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Assay conditions of a plasma-based ecarin clotting time (ECT) were evaluated and the precision of the ECT in monitoring plasma levels of r-hirudin assessed. The snake venom enzyme ecarin converts prothrombin to meizothrombin possessing only weak coagulant but strong esterase activity. In r-hirudin containing plasma samples, meizo thrombin is rapidly neutralized by r-hirudin resulting in a dose-dependent prolongation of the clotting times. Among the different assay conditions tested, addition of 50 microliters of ecarin (4 U/ml) to 100 microliters of undiluted citrate-anticoagulated plasma gave optimal results with respect to precision and reproducibility. The measuring range of the ECT performed in this way is about 0.02-5.0 micrograms/ml r-hirudin. In vitro studies performed on r-hirudin-spiked plasma samples of 50 healthy individuals demonstrate remarkably low interindividual differences in r-hirudin responsiveness as indicated by CV-values below 5% and 7% at r-hirudin concentrations between 0-3 micrograms/ml and 4-5 micrograms/ml, respectively. The specificity for r-hirudin of the ECT is further demonstrated by the strong correlation (r = 0.94) between the results of a chromogenic assay and the ECT-measured r-hirudin concentrations obtained on 67 ex vivo blood samples. Depending on the concentration of r-hirudin the ECT is sensitive to plasma levels of prothrombin. In the absence of r-hirudin the critical prothrombin concentration was found to be 20% but increasing to 60% in the presence of 2.0 micrograms/ml r-hirudin. A fibrinogen concentration of 50 mg/dl was found to be the minimal concentration independent of the r-hirudin concentration. The precision of the ECT in measuring plasma levels of r-hirudin is not influenced by treatment with heparin, aprotinin or oral anticoagulants. The data demonstrate that the ECT is a rapid and easily perfor mable clotting assay which allows accurate measurement of r-hirudin plasma levels. ECT-monitored hirudin treatment will help to establish optimal dose regimens that are more efficacious but still as safe as heparin.

A titration assay for the determination of the fast acting plasminogen activator inhibitor capacity (PAI-cap) was developed. Most of the hitherto published assays for fast acting PAI are reported to have the disadvantage of non-parallel titration curves of tissue plasminogen activator (t-PA) in buffer and plasma. In the assay reported here, this problem has been overcome by adequate predilution of the samples, thus achieving parallel titration curves regardless of the individual PAI-cap of a sample. Provided that values are calculated from parallel titration curves, reproducible PAI-cap values at different dilutions of a sample are obtained. This assay can be applied for the determination of PAI-cap in plasma, serum and other biological fluids as platelet releasates and endothelial cell conditioned medium. PAI-cap of plasma of 10 healthy male volunteers ranged from 15.3 to 32.3 arbitrary inhibitor units AU/ml (24.5 +/- 5.2, mean +/- SD). The alternative use of three different anticoagulants (citrate, EDTA, heparin) had no influence on PAI-cap determinations. Serum generated from blood contained a mean of PAI-cap of 129% in comparison to the plasma of the same donor indicating the release of PAI from cells during clotting. Plasma PAI-cap changed during the day with a constant decrease from the highest levels in the morning (100%) to low levels in the evening (62.9%). Platelets aggregated by thrombin released plasminogen activator inhibitor amounting to a mean PAI-cap of 4.33 +/- 2.98 AU per 2.5 X 10(8) cells.

Sodium citrate is almost always used to anticoagulate blood for the preparation of washed platelet suspensions. Several adverse effects of citrate on platelet functional responses have been reported. We investigated the extent of activation of platelets and plasmatic coagulation during the preparation of washed platelets from human blood to which no anticoagulant was added. Washed platelets from native blood (PNB) were prepared by passing freshly-drawn human blood rapidly through a mixed Sephadex G-25/G-50 column to remove divalent cations. Gel-filtered blood (GFB), diluted by the elution medium containing 0.35% albumin and 2U/ml apyrase, was obtained within 5 minutes of venepuncture. Using CaCl2, the GFB was found to contain a mean of 1.65 X 10 mM calcium. Fibrinopeptide A measurements indicated less activation of plasmatic coagulation in GFB than in citrated blood. Measurements of beta-thromboglobulin and platelet factor 4 during the various stages of the preparation of PNB showed no platelet activation. No platelet aggregates, as measured by the platelet aggregate ratio method, were observed in GFB. Transmission electron microscopy showed intact discoid platelets similar to those in platelet-rich plasma. The reduced activation of platelets and of plasmatic coagulation was due to the more effective removal of calcium ions from the blood by gel filtration than chelation by citrate. PNB may thus provide a model for studying the requirements for calcium in platelet function in the absence of any citrate-platelet interactions.

Monomeric 125I-desAA-fibrin and 125I-desAABB-fibrin were prepared by treating 125I-fibrinogen with thrombin. Preactivated factor XIII was added to 125I-fibrin/131I-fibrinogen mixtures, and after incubation for 2 and 6 hours, samples were investigated by SDS polyacrylamide gel electrophoresis under reducing conditions. The electrophoresis pattern showed a gamma-gamma band containing both 125I-fibrin and 131I-fibrinogen. These experiments indicate that a fibrin molecule polymerizes with a fibrinogen molecule in a similar way as a fibrin molecule polymerizes to a second fibrin molecule. This type of polymerization results in conformational changes of the molecules involved thus enabling FXIIIa for specific crosslinking reaction. The polymerization of fibrin and fibrinogen molecules as well as the crosslinking of fibrin to fibrinogen molecules seem to represent a mechanism for interrupting the process of further fibrin polymerization.

DesAA-fibrin Sepharose was produced by treating fibrinogen-Sepharose with batroxobin. DesAA-fibrin Sepharose was mixed with different concentrations of fibrinogen and at different ratios, and incubated with preactivated FXIII. After 2 hours at 37 degrees C, the Sepharose beads were separated by centrifugation and non-crosslinked fibrinogen was removed by twice times washing with guanidinium chloride, pH 4.1. Under these experimental conditions specific crosslinking of fibrinogen to immobilized desAA-fibrin by FXIIIa was found. These results support the concept of a specific interaction between fibrinogen and fibrin involving polymerization which enables FXIIIa to crosslink fibrin to fibrinogen being in an half-staggered overlap position but not in DD-long contact.

The glycosylated component of tissue factor from human brain which totally retained on Concanavalin A-Sepharose was isolated by a four step procedure. The final product (6-7 S) exhibited an apparent molecular weight of approximately 45,000 under reducing conditions on SDS-gels corresponding with procoagulant activity of tissue factor after detergent removal. The optimal ratio of lipid to protein in reconstituting tissue factor activity was 500:1 (w/w) amounting to 240,000 U/mg. Peptidase activity was not detectable in the purified apoprotein or in the relipidated protein. The procoagulant activity of tissue factor was dependent on Factor VII but not on Factors VIII or IX, since in the absence of Factor VII clotting times were significantly prolonged. The activity of the reconstituted tissue factor could be reduced by a factor of 15 upon incubation with an antibody against the purified glycosylated apoprotein that was raised in the rabbit. The IgG-fraction of the antiserum neutralized the procoagulant activity of tissue factor in saline extracts from human brain and placenta in a time- and concentration-dependent manner indicating common antigenic determinants on the cofactor protein from both tissues.

The physiological role of coagulation cofactor Protein S (PrS) for activated Protein C (APC) has recently been appreciated by the description of patients with PrS-deficiency, suffering from thromboembolism. The present study introduces a one-stage clotting assay for the assessment of PrS functional activity in plasma samples. The assay procedure is based on a factor Xa-initiated clotting test utilizing a mixture of AL(OH)3-adsorbed substrate plasma and patient's plasma supplemented with purified prothrombin (0.15 microM) and APC (0.05 microM), with phospholipids and CaCl2. Owing to the varying concentration of PrS in the sample plasma, clotting times were prolonged up to 25 seconds in the presence of APC, whereas no prolongation occurred in its absence. The test procedure proved to be specific for PrS, since preincubation with monospecific antibodies against PrS abolished the prolongation of clotting time, while reconstitution of adsorbed plasma with purified PrS restored its cofactor activity completely. The functional assay showed an inter-assay and intra-assay variation in the normal range of 11.7% and 10.1%, respectively (n = 20). PrS activity in a group of unselected patients (n = 34), revealing no abnormalities in global coagulation tests, amounted to 95.8 +/- 16.5% (mean +/- S.D.) with a range from 67% to 136% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Patients (n = 32) undergoing oral anticoagulant therapy presented 21.1 +/- 10.8% residual PrS-activity accompanied by a concomitant decrease in PrS-antigen levels to 69.9 +/- 21.2%. The assay described is sensitive, it can be performed on routine basis and allows the detection of patients with PrS-deficiency.

The activation of fibrinolysis during bicycle ergometry was studied in two pairs of age-matched groups. Group A: 18 healthy male competitive athletes (23 +/- 3.5 years of age, mean +/- SD), Group B: 18 healthy male volunteers (25.7 +/- 2.7) not engaged in any sports, Group C: 17 healthy male volunteers (50.6 +/- 7.7) regularly practicing sports, and Group D: 18 male survivors from myocardial infarction (MI-patients, 54.2 +/- 7.9) who took part in a rehabilitation sports program. Before ergometry, healthy participants with regular sporting activities showed significantly lower plasma plasminogen activator inhibitor capacities (PAI cap) than the members of the respective age-matched control groups: Group A 13.9 +/- 2.6 AU/ml, Group B 18.5 +/- 5.5, p less than 0.005; Group C 15.2 +/- 2.9, Group D 20.7 +/- 5.5, p less than 0.05. During ergometry the release of tPA antigen did not differ significantly between the age-matched groups, however, tissue plasminogen activator (tPA) activities after ergometry were higher in groups presenting lower pre-test PAI cap values. Group A 5.5 +/- 6.4 AU/ml, Group B 1.1 +/- 2.9 AU/ml, p less than 0.05; Group C 2.9 +/- 3.3, Group D 0.2 +/- 0.7, p less than 0.05. Levels of the fibrin split product (D-dimer) did not change in any of the groups. This investigation indicates that (1) regular vigorous sporting activities enhance blood fibrinolysis by reducing blood PAI cap in healthy individuals, (2) rehabilitation sport is not capable of reducing blood PAI cap in MI-patients to values measured in age matched healthy individuals regularly practicing sports and (3) the activation of fibrinolysis during physical exercise has no systemic fibrinolytic effect.