The interaction between plasminogen and antiplasmin variants as studied by surface plasmon resonance.

The interaction between immobilized plasminogen or an elastase-degradation product from plasminogen, constituting "kringles" 1-3 and different purified variants of antiplasmin has been studied by surface plasmon resonance utilising a BIAcore. The antiplasmin variants studied are wild-type, K429E, K436E, E443G, D444G, K452E and K452T. It is shown that the two mutants K452T and K452E react in quite a similar way as wt-antiplasmin, suggesting that Lys452 is not involved in the lysine-binding site interaction between plasminogen and antiplasmin.

Structures of importance for the stability of antiplasmin as studied by site-directed mutagenesis.

Human antiplasmin, a fast-acting inhibitor of plasmin in plasma, belongs to the serpin super-family of proteins. Like other members of this family, antiplasmin has a scissile peptide bond exposed within a reactive centre loop, typically present at the surface of the molecule. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated.

The complex between tPA and PAI-1: risk factor for myocardial infarction as studied in the SHEEP project.

Introduction: The tPA/PAI-1 complex seems to be an important biochemical marker for myocardial reinfarction. Therefore we explored the distribution, correlation and interaction of plasma concentrations of tPA/PAI-1 complex in all available patients and matched controls in the Stockholm Heart Epidemiology Program (SHEEP).

Methods And Patients: The SHEEP study is a case control study of 2246 patients with a first myocardial infarction (MI), of which 1267 surviving patients were subjected to blood sampling about 3 months after MI and compared with a control group, matched for age, sex and living area within the Stockholm County.

Inactivation of antiplasmin at low pH: evidence for the formation of latent molecules.

Several serine proteinase inhibitors (serpins) are metastable proteins which under certain conditions may undergo conformational changes resulting in the insertion of the reactive centre loop into the so-called Abeta-sheet and hence forming latent molecules. Here we have studied the inactivation of antiplasmin as a function of pH and temperature with time. At decreased pH (4.

Identification of amino acids in antiplasmin involved in its noncovalent 'lysine-binding-site'-dependent interaction with plasmin.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule.

Prognostic importance of the uPa/PAI-1 complex in breast cancer.

The complex between urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 (plasminogen activator inhibitor 1) has been prognostically evaluated in patients with breast cancer. The concentrations of uPA antigen, PAI-1 antigen and the uPA/PAI-1 complex were analysed in extracts from breast cancer tumours from 233 patients (median follow-up of patients: 71months). The uPA/PAI-1 complex typically constituted about 5% of the uPA antigen (total uPA).

Criteria for the Specific Measurement of Plasmin Inhibitor Activity Using an Enzymatic Procedure.

There is a lack of well-established criteria for the specific measurement of fibrinolytic parameters. On behalf of the SSC, the subcommittee on Fibrinolysis started a process to develop criteria for the specific measurement of fibrinolytic variables. This report describes the criteria for the specific measurement of plasmin inhibitor activity.

The Bbeta-sheet in the PAI-1 molecule plays an important role for its stability.

We have investigated the B beta-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His(219) to Tyr(241) (except for Gly(230) and Pro(240)), covering the s2B and s3B strands, and in addition His(185) and His(190)) were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E.

PAI-1 stability: the role of histidine residues.

The role of the 13 histidine residues in plasminogen activator inhibitor 1 (PAI-1) for the stability of the molecule was studied by replacing these residues by threonine, using site-directed mutagenesis. The generated mutants were expressed in Escherichia coli, purified and characterized. All variants had a normal activity and formed stable complexes with tissue-type plasminogen activator.

The fibrinolytic enzyme system. Basic principles and links to venous and arterial thrombosis.

This article briefly describes some important aspects of the fibrinolytic system, its regulation, and possible disturbances of this system in connection with deep vein thrombosis and myocardial infarction.

Angiostatin fragments in urine from patients with malignant disease.

Angiostatin, a family of fragments originating from the NH2-terminal portion of plasminogen, has been described as a potent inhibitor of angiogenesis. In order to examine to what extent angiostatin can be detected in cancer patients, urine was collected from 117 patients with different types of malignancies and subjected to Western blot analysis, utilizing antibodies raised against "kringles" 1-3 in plasminogen. A heterogeneous mixture of fragments was observed, with patterns that also varied between patients.

Predictive value of fibrinolytic factors in coronary heart disease.

An impaired fibrinolytic function, as evidenced by increased plasma concentrations of PAI-1, tPA antigen and tPA/PAI-1 complex, or a decreased capacity to release active tPA on exercise, is more common in individuals suffering from myocardial infarction. These factors, especially the tPA/PA-1 complex, also predict myocardial infarction in patients with manifest coronary heart disease, such as angina pectoris or a previous myocardial infarction. There is a highly statistically significant correlation between tPA/PAI-1 complex and both PAI-1 and tPA antigen.

Tissue plasminogen activator (tPA) antigen in plasma: correlation with different tPA/inhibitor complexes.

In many studies, tPA antigen has been a strong predictor of myocardial infarction. However, only a few percent of the total tPA antigen present in plasma samples taken at rest constitutes active tPA. The rest is enzymatically inactive and consists of a heterogeneous mixture of tPA in complex with inhibitors such as PAI-1, antiplasmin and C1 inhibitor.

Effects on the hemostatic system and liver function in relation to Implanon and Norplant. A prospective randomized clinical trial.

In this prospective randomized clinical trial, two long-term contraceptive implants were studied with respect to hemostasis and liver function in 86 healthy young women. The two implants used were Implanon, containing the progestagen etonogestrel (the biologically active metabolite of desogestrel) and Norplant, the implant containing the progestagen levonorgestrel. The results of the trial showed that both implants had similar small effects on the hemostatic system that are not suggestive of a tendency towards thrombosis.

Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule.

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coli and purified by chromatography on heparin-Sepharose and on anhydrotrypsin-agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability.

Stability of plasminogen activator inhibitor-1: role of tyrosine221.

Using site-directed mutagenesis, changes of Tyr221 in plasminogen activator inhibitor-1 (PAI-1) have provided mutants with normal activity, but with increased stability. At physiological conditions, the transition of the PAI-1 mutants Tyr221His and Tyr221Ser to the latent form was significantly prolonged (half-lives 14.8 and 4.

Plasminogen activator inhibitor-1 (PAI-1) content in platelets from healthy individuals genotyped for the 4G/5G polymorphism in the PAI-1 gene.

We have studied PAI-1 activity and antigen content in platelets and in plasma from 37 healthy individuals who were also genotyped for the 4G/5G polymorphism in the PAI-1 promoter region. The PAI-1 data obtained were compared with the vitronectin and the beta-thromboglobulin contents in platelets from the same individuals. A highly significant correlation between PAI-1 activity and PAI-antigen was obtained, both in the plasma samples (p < 0.

Plasminogen activator inhibitor 1 in thrombotic disease.

Impaired fibrinolytic function, mainly due to an elevation of plasma plasminogen activator inhibitor 1 concentration, is a common finding in patients with thrombotic disease. In patients subjected to hip surgery, preoperatively increased levels of plasminogen activator inhibitor 1 seem to be predictive of postoperative deep venous thrombosis. Several prospective studies of patients with angina pectoris or a past myocardial infarction have shown strong correlation between plasmatic plasminogen activator inhibitor 1 elevation and future cardiovascular events.

Plasminogen activator inhibitor 1 (PAI-1) in plasma: its role in thrombotic disease.

Impaired fibrinolytic function, mainly due to an elevation of the plasma PAI-1 concentration, is a common finding in patients with thrombotic disease. Unfortunately, regarding patients with idiopathic deep vein thrombosis (DVT), no reliable prospective or genetic studies have been published. Concerning postoperative DVT, preoperatively increased PAI-1 level seems to predict a postoperative DVT in patients subjected to hip surgery.

Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis.

The native fibrin gel structure formed in vitro from plasma samples was examined by liquid permeation of the hydrated fibrin gel networks in 18 men who had suffered a myocardial infarction before the age of 45 years and in 20 control subjects. Patients with an elevated plasma fibrinogen concentration had a considerably lower fibrin gel porosity (permeability coefficient, Ks) compared with patients with a normal plasma fibrinogen level and with controls. The calculated fiber mass-length ratio of the fibrin gel networks was decreased in both patient groups.

On laboratory problems in diagnosing mild von Willebrand's disease.

By providing some examples of variations in the levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor, and Factor VIII during one month, the authors wish to emphasize the difficulty of diagnosing mild forms of von Willebrand's disease (vWD), especially type I. In three of 15 normal female volunteers the vWF:Ag levels, on some sampling occasions, were so low (0.25-0.

Proteolytic activity during storage of platelets in plasma.

Proteolytic activity was studied in platelet concentrates (PC) stored in plasma at 22 degrees C. In experiment 1, two PC with a higher (A) and a lower (B) white cell concentration were prepared from each of nine donors by centrifugation. Aliquots of the cell-free plasma, PPP, were stored as a control.