A new euglobulin clot lysis assay for global fibrinolysis

Plasma fibrinolytic activity has been measured by the euglobulin clot lysis time (ELT) since the late 1950s. The euglobulin clot lysis assay (ECLA) method has been modified using a computerized kinetic spectrophotometric microtiter plate reader and measures optical density changes of recalcified euglobulin fraction of plasma samples over time. This method has been applied to normal healthy adults, children, pregnant women and newborn infants, which represent physiologic extremes of the ELT. The ECLA method adds measurements of maximum absorbance (Max Abs), area under the curve (AUC) and mean velocity to the standard clot lysis time. The resulting curves are unique to this method and have been analyzed and compared in order to establish normal ranges. Fibrinogen levels, plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity and thrombin activatable fibrinolytic inhibitor (TAFI) antigen levels were measured in each individual of the four groups. Each protein measured within each study group except TAFI correlated with the lysis time, maximum absorbance and area under the curve. Considering all four groups together, PAI correlates most highly with lysis time, fibrinogen correlates the highest with Max Abs; fibrinogen and PAI-1 antigen have equally high correlations to AUC. Area under the curve is highly correlated with all coagulation parameters measured; the most significant contributor is fibrinogen. These observations are interesting, but at this time, it cannot be said that any of the test parameters are better than lysis time in distinguishing between these normal physiologic states.     

Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and ¹²⁵I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.     

Influence of factor XIIIa activity on human whole blood clot lysis in vitro

We studied the influence of Factor XIIIa (F XIIIa) activity on the lysis rate of fresh whole human blood clots, without using anticoagulants. Clotting was induced by exogenous thrombin, lysis by tissue-type Plasminogen Activator (t-PA) added before clotting. After various periods of time, lysis rates were determined by measuring the radioactivity in the supernatant of the clot originating from 125I-Fibrinogen added before clotting. Lysis rates were determined in the presence of endogenous F XIIIa and compared with those obtained after specific inhibition of F XIIIa activity. We used an IgG fraction of an antiserum quenching the F XIIIa activity. Addition of increasing amounts of the antibodies to normal blood resulted in a dramatic increase in clot lysis rate, concomitant with loss of F XIII activity. Lysis of blood clots from a patient with a congenital, homozygous, functional alpha 2-Antiplasmin (alpha 2-AP) deficiency (alpha 2-AP-Enschede) was not or slightly increased by the anti F XIII antibodies indicating that fibrin-fibrin crosslinking per se does not contribute essentially to resistance of the blood clot against fibrinolysis. Both active alpha 2-AP and F XIIIa are required for the major part of the F XIII-dependent resistance of whole blood clots against lysis.     

Protraction of whole blood and plasma clot lysis in patients with high levels of an inhibitor of tissue-type plasminogen activator

The influence of the newly discovered, fast-acting inhibitor of tissue-type plasminogen activator (t-PA) on the lysis time of plasma clots was studied by visual observation of lysis of clotted citrated plasma after addition of purified t-PA. To a series of plasma samples with various concentrations of naturally occurring PA-inhibitor purified t-PA was added to a final concentration, which in pooled normal plasma is sufficient to induce clot lysis within a few hours. In those plasma samples with a high free inhibitor level, determined by measuring the recovery of the activity of added purified t-PA, clot lysis was retarded. Whole blood clots were made by clotting freshly collected non-anticoagulated blood with thrombin after admixture of a trace amount of radiolabeled fibrinogen and a fixed amount of t-PA. Lysis rate, read from the appearance of radioactivity in the serum after centrifugation, was significantly lower in clots obtained from subjects with a high free inhibitor level than in those with a low inhibitor level. It is concluded that the PA-inhibitor protracts clot lysis and may be relevant for physiological fibrinolysis.     

A precise and rapid microtitre plate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear to influence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

尿激酶溶凝时效性和量效性的体外研究

目的探讨不同剂量尿激酶溶解体外血凝块的效果,寻找最佳溶凝时间和用药剂量,为临床上合理应用尿激酶溶解液化颅内血肿提供实验依据。方法参照临床常见的高血压脑内血肿出血量,取180例健康志愿者静脉血60m1,加尿激酶0．5、1、2、5、10及15万U,测量加药后1、2、3、4、5、6h的溶凝效果,分析尿激酶最佳溶凝时间和用药剂量。结果尿激酶溶凝作用随时间延长逐渐降低,不同尿激酶剂量在加药后1、2、3、4h均有显著性差异（P〈0．05）;4h后无明显差异（P〉0．05）。各时间点,0．5、1、2万U尿激酶无显著性差异（P〉O．05）,5、10及15万U元显著性差异（P〉0．05）,但0、5、1、2万U和5、10、15万u间均有显著性差异（P〈0．05）。结论本研究提示尿激酶溶凝最佳作用时间为4h,尿激酶首次最佳用量为1万U／10ml。     

Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner. Study of seven plasminogen activators in an internal clot lysis model

TAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombo-modulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI.     

A history of early stent thrombosis is associated with prolonged clot lysis time

It has been demonstrated that formation of compact plasma fibrin clots resistant to plasmin-mediated lysis characterises patients following in-stent thrombosis (IST). The relationship between defective fibrinolysis, reflected as prolonged clot lysis time (CLT) and IST is unclear. We sought to investigate whether patients with acute and subacute IST have impaired fibrinolytic capacity. We studied 41 definite IST patients, including 15 with acute and 26 with subacute IST experienced 2-73 months prior to enrollment, versus 41 controls matched for demographics, cardiovascular risk factors, concomitant treatment and angiographic/stent parameters. CLT, reflecting lysis of a tissue factor-induced plasma clot by exogenous tissue plasminogen activator, together with plasminogen activator inhibitor-1 (PAI-1) antigen and activity, thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and activity, thrombomodulin (TM), plasminogen and α2-antiplasmin (α2AP) were measured. There were no inter-group differences in angiographic parameters, indication to the first PCI, culprit vessel or a type of stent. Patients with IST had 11% longer CLT (p=0.005) and 13% higher PAI-1 antigen (p=0.04) compared to controls. There were positive correlations in both groups between CLT and PAI-1 antigen and TAFI activity (all p<0.001). Multiple regression analysis showed that CLT (odds ratio [OR]=1.04 per 1 minute, 95% CI 1.01-1.08, p=0.02) and platelet count (OR=1.01 per 1,000/μl, 95% CI 1.00-1.02, p=0.034) were independent predictors of IST (R(2)=0.28, p<0.05). Concluding, impaired fibrinolytic potential, that is in part determined by plasma PAI-1 antigen and TAFI activity, characterises patients with a history of acute and subacute IST, which might help identify patients at higher risk of IST.     

A method of testing platelet aggregation in native whole blood

A method of testing collagen induced platelet aggregation and ATP release in native (= non anticoagulated) whole blood by monitoring the electrical impedance in the Chrono Log Whole Blood Aggregometer is reported. It is the first simple method by which aggregation of human platelets can be measured in their natural environment. In normal individuals lower threshold collagen concentrations could induce platelet aggregation as determined with this method than in the other tested methods (impedance method with citrated blood, optical method in platelet rich plasma). The aggregation response was not inhibited by hirudin or heparin in therapeutic dose. The luminescence channel of the Whole Blood Aggregometer permits measurements of ATP release in native whole blood.     

A quantitative method for analysis of in vitro neurite outgrowth

The adult mammalian CNS is extremely limited in its ability to regenerate axons following injury. Glial scar, neuroinflammatory processes and molecules released from myelin impair axonal regrowth and contribute to the lack of neural regeneration. An in vitro assay that quantitates neurite outgrowth from cultured neurons as a model of neuronal regenerative potential is described. Specifically, the neurite outgrowth from primary neurons (rat cerebellar granule neurons; CGNs) and a neuronal cell line (NG108-15) were quantitatively measured after optimization of culture conditions. After cultures were fixed and immunostained to label neurons and nuclei, microscope images were captured and an image analysis algorithm was developed using Image-Pro Plus software to allow quantitative analysis. The algorithm allowed the determination of total neurite length, number of neurons, and number of neurons without neurites. The algorithm also allows for end-user control of thresholds for staining intensity and cell/nuclei size. This assay represents a useful tool for quantification of neurite outgrowth from a variety of neuronal sources with applications that include: (1) assessment of neurite outgrowth potential; (2) identification of molecules that can block or stimulate neurite outgrowth in conventional culture media; and (3) identification of agents that can overcome neurite outgrowth inhibition by inhibitory substrates.     

Fibrinogen-fibrin related antigen pattern in human blood. Incomplete lysis of whole blood clots by Urokinase

The immunochemical pattern of Urokinase-induced lysis of whole blood clots (intrinsic lysis) in normals and patients have been studied. Despite 24 hours incubation at 37.5°C following clot lysis, further proteolysis seems to be markedly inhibited and degradation ends with one main precipitin line (probably representing a mixture of different fragments) and an additional anodic component, the latter sharing common antigen(s) with the other one. This electrophoretic pattern was qualitatively very similar in the majority of normals and in patients with acquired hemostatic disturbancies.     

Sensitive and quantitative, 10-min immunofluorometric assay for D-Dimer in whole blood

INTRODUCTION: Normal concentrations of D-Dimer can be used to exclude venous thromboembolism (VTE). However, methods for sensitive and quantitative D-Dimer measurements at the point-of-care (POC) are still limited. MATERIALS AND METHODS: We developed a 10-min, non-competitive immunofluorometric assay for D-Dimer in citrated whole blood and plasma using pre-dispensed reagents dried in single assay wells. The simple, automated assay procedure comprises a 1:50 sample dilution, one-step incubation, washing, and time-resolved fluorometric measurement directly from the wet well surface. RESULTS: The limits of detection (background + 3SD) and quantification (CV <15%) were 0.05 and 0.2 mg/L D-Dimer, respectively, and the assay was linear up to 400 mg/L. Correlations to Roche TinaQuant (r=0.726, n=200) and Biopool Auto.Dimer (r=0.190, n=149) were carried out using citrated plasma. Diagnostic sensitivity, specificity, and negative (NPV) and positive (PPV) predictive values were 98.7%, 64.4%, 99.1% and 55.1%, and 92.2%, 81.0%, 95.9% and 68.3%, respectively, using cut-off values of 0.6 and 1.0 mg/L, respectively, in outpatients with deep vein thrombosis (DVT) and/or pulmonary embolism (PE) (n=77) compared with outpatients with various other diseases (n=174). The within- and between-run CVs near the cut-off values were < or =10% in both whole blood and plasma. The 95th percentile upper range in apparently healthy individuals was 0.68 mg/L of whole blood (n=101). CONCLUSIONS: The high sensitivity and NPV suggest that the rapid immunofluorometric assay could be valuable for rapid exclusion of VTE in outpatients. With appropriate cut-offs, the assay could potentially be used as a stand-alone test or combined with clinical probability assessment, but further studies are required.     

A modified temporal self-correlation method for analysis of fMRI time series

Temporal self-correlation has recently been proposed as a measure for fMRI-activation detection. In this paper, a modified temporal self-correlation method is introduced. The modified temporal self-correlation is based on the expectation value and standard deviation of the correlation coefficients between all pairs of epochs, while the original temporal self-correlation method is only based on the expectation value. Performance of the proposed method is evaluated on both simulated and in vivo fMRI data. Compared with the original temporal self-correlation method, the proposed method shows a significant improvement. In addition, a technique for quantitative comparison of different fMRI data analysis methods is proposed.     

A new method for the determination of plasma activators fibrinolysis

The term "effective activator" of plasminogen is proposed, to denote the resultant of activator-antiactivator interaction, and a method for the determination of the level of these activators is described. By adding axcess plasminogen to the euglobulin fraction of plasma the influence of the level of endogenous plasminogen and of the antiplasmin is eliminated. It is shown that the level of fibrinogen has very little bearing on the results. An effective activator unit is defined as equal to 1 CTA unit of urokinase activity on a fibrinogen-plasminogen substrate.     

Dependence of blood clot lysis on the mode of transport of urokinase into the clot--a magnetic resonance imaging study in vitro

Magnetic resonance imaging was employed to study the dependence of clot lysing patterns on two different modes of transport of urokinase into whole blood clots. In one group of clots (nonperfused clots, n1 = 10), access of urokinase to the fibrin network was possible by diffusion only, whereas in the other group (perfused clots, n2 = 10) bulk flow of plasma containing urokinase was instituted through occlusive clots by a pressure difference of 3.7 kPa (37 cm H2O) across 3 cm long clots with a diameter of 4 mm. It was determined separately that this pressure difference resulted in a volume flow rate of 5.05 +/- 2.4 x 10(-2) ml/min through occlusive clots. Perfused clots diminished in size significantly in comparison to nonperfused ones already after 20 min (p less than 0.005). Linear regression analysis of two-dimensional clot sizes measured by MRI showed that the rate of lysis was more than 50-times faster in the perfused group in comparison to the nonperfused group. It was concluded that penetration of the thrombolytic agent into clots by perfusion is much more effective than by diffusion. Our results might have some implications for understanding the differences in lysis of arterial and venous thrombi.     

A method of automated quantitative analysis of brain slices microphotographs

Automated quantitative analysis allows us to save the time necessary for data analysis after immunohistochemical staining of biological material in routine experiments. In this study, we present a method of quantitative analysis that was developed for the processing of brain slices stained immunohistochemically for glial fibrillary acid protein. We also showed some experimental examples of application of this method.     

A study of transformations of blood platelets intracellular granules during clot formation by means of fluorescent marker method

Fluorescent basic drugs have been shown to selectively accumulate in granules of blood platelets. The transformation kinetics of these organelles was studied in the course of clot formation. Simultaneous recording of fluorescence intensity and light transmission demonstrated that processes of fibrin filaments sedimentation and fibrin-blood platelet interaction were followed by the “collapse” of cells and liberation of the fluorescent marker. The platelet degranulation takes place due to contractile properties of platelets. This process was found to be inhibited by cytochalasin B and colchicine. Redistribution of platelets into aggregated, caused by the addition of ADP and stirring, led to the inhibition of the liberation. These experiments provide the evidence that secretion by platelets is realized by different mechanisms. Fluorescent basic drugs are a useful tool for investigating the function of the granules of blood platelets.