Björn Wiman

Plasminogen activator release during venous stasis and exercise as determined by a new specific assay

A method for the specific determination of tissue plasminogen activator in plasma samples has been developed. The method is based on a recently described parabolic rate assay for tissue plasminogen activator (Rånby and Wallén, 1980) measuring plasmin activity by a chromogenic tripeptide-paranitroanilide substrate. The potential interference by plasmin inhibitors in plasma is overcome by acidification of the plasma samples to pH 4 for 15 min prior to the analysis. The baseline activity (after 10 min of rest) in 22 healthy individuals was determined to be 0.05 +/- 0.03 (SD) IU/ml (0.2 +/- 0.1 ng/ml). After venous occlusion for 10 min at 100 mm Hg the activator concentrations had increased to 1.2 +/- 1.2 (SD) IU/ml (5.2 +/- 5.2 ng/ml). Furthermore, prior to venous occlusion almost no plasmin-alpha 2-antiplasmin complex was found in the samples, whereas a pronounced increase in this complex was noted in 90% of the subjects after venous occlusion. During exercise, a gradual work-load dependent increase of tissue plasminogen activator concentration was observed. At maximal work-load the concentration was 2.3 +/- 1.9 (SD) IU/ml (9.9 +/- 8.1 ng/ml active enzyme), which after 10 min rest decreased to 26 +/- 12 (SD) % of the values at maximal work-load. In these samples only small amounts of plasmin-alpha 2-antiplasmin complex were detected, although the activator content was of the same order of magnitude as after venous occlusion.

The specific interaction between plasminogen and fibrin. A physiological role of the lysine binding site in plasminogen

Abstract By chymotryptic fragmentation of plasminogen or partially reduced and S-carboxymethylated plasmin A-chain it is possible to obtain peptides in the molecular weight region of less than 10 000, which still seems to have an intact lysine-binding site. These peptides have been purified by chromatography on lysine·Sepharose and Sephadex G-75. Amino acid analysis indicates that they originate from the NH 2 -terminal part of the plasmin A-chain. The interaction between plasminogen and fibrin is studied by affinity chromatography on insolubilized fibrin or fibrinogen using gradient elution with 6-aminohexanoic acid. “Glu-plasminogen”, “Lys-plasminogen” and plasmin A-chain were all eluted in two peaks, very similar to the results obtained on lysine·Sepharose. All chymotryptic fragments obtained from plasminogen which had affinity for lysine·Sepharose were also completely adsorbed to fibrin·Sepharose, while the peptides with no affinity for lysine·Sepharose neither were adsorbed to fibrin·Sepharose. Thus it is concluded that the lysine binding site in plasminogen is responsible for its interaction with fibrin.

On the biological significance of the specific interaction between fibrin, plasminogen and antiplasmin.

Abstract The influence of antiplasmin on the interaction between fibrin and plasminogen was studied in plasma and in a purified system. The amount of plasminogen bound to fibrin was quantitated using trace amounts of 125I-labeled Glu-plasminogen (plasminogen with NH2-terminal glutamic acid) or 125I-labeled Lys-plasminogen (NH2-terminal lysine). When whole plasma was clotted, 5.2% of Glu-plasminogen was associated with the fibrin clot. In plasma clotted in the presence of 20 mM 6-amino-hexanoic acid only 1.4% of the plasminogen was bound to fibrin, indicating that about 4% of the plasma plasminogen specifically binds to fibrin. With Lys-plasminogen these values were approximately twice as high. When antiplasmin-depleted plasma was used, only slightly higher amounts of both types of plasminogen were associated with the fibrin. The adsorbed plasminogen was not significantly eluted with plasma or with purified antiplasmin at physiological concentrations. These findings indicate that antiplasmin does not play a significant role in the inhibition of the binding of plasminogen to fibrin or the dissociation of the plasminogen · fibrin complex. These observations in conjunction with previous findings on the kinetics of the plasmin-antiplasmin reaction suggest that the lysine-binding site of plasminogen, which is responsible both for its interaction with fibrin and its interaction with antiplasmin, plays an important role in the very fast neutralization of plasmin formed in circulating blood and serves to attach plasminogen to fibrin and thereby sequestrate plasmin formed in loco from circulating antiplasmin.

On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or α2-antiplasmin

The reaction between plasmin and aprotinin occurs in a two-step reaction: A fast reversible second-order reaction followed by a slower first-order transition. The dissociation constant of the initial complex and the rate constants of the two steps have been determined. It is also demonstrated that plasmin-streptokinase complex is inactivated by aprotinin but about 100-fold slower than plasmin. The rate constant was determined as 1.1 × 104 M−1·s−1. There are no significant inhibitors of plasmin-streptokinase complex in plasma. However, after addition of aprotinin (1 μmol/1) to normal plasma a progressive inactivation takes place (t12 = 250 s). It is suggested that aprotinin may be used to control bleeding complications during thrombolytic therapy with streptokinase. In contrast to plasmin the streptokinase-plasmin complex reacts very slowly with α2-antiplasmin (rate constant 0.8 M−1·s−1). This is at least 2 × 107 fold slower than the reac tion between plasmin and α2-antiplasmin, indicating a pronounced stability of the streptokinase-plasmin complex. The reaction rate of the plasmin-streptokinase reaction is demonstrated to be extremely rapid. A value of 4.7 × 107 M−1·s−1 is obtained for the rate constant.

Fibrinolytic activity in plasma and deep vein thrombosis after major abdominal surgery

In a prospective study of the frequency of deep vein thrombosis (DVT) in 45 patients subjected to major abdominal surgery, 17 patients showed signs of DVT as assessed by the 125I-fibrinogen test. In 15 of the patients the DVT was diagnosed during the first four postoperative days. Blood samples were taken pre- and postoperatively and analysed for fibrinogen, prothrombin complex, APTT, platelet-count, plasminogen, alpha 2-antiplasmin, fibrin(ogen) degradation products, and plasmin-alpha 2-antiplasmin complex (PAP). The latter was used in order to reflect the fibrinolytic activity. Preoperatively, and postoperatively on day 3, the levels of PAP were significantly higher in patients without postoperative DVT. The data suggests that patients subjected to major abdominal surgery, who have enhanced fibrinolytic activity preoperatively, have a lesser tendency to develop postoperative DVT. Patients with postoperative DVT may have decreased fibrinolytic ability. From the data of the other parameters it is concluded that patients with DVT can have increased levels of FDP at the time of development of thrombosis.

Studies on a form of α2-antiplasmin in plasma which does not interact with the lysine-binding sites in plasminogen

A method for the specific determination of a form of alpha 2-antiplasmin which does not interact with the lysine-binding sites in plasminogen (NPB-AP) has been elaborated. Basically, the method is an electroimmnoassay utilizing an intermediate gel, which adsorbs the plasminogen-binding form of alpha 2-antiplasmin (PB-AP). The plasma concentration of NPB-AP in healthy individuals was determined as 0.40 mumol/1 +/- 0.14 (SD), constituting 35% +/- 11 (SD) of the total plasma alpha 2-antiplasmin concentration. During extensive plasmapheresis of two pregnant severely D-immunized women the NPB-AP form decreased significantly, while the PB-AP form increased, thus maintaining the total alpha 2-antiplasmin at an almost constant level. The increased biosynthesis rate of alpha 2-antiplasmin during the extensive plasmapheresis is thus accounted for by PB-AP indicating this form to be the one primarily synthesized and that the non-binding form is formed in plasma from PB-AP secondarily.

Purification and characterization of human ci-esterase inhibitor

A new purification method for C1-esterase inhibitor is described, which is essentially a three-step procedure: precipitation with poly(ethylene glycol), chromatography on DEAE-cellulose and hydrophobic interaction chromatography on hexyl-Sepharose. The final product is a single-chain glycoprotein with a molecular weight of about 100 000 and NH2-terminal asparagine. The molecule is fully active as judged by complex formation with C1s. Two of its three disulphide bridges can be easily reduced and S-carboxymethylated under non-denaturing conditions without loss of activity. However, at high dithioerythritol concentration the third disulphide bridge is also cleaved and accompanied by loss of the activity, indicating that this disulphide bridge is involved in maintaining the conformation around the reactive site in the inhibitor.

On the structure of the stable complex between plasmin and α2-antiplasmin

Many of the protease inhibitors in plasma form extremely stable complexes with proteolytic enzymes, which cannot be dissociated by strong denaturating agents [ 1-4]. Many of these complexes are, however, dissociated by nucleophilic agents; a covalent bond may be involved in their stabilization [1,2]. This has also been suggested for inhibitors such as soybean trypsin inhibitor and aprotinin in their complexes with proteases [5,6]. The reaction between plasmin and ct2-antiplasmin was investigated by a number of kinetic [7-9] and structural studies [ 10]. The kinetic data have revealed a two-step reaction: (i) a very fast reversible second-order reaction; followed by (ii) a slower irreversible transition [7-9]. The structural data have indicated that the reactive site in a2-antiplasmin constitutes a specific leucyl-methionyl residue in the carboxy-terminal portion of the molecule [ 10]. a2-Antiplasmin and its interactions are reviewed in [11]. This study was done to investigate whether the reactive site in az-antiplasmin is cleaved as a result of complex formation with plasmin. Part of this work has been presented in [12].

A new simple method for determination of C1̄-esterase inhibitor activity in plasma

A convenient method for the determination of C1-esterase inhibitor activity in plasma samples is described. The method is based on addition of purified C1s to plasma and measuring excess C1s with a new chromogenic tripeptide-p-nitroanilide substrate with a recording spectrophotometer. Addition of C1s to C1-esterase inhibitor-depleted plasma did not result in any appreciable inactivation of the enzyme for three hours. The concentration of C1-esterase inhibitor in 19 healthy individuals was estimated as 1.63 +/- 0.27 (SD) mumol/l. The correlation with C1-esterase inhibitor antigen in these individuals and 19 patients with varying concentrations of C1-esterase inhibitor was excellent. The correlation with an antikallikrein assay was found to be poor.

Circular dichroism studies on α2-antiplasmin and its interactions with plasmin and plasminogen

Spectropolarimetric studies of alpha 2-antiplasmin in the far ultraviolet region indicates a content of 16% alpha-helix, 18% beta-structure and 66% random coil. Two of its three disulphide bridges are reduced under non-denaturing conditions without major changes in conformation of functionality. Cleavage of the third disulphide bridge requires denaturing agents and is accompanied by complete loss of activity. The interaction of alpha 2-antiplasmin with plasminogen or fragments derived from plasminogen by elastase digestion has been studied by circular dichroism analysis in the near ultravoilet region. The results indicate that the fragment of plasminogen constituting the three NH2-terminal triple-loop structures contains at least two lysine-binding sites: one with high affinity and one with low affinity. One of these sites, probably the high-affinity site, is involved in the interaction with alpha 2-antiplasmin. This site seems also to be exposed in the intact plasminogen molecule. The formation of the stable complex between plasmin (EC 3.4.21.7) and alpha 2-antiplasmin is also accompanied by conformational changes.