Sixtus Thorsen

Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation influence of ω-aminocarboxylic acids

Pretreatment of native plasminogen with plasmin or activators resulted in a pronounced increase in the binding of plasminogen to fibrin. The pretreated plasminogen was considered to be identical to the proteolytically degraded proenzyme with NH2-terminal lysine, valine or methionine, which is formed as an intermediate stage during activation of plasminogen. Bound plasminogen could be extracted by 6-aminohexanoic acid indicating a reversible binding between plasminogen and fibrin. Adsorption of pretreated plasminogen decreased when increasing concentrations of 6-aminohexanoic acid or trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA) were present during fibrin formation. The concentration of amino acid producing a decrease in the binding of pretreated plasminogen to 0.5 of the amount bound in the absence of amino acid was 8.0-10(-5) M with 6-aminohexanoic acid and 1.7.10-5 M with t-AMCHA. The decrease in binding is most likely related to an effect of the amino acids on plasminogen, since agarose gel electrophoresis of pretreated plasminogen in the presence of 6-aminohexanoic acid or t-AMCHA showed a cathodic shift in mobility at the same range of concentrations of amino acid, which produced the decrease in binding of plasminogen to fibrin. Evidence is provided that the decrease in binding of proteolytically degraded plasminogen may result in an inhibition of fibrinolysis caused by activators.

Fibrinogenolysis and Fibrinolysis with Tissue Plasminogen Activator, Urokinase, Streptokinase-Activated Human Globulin, and Plasmin

Summary In the presence of porcine tissue plasminogen activator, fibrinolysis is greatly enhanced in comparison with fibrinogenolysis. In contrast, other activators (UK, SK-activator) and plasmin showed no, or moderate, differences between lysis of fibrinogen and fibrin. The enhanced tissue activator effect is related to the activation of plasminogen. The observations add a specific mechanism of enhancement of fibrinolysis by tissue activator to that caused by the influence of inhibitors. The process may help to enhance localized fibrinolysis following tissue injury without affecting fibrinogen.

Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor.

Plasminogen activator-inhibitor complexes were analyzed by SDS-polyacrylamide gel electrophoresis and enzymography. The complexes appeared as fibrinolytically active bands in the fibrin-indicator gel. A high-molecular-weight t-PA form comigrating with a t-PA-inhibitor complex (Mr 95 000-135 000) from cultured human endothelial cells was purified from plasma by immunoadsorption on anti-t-PA-Sepharose followed by gel filtration on Sephadex G-150. The high-molecular-weight t-PA form was fibrinolytically inactive when assayed by the fibrin-plate method. It was converted to a form with the same electrophoretic mobility as t-PA (Mr 72 000) when treated with 1.5 M NH4OH/39 mM SDS. These observations suggested that the plasma high-molecular-weight t-PA form was an enzyme-inhibitor complex. The complex did not show immunological cross-reactivity with a number of known plasma serine proteinase inhibitors. Both t-PA and u-PA rapidly formed complexes with an inhibitor which was present in plasma in pmolar concentrations. p-Aminobenzamidine blocked the reaction, indicating that the active center of the activator was indeed implicated in complex formation. The complex between the plasma inhibitor and t-PA and the high-molecular-weight t-PA had the same electrophoretic mobilities. The rapid plasminogen activator inhibitor in plasma showed remarkable similarity to a plasminogen activator inhibitor from cultured human endothelial cells. In addition to the high-molecular-weight t-PA form described above, three other t-PA forms were isolated from plasma. Our results indicated that they represented free t-PA and t-PA in complex with respectively C1-esterase inhibitor and alpha 2-antiplasmin.

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95 000-135 000). This t-PA form was converted to Mr-72 000 t-PA by 1.5 M NH4OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and Mr-72 000 t-PA comigrated with high-molecular-weight t-PA. From the increase in Mr of t-PA or u-PA upon complex formation, the Mr of the endothelial cell component was estimated to be 50 000-70 000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH4OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involved in the complex formation. It was further noted that serum-free conditioned medium or endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.

A novel missense mutation in the human plasmin inhibitor (alpha2-antiplasmin) gene associated with a bleeding tendency.

Heterozygosity for a Gu2003→u2003A mutation converting Val384(GTG) to Met(ATG) associated with plasmin inhibitor (alpha2‐antiplasmin) deficiency was identified in three family members with bleeding tendency. The proband had traumatic breast haematoma and per‐/postoperative bleeds. An affected daughter required a blood transfusion after a normal delivery and a son had prolonged bleeding after tooth extraction. The plasma plasmin inhibitor activities of the affected family members were reduced to 49–66% of normal. The antigenic concentrations determined by electroimmunoassay were reduced to 57–68% of normal. Crossed immunoelectrophoresis of plasma from the proband showed a normal pattern. The amino acid Val384 is located eight residues C‐terminal (P8′) of the P1 residue (Arg376) in the reactive site. The P8′ residues of bovine and mouse plasmin inhibitor are both Val. Among other serpins the P8′ residue is unconserved. The mutation was not present in the non‐affected family member or 30 blood donors. In addition to the Val384Met mutation two new polymorphisms Ala‐26(GCG)/Val(GTG) and Arg407(AGG)/Lys(AAG) and one previously reported polymorphism Arg6(CGG)/Trp(TGG) were identified in the plasmin inhibitor gene of the family. The allelic frequencies among 30 blood donors with normal values of plasma plasmin inhibitor (functional) were 0.84/0.16 for C/T in codon −26, 0.81/0.19 for C/T in codon 6 and 0.83/0.17 for G/A in codon 407.

The application of the Reaction Rate Analyser LKB 8600 as an automatic coagulometer

The application of the Reaction Rate Analyser LKB 8600 (RRA) as an automatic coagulometer is described. The RRA was slightly modified without interfering with its function as an enzyme reaction rate analyser. The endpoint of coagulation was recorded when the increase in absorbance exceeded 0.047 at lambda = 340 nm. The coagulation time was monitored by a counter with automatic print-out or by a recorder. The analytical dispersion, s(x)x, ranged between 0.01 and 0.05. Results (x, y) of the determination of the quantity of plasma coagulation factors (II + VII + X) by RRA and manually by visual recording could be expressed by y = 0.93x + 0.04, r = 0.96, n = 66 (method of Owren & Aas) or by y = 1.00x + 0.02, r = 0.98, n = 49 (Normotest). Similarly, plasma activated partial thromboplastin time (Activated Thrombofax) could be expressed by y = 0.99x + 5.00, r = 0.99, n = 23.

Purification of a Plasminogen Activator Inhibitor Indistinguishable from α1-Antitrypsin and an Urokinase Inhibitor in Pregnancy Plasma

An inhibitor of urokinase present in increased concentrations in plasma during pregnancy was purified. The inhibitor was identified by its pronounced effect on urokinase-induced fibrinolysis as opposed to the absence of effect on fibrinolysis induced by a tissue plasminogen activator. The inhibitor progressively inactivated urokinase upon incubation and was found to be indistinguishable from alpha1-antitrypsin.

Biphasic Inhibition of Urokinase-Induced Fibrinolysis by ∊-Aminocaproic Acid; Distinction from Tissue Plasminogen Activator∗

Summary Inhibition of porcine tissue plasminogen activator by ∊-aminocaproic acid (EACA), trans-4-aminomethylcyclohexancarboxylic acid (trans-AMCHA) and p-axmnomethylbenzoic acid (PAMBA) increased uniformly with increasing concentrations of inhibitor. In contrast, with human urokinase an early phase of inhibition at low inhibitor concentrations was followed by a phase of enhancement of fibrinolysis turning into a second phase of inhibition at inhibitor concentrations 300 times higher than required in the first phase. This biphasic inhibition of urokinase distinguishes it chemically from tissue plasminogen activator.

Differences in the Reactivities of Human Urokinase and the Porcine Tissue Plasminogen Activator

It has previously been shown, that large differences exist between the effects of 6-aminohexanoic acid or alpha1-antitrypsin on fibrinolysis caused by a porcine tissue plasminogen activator or by human urokinase, while insignificant differences exist between the effects of a number of natural protease inhibitors on fibrinolysis caused by the two types of plasminogen activator. The present study shows that changes in substrate composition (pH, ionic strength fibrinogen concentration, plasminogen concentration) may influence to different degrees the fibrinolytic activities of human urokinase and the porcine tissue plasminogen activator. It is suggested, that this finding is partly related to marked differences in affinity for fibrin of the two activators.