Quantitation of the factor VII- and single-chain plasminogen activator-activating protease in plasmas of healthy subjects.

Plasma samples of 189 healthy subjects were investigated for antigen levels of the recently reported factor VII- and single-chain plasminogen activator-activating protease (FSAP) and the corresponding pro-urokinase activating potencies. While the age of donors had no significant effect on the investigated parameters, female plasmas revealed a trend to higher antigen contents and activity levels. Surprisingly, as much as 9% of all samples contained significantly reduced single-chain urinary plasminogen activator activating potential, whereas antigen concentrations were normal. Additionally, 1% of the plasmas was found to decrease in both FSAP antigen and activity contents. FSAP of three subjects displaying reduced activities throughout a follow-up period of 6 months were purified from plasmas and were characterized. As compared with pool plasma derived FSAP, investigation of the individual preparations confirmed their reduced potency to activate pro-urokinase. However, factor VII activation was not affected. It is speculated that the FSAP binding site for single-chain plasminogen activators is affected, potentially by as yet unknown polymorphism(s) or mutation(s).

Factor VII and single-chain plasminogen activator-activating protease: activation and autoactivation of the proenzyme.

Structural and biological characteristics of a recently described plasma serine protease, which displayed factor VII as well as pro-urokinase-activating properties in vitro, indicated a dual role for this factor VII-activating protease (FSAP) in hemostasis. Only the active protease (two-chain FSAP) has been isolated from plasma and from a prothrombin complex concentrate, whereas activators of the proenzyme have not been identified so far. After purification of the FSAP proenzyme from cryo-poor plasma by adsorption to an immobilized mAb and subsequent ion-exchange chromatography, activation to generate two-chain FSAP was followed by a direct chromogenic assay as well as by the ability of two-chain FSAP to activate pro-urokinase. Purified single-chain FSAP underwent autoactivation leading to the typical protease two-chain pattern and subsequent degradation products, as demonstrated by Western-blotting analysis using a site-specific mAb. This autoactivation was significantly enhanced in the presence of heparin, whereas Ca2+ ions stabilized single-chain FSAP (the proenzyme) resulting in slower autoactivation kinetics. Correspondingly, the heparin-augmented reaction, which was associated with autodegradation particularly of the protease domain, was slowed down by co-incubation with Ca2+. Of the other proteases and cofactors tested, only urokinase (uPA) was able to generate the typical two-chain FSAP pattern. Studies with different forms of uPA suggest that the catalytic activity of pro-urokinase/uPA is needed to activate single-chain FSAP, indicating that it is the only hemostatic protease that can act as a physiological activator of FSAP.

A protease isolated from human plasma activating factor VII independent of tissue factor.

An activity detected in a prothrombin complex concentrate, termed 'thrombin-like' due to its amidolytic properties, was recently reported by another working group. This serine-protease revealed partial structural homology with a 'hepatocyte growth factor activator'. An impact of this protease on coagulation has not yet been described. The protease was isolated from plasma fractions by ion exchange chromatography and adsorption to immobilized heparin and/or aprotinin. Clotting tests including the FVIIa-rTF assay were performed employing coagulometry. A monoclonal antibody-derived F(ab')2 to FVIII was used to investigate the FVIII bypassing activity (FEIBA). The identity of the-protease with the so-called 'thrombin-like' protease was supported by sequencing of the amino-termini. Its amidolytic activity was significantly enhanced in the presence of calcium and/or heparin. Incubation with purified FVII revealed the generation of FVIIa, but was prevented by pre-incubation of the protease with aprotinin. In contrast, purified FV and FVIII were inactivated. Studying coagulation parameters, clotting times like plasma recalcification times and the prothrombin times were found to be shortened by addition of the protease. Employing a FVIII-inhibitory F(ab')2 and enhancing clotting times significantly, FEIBA of the protease was found. We demonstrated that the isolated protease activates FVII independent of tissue factor. Net acceleration of coagulation was found in several global clotting assays resulting in an in vitro FEIBA. The physiological relevance of these findings deserves further investigation.

Inhibition of in vitro clot growth by r-hirudin is more effective and longer sustained than by an analogous peptide.

The specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D-FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests. r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D-FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini.

Mutations were directed to specific regions of the human tissue-type plasminogen activator (t-PA) gene in an effort to better define structure-function relationships of the enzyme. Three types of modifications were effected by in vitro mutagenesis: elimination of glycosylation sites; substitutions of amino acids at the cleavage site for conversion of single-chain t-PA to two-chain t-PA; and truncations of the N- and C-termini. Thirteen variants were purified from permanent CHO cell lines and analyzed for specific activity, fibrin stimulation, fibrin binding, inhibition by plasminogen activator inhibitor-2 (PAI-2) and half-life. The results of these analyses are: (i) variants with carbohydrate-depleted kringle domains possessed higher specific activities than wild-type t-PA; (ii) a cleavage site variant substituted at Arg275 with Gly had greatly reduced specific activity; (iii) two variants substituted at Lys277 exhibited altered interactions with PAI-2; (iv) the variant with a truncated C-terminus had reduced activity in the absence of fibrin; and (v) no variants had significantly altered half-lives. In order to test the effects of combining mutations, four additional variants were produced. Each combination variant retained at least one of the altered properties observed in the original variants, and in three of the variants the diverse properties were additive.

Study on the mechanism of action of heparin and related substances on the fibrinolytic system: relationship between plasminogen activators and heparin.

It is now generally well accepted that heparin and related substances increase the fibrinolytic activity in vivo. The stimulation of the amidolytic, plasminogenolytic and fibrinogenolytic activity of tissue plasminogen activator and urinary plasminogen activator through heparin was investigated in vitro. A concentration-dependent stimulation of the plasminogenolytic and fibrinogenolytic activity of both urinary and tissue-type plasminogen activators was observed in the presence of heparin. No heparin dependence was observed in the amidolytic assay. Heparin stimulates the plasminogenolytic activity of tissue plasminogen activators in the same manner as fibrin. Both activators form complexes with heparin; the heparin-binding-site seems to be identical or related with the fibrin-binding-site of tissue plasminogen activator. The physiological role of these interactions is discussed.