tests.
The synergistic effect of antithrombin III (ATIII), activated protein C (APC) and heparin on the tissue thromboplastin (TP)-mediated coagulation cascade was studied. APC prolonged the activated partial thromboplastin time of human plasma with increasing APC concentration but affected the prothrombin time only slightly. Neither ATIII nor heparin prolonged the prothrombin time by itself, while a mixture of APC and heparin strongly inhibited the coagulation. When the effects of APC, heparin and ATIII on the TP-mediated coagulation were examined with a solution consisting of prothrombin, factor V, factor VII, factor X and fibrinogen at physiological concentrations, the coagulation time was prolonged only slightly by the APC-heparin or ATIII-heparin mixture. However, the coagulation time was prolonged markedly by simultaneous addition of APC, ATIII and heparin to the solution. Inhibition of thrombin activity by the ATIII-APC-heparin mixture was weak as compared with that by the ATIII-heparin mixture after a 1-min incubation, but after a 2-min incubation the inhibitory activities were equal. Suppression of thrombin activity by the ATIII-APC-heparin mixture was supposed to be due to the inhibition of the interaction between ATIII and heparin on thrombin by APC because the APC-ATIII-heparin complex was detected by crossed immuno-electrophoresis but not by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. When the inhibitory effect of APC alone or the APC-heparin mixture on the platelet prothrombin converting activity (PPCA) was examined, heparin accelerated the PPCA inhibition by APC.(ABSTRACT TRUNCATED AT 250 WORDS)
OBJECTIVE: To evaluate a point-of-care coagulation analyzer (PCCA) in dogs with coagulopathies and healthy dogs. ANIMALS: 27 healthy and 32 diseased dogs with and without evidence of bleeding. PROCEDURE: Prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were determined, using a PCCA and standard methods. RESULTS: Using the PCCA, mean (+/- SD) PT of citrated whole blood (CWB) from healthy dogs was 14.5+/-1.2 seconds, whereas PT of nonanticoagulated whole blood (NAWB) was 10.4+/-0.5 seconds. Activated partial thromboplastin time using CWB was 86.4+/-6.9 seconds, whereas aPTT was 71.2+/-6.7 seconds using NAWB. Reference ranges for PT and aPTT using CWB were 12.2 to 16.8 seconds and 72.5 to 100.3 seconds, respectively. Activated clotting time in NAWB was 71+/-11.8 seconds. Agreement with standard PT and aPTT methods using citrated plasma was good (overall agreement was 93% for PT and 87.5% for aPTT in CWB). Comparing CWB by the PCCA and conventional coagulation methods using citrated plasma, sensitivity and specificity were 85.7 and 95.5% for PT and 100 and 82.9% for aPTT, respectively. Overall agreement between the PCCA using NAWB and the clinical laboratory was 73% for PT and 88% for aPTT. Using NAWB for the PCCA and citrated plasma for conventional methods, sensitivity and specificity was 85.7 and 68.4% for PT and 86.7 and 88.9% for aPTT, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The PCCA detected intrinsic, extrinsic, and common pathway abnormalities in a similar fashion to clinical laboratory tests.
The purpose of the study was to evaluate whether the infusion of N-acetylcysteine decreased the measurement of prothrombin time and activated partial thromboplastin time (APTT) in healthy persons. N-acetylcysteine was administered intraveneously 10 mg kg-1 as a loading dose and then at a rate of 10 mg kg-1 h-1 for 32 h in six male subjects. The intrinsic, extrinsic and common pathway of coagulation were monitored with activated partial thromboplastin time (APTT), and prothrombin time, respectively. In addition, the extrinsic coagulation pathway was monitored with the clotting activity of single factors II, VII, and X. No effect on the intrinsic coagulation pathway was observed. There was a significant and rapid decrease in prothrombin time. Coagulation factors II, VII and X, the three components of prothrombin time, decreased significantly to different degrees. We conclude that infusion of N-acetylcysteine intraveneously decreases the prothrombin time in healthy subjects. Thus, one should not make conclusions which are too far-reaching based on prothrombin time alone in patients who have been treated recently with N-acetylcysteine intraveneously.
In a double-blind study 18 patients were randomized to receive a daily dietary supplement of concentrated ethyl ester compound of n-3 fatty acids or placebo (corn oil) for at least 6 weeks before coronary bypass surgery. Three-fold increase of serum eicosapentaenoic acid and 20% reduction of triglyceride levels were found preoperatively in the n-3 group, while the two groups were similar as regards monocyte and platelet counts, mean platelet volume and monocyte activation as expressed by thromboplastin activities. For determination of transcardiac gradients, coronary sinus and aortic blood were sampled preoperatively 5, 10 and 30 minutes after release of the aortic cross-clamp. In both patient groups the monocyte count was lower in coronary sinus than in aortic blood at 5 and 10 minutes, but the differences were not significant. The platelet counts showed no significant change. In vitro stimulation of monocytes, however, evoked significantly (p < 0.05) less thromboplastin activity in coronary sinus blood than in aortic blood at all three sampling times, without significant intergroup difference. The monocytes most sensitive to activation presumably were trapped in the reperfused myocardium, and this sequestration was not hindered by pretreatment with n-3 fatty acids.
As part of an international co-operative study of the International Committee for Standardisation in Haematology, human brain thromboplastins prepared by the acetone-extracted and phenol-saline extraction techniques have been compared as to the yield, the proportion of reagent discarded and wasted, and the sensitivity related to an international standard, the British Comparative Thromboplastin standard (BCT). The phenol-saline extract showed greatly improved sensitivity with greatly reduced batch-to-batch variation as compared with the acetone-dried preparation. In addition, the sensitivity of this reagent conformed to that of the BCT. The implications of the use of such a standardised reagent to the therapeutic management of oral anticoagulant therapy are: (i) standardised, reproducible results; (ii) results comparable with those carried out by other laboratories using a similarly standardised reagent; and (iii) stable therapeutic range.
In experiments with rats, antithrombin III (a natural inhibitor of fibrinolysis) was shown to decrease the activation of the anticoagulation system either in prophylactic administration or during the development of thrombogenesis, caused by intravenous administration of tissue thromboplastin. The phenomenon led to the maintaining of the more high content of fibrinogen in blood of experimental animals, to the shortening of the thrombin time and to the increase in content of alpha2-macroglobulin and alpha1-antitrypsin (in-hibitors of fibrinolysis) as compard with control animals, administered with the only thrombolastin. Effect of antithrombin III depended on periods of administration of the prearation and on injection of tissue thromboplastin.
Prothrombin time (PT) assays are clotting methods that measure the activity of vitamin K-dependent coagulation factors (F) II, VII, and X. There are three main types of PT assays in general usage, namely the Quick assay, Owren's assay and PT dry chemistry test cards. PT assays were initially developed to monitor dose-adjustments of vitamin K antagonists such as warfarin. The aim of the present study was to investigate whether commercially available PT assays are suitable for evaluating the anticoagulant activity of direct thrombin inhibitors. Melagatran, a reversible direct thrombin inhibitor, was added to human plasma at concentrations ranging from 0.1 to 2.0 micromol/l. Seventeen different commercially available PT kits were used, including thirteen Quick reagents, two Owren reagents and two PT test cards. The sensitivity of the different reagents, expressed as the concentration of melagatran that doubled the prothrombin time (IC50) varied widely, with Thromboplastin S and Thromboplastin HS being the most sensitive (IC50 = 0.9 micromol/l). The reagents with apparently the lowest sensitivity were the two Owren reagents Nycotest PT and SPA 50 with an IC50 of 2.2 and 2.9 micromol/L, respectively. This is most likely due to a higher dilution of melagatran in these assays compared to the dilution in the Quick assays. The results were also dependent on the International Sensitivity Index (ISI) of each reagent. The concentration of melagatran that produced an International Normalized Ratio (INR) of 2 was calculated from dose-response curves for each assay, and these results revealed that reagents with a high ISI value gave an INR of 2 at much lower concentrations of melagatran (0.5-0.7 micromol/L) than those with an ISI-values around one (0.9-1.2 micromol/L). It was found that INR depends not only on the plasma concentration of melagatran, but also on the sensitivity of the PT reagent and on the final dilution of the plasma sample in the prothrombin time assay. Thus, since the same melagatran concentration can be associated with widely varying PT/INR results depending on the specific assay used it is concluded that PT assays and INR can not be used to monitor melagatran activity.
The GAIT (Genetic Analysis of Idiopathic Thrombophilia) Project is a family-based study dedicated to elucidating the genetic basis of hemostasis-related phenotypes and thrombosis risk. In this paper, we have examined several lesser-studied hemostasis-related phenotypes in the 21 GAIT families: levels of vitamin B 12, serum folate, whole blood folate, alpha2-antiplasmin, prekallikrein, beta2-glycoprotein I, soluble P-selectin, factor XIII A and B subunits and a new coagulation measurement based on thromboplastin time in the presence or absence of thrombomodulin. Using the variance component method, we estimated the relative contributions of genetic and environmental influences on these phenotypes. In addition, we calculated the genetic correlations between thrombosis risk and each of these phenotypes. All 12 phenotypes showed significant genetic contributions with genes accounting for 22% to 78% of the variance after correction for covariate effects. Four phenotypes (three traits involving thromboplastin-thrombomodulin mediated coagulation time and serum folate) exhibited significant genetic correlations with thrombosis. Thus, some of the genes that influence quantitative variation in these physiological phenotypes also influence the risk of thrombosis. The high heritabilities and significant genetic correlations between thrombosis and some risk factors suggest that joint consideration of correlated quantitative phenotypes will aid in identifying susceptibility genes.
The most appropriate combination of tests for lupus anticoagulants (LA) is unknown. The standard double centrifugation method to prepare plasma was inadequate for platelet elimination, interfering with kaolin clotting time and mixing studies. In the present study, the percentage correction of activated partial thromboplastin time (APTT) and Russell's viper venom time (RVVT) by high compared with low concentrations of phospholipid was used for both screening and confirmation of LA. Abnormality in either one was reported as positive. The specificity of the tests in 122 individuals without LA was 100 per cent for RVVT and 96.7 per cent for APTT, which yielded false positive by heparin. The mixing study was omitted from the authors' strategy without decreasing the specificity. In 795 patients with thrombosis, LA was detectable in 6.03 per cent. The sensitivity of diluted activated partial thromboplastin time (dAPTT) and diluted Russell's viper venom time (dRVVT) alone, compared with the combination of the two was 83.3 per cent and 79.2 per cent respectively. Therefore, this new test scheme is a simple, inexpensive and efficient method for Thai patients.
Dynamics of protein C concentration was studied in rat blood after administration of thrombin and thromboplastin. Administration of 0.5 ml 1% thromboplastin caused fast decrease of protein C concentration, down to 60% of the initial level, within 3 min, while activity of factor V reached the minimal rate (30%) within 5 min. Content of protein C returned to the initial level in blood within 2-2.5 hrs and of factor V--within 6 hrs. After administration of thrombin 3 NIH in content of protein C was decreased to 91.3% whereas heparin was released only after injection of 6 NIH. The data obtained suggest that the protein C system responded earlier to occurrence of thrombin in circulation as compared with the neurohumoral regulators of the anticoagulation system; the protein C system is one of primary mechanisms of the antithrombosis defence.