Human Plasminogen Activator From Heart And Vascular Endothelium

Plasminogen activators play a central role in fibrinolysis. At present little is known about the interrelationships and molecular properties of these protein(s). There have been conflicting reports on the number of polypeptide chains present in the active molecules; plasminogen activators, purified from human uterus and human plasma after venous occlusion, are reported to consist of two chains, each of approximately 30,000 MW, linked by disulphide bonds. In contrast, the protein purified from human vascular endothelium consists of a single chain of 67,000 MW. Evidence for proteolytic activation of a single chain precursor to a two-chain form has been obtained by Wallen, in that the protein could be isolated in the single chain form only if inhibitors of proteolysis were present throughout purification. The two-chain form was found to possess significantly greater activity. By analogy with other plasma systems, generation of an active two-chain form from a single-chain precursor may serve a regulatory function, but the role of limited proteolysis of plasminogen activator remains to be elucidated.Using modifications of reported methods, we have obtained improved purification of plasminogen activator from human heart and vascular endothelium by similar procedures. This has allowed detailed comparison of the enzyme from these two sources, in terms of chain structure, active site labelling and carbohydrate content. In particular, the effect of variation of conditions during purification on the chain structure of the isolated molecule has been studied.

CHARACTERIZATION OF LIMITED PROTEOLYTIC DIGESTS OF TISSUE PLASMINOGEN ACTIVATOR

10.1055/s-0038-1644375 ◽

1987 ◽

Author(s):

G Teshima ◽

R Harris ◽

R Keck ◽

A Meunier ◽

J Burnier ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Fibrinolytic Activity ◽

Limited Proteolysis ◽

Single Chain ◽

Amidolytic Activity ◽

Amino Terminal ◽

Tissue Plasminogen ◽

Limited Digestion ◽

Tissue plasminogen activator (tPA) is a single chain glycoprotein of 527 amino acids consisting of structural domains homologous to other plasma proteins ("finger","epidermal growth factor", "kringles" and "protease"). Unlike zymogens of other serine proteases, tPA in the single chain form (1-527), has amidolytic and fibrinolytic activity. However, the amidolytic activity is enhanced when tPA is cleaved by plasmin at the Arg275-Ile276 bond to yield the disulfide bonded two chain form. We used trypsin to study the structure and function of tPA by limited digestion. Aliquots of tPA (1 mg/ml) were digested at pH 7 with varying amounts of trypsin (1:10,000, 1:1000, 1:100 and 1:10; enzyme to substrate ratio). The dilute solutions of trypsin (1:10,000) were effective at completely converting one chain tPA to the two chain form, but little additional proteolysis was observed on SDS-PAGE. The proteolytic fragments of tPA were isolated by reduction and carboxymethylation (RCM), SDS gel electrophoresis and reversed phase HPLC. The RCM polypeptides were identified by amino acid composition and sequence. Specific antisera were prepared against peptide antigens of tPA including (1-27), (1-275), (276-527) and (502525). Immunoblotting experiments with the tryptic digests of tPA indicated that the region (1-275) is more susceptible to proteolytic attack than the protease (275-527). Specific cleavage sites were identified at positions 7, 10, 27 and 40. Partially digested tPA preparations were tested for enzymatic activity as determined by hydrolysis of the peptide substrate S-2288 or by clot lysis. Limited proteolysis at the amino terminus was correlated with significant loss of fibrinolytic . activity but minimal effect on the amidolytic activity. Increased tryptic digestion resulted in complete loss of amidolytic activity and significant reduction in antigenic activity as determined by polyclonal anti-tPA ELISA. These results areconsistent with the amino terminal "finger" domain being in part responsible for the fibrin-binding specificity of tPA. Limited tryptic digest of tPA, cleaves first at Arg-275, then subsequently cleaves the "finger" with associated loss of fibrinolytic activity.

On The Fibrinolytic Properties Of Single-Chain And Two- Chain Human Tissue Plasminogen Activator

10.1055/s-0038-1651973 ◽

1981 ◽

Cited By ~ 2

Author(s):

D C Rijken ◽

M Hoylaerts ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Gel Electrophoresis ◽

System Analysis ◽

Molecular Forms ◽

Regulatory Function ◽

Single Chain ◽

Tissue Plasminogen ◽

Dodecyl Sulfate ◽

Tissue plasminogen activator from pig hearts may be isolated as a single-chain or as a two-chain molecule (Wallén et al., 1980). The present report deals with the two molecular forms of a plasminogen activator (tissue plasminogen activator-like) secreted by human melanoma cells in culture. A single-chain form was prepared by addition of aprotinin to the culture media and during the purification procedure while a two-chain form or a mixture of both was obtained in the absence of aprotinin.The fibrinolytic activities of the two forms were comparable on fibrin plates and in a clot lysis time system. Analysis of the molecular structure of 125I-labeled plasminogen activator by dodecyl-sulfate-gel electrophoresis, revealed that the single-chain form was converted into a two- chain form during the lysis of fibrin. The plasminogen activating properties of the two molecular forms were therefore measured in the presence of aprotinin, which prevents the conversion. Aprotinin (1,000 KIU/ml) was incorporated in a fibrin clot (1 mg/ml) containing plasminogen activator (20 ng/ml), 125I-labeled Glu-plasminogen and varying amounts of unlabeled Glu-plasminogen. The amount of plasmin formed after 30 min was quantitated by measuring the radioactivity migrating in the position of the plasmin B-chain on dodecyl-sulfate-gel electrophoresis under reducing conditions. The rate of plasmin formation obeyed Michaelis-Menten kinetics with Km = 2.5 μM and kcat = 0.6 s-1 for the single-chain form and Km = 1.1 μM and kcat = 0.3 s-1 for the two-chain form.Although the conversion of the single-chain tissue plasminogen activator into a two-chain form during fibrinolysis might have a regulatory function, these kinetic parameters of the plasminogen activation do not support this hypothesis.

Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

10.1055/s-0038-1647039 ◽

1988 ◽

Vol 60 (02) ◽

pp. 247-250 ◽

Cited By ~ 1

Author(s):

H R Lijnen ◽

L Nelles ◽

B Van Hoef ◽

F De Cock ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Rate Constants ◽

Plasminogen Activators ◽

Second Order ◽

Tissue Type ◽

Single Chain ◽

Chain Molecules ◽

Order Rate ◽

Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

10.1055/s-0038-1648982 ◽

1994 ◽

Vol 72 (06) ◽

pp. 906-911 ◽

Cited By ~ 8

Author(s):

D C Rijken ◽

E Groeneveld ◽

M M Barrett-Bergshoeff

Keyword(s):

Plasminogen Activator ◽

Half Life ◽

Polyacrylamide Gel Electrophoresis ◽

Tissue Type ◽

Single Chain ◽

Tissue Type Plasminogen Activator ◽

Sds Page ◽

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

10.1055/s-0038-1649685 ◽

1993 ◽

Vol 70 (05) ◽

pp. 867-872 ◽

Cited By ~ 21

Author(s):

Dingeman C Rijken ◽

Gerard A W de Munk ◽

Annie F H Jie

Keyword(s):

Molecular Weight ◽

Ionic Strength ◽

Plasminogen Activator ◽

Plasminogen Activators ◽

Fibrinolytic System ◽

Tissue Type ◽

Single Chain ◽

Physiological Conditions ◽

Amino Terminal ◽

Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

10.1055/s-0038-1651634 ◽

1993 ◽

Vol 69 (05) ◽

pp. 466-472 ◽

Cited By ~ 7

Author(s):

M Colucci ◽

L G Cavallo ◽

G Agnelli ◽

A Mele ◽

R Bürgi ◽

...

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Cleavage Site ◽

Plasminogen Activators ◽

Tissue Type ◽

Low Molecular Weight ◽

Single Chain ◽

Kringle 2 ◽

Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Blood ◽

10.1182/blood.v82.5.1522.1522 ◽

1993 ◽

Vol 82 (5) ◽

pp. 1522-1531 ◽

Cited By ~ 20

Author(s):

SE Lind ◽

JR McDonagh ◽

CJ Smith

Keyword(s):

Plasminogen Activator ◽

Active Site ◽

Serine Proteases ◽

Plasminogen Activators ◽

Single Chain ◽

Site Specific ◽

Oxidative Inactivation ◽

Urokinase Type Plasminogen Activator ◽

Specific Oxidation

Abstract Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2′:6′,2″- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

The human fibrinolytic system is a target for the staphylococcal metalloprotease aureolysin

Biochemical Journal ◽

10.1042/bj20070650 ◽

2008 ◽

Vol 410 (1) ◽

pp. 157-165 ◽

Cited By ~ 29

Author(s):

Nathalie Beaufort ◽

Piotr Wojciechowski ◽

Christian P. Sommerhoff ◽

Grzegorz Szmyd ◽

Grzegorz Dubin ◽

...

Keyword(s):

Plasminogen Activator ◽

Active Form ◽

Catalytic Efficiency ◽

Plasminogen Activator Inhibitor ◽

Opportunistic Pathogen ◽

Fibrinolytic System ◽

Growth Media ◽

Single Chain ◽

Plasminogen Activator Inhibitor 1 ◽

Proteolytic Activation

The major opportunistic pathogen Staphylococcus aureus utilizes the human fibrinolytic system for invasion and spread via plasmin(ogen) binding and non-proteolytic activation. Because S. aureus secretes several proteases recently proposed as virulence factors, we explored whether these enzymes could add to the activation of the host's fibrinolytic system. Exposure of human pro-urokinase [pro-uPA (where uPA is urokinase-type plasminogen activator)] to conditioned growth media from staphylococcal reference strains results in an EDTA-sensitive conversion of the single-chain zymogen into its two-chain active form, an activity not observed in an aureolysin-deficient strain. Using purified aureolysin, we verified the capacity of this thermolysin-like metalloprotease to activate pro-uPA, with a 2.6×103 M−1·s−1 catalytic efficiency. Moreover, activation also occurs in the presence of human plasma, as well as in conditioned growth media from clinical isolates. Finally, we establish that aureolysin (i) converts plasminogen into angiostatin and mini-plasminogen, the latter retaining its capacity to be activated by uPA and to hydrolyse fibrin, (ii) degrades the plasminogen activator inhibitor-1, and (iii) abrogates the inhibitory activity of α2-antiplasmin. Altogether, we propose that, in parallel with the staphylokinase-dependent activation of plasminogen, aureolysin may contribute significantly to the activation of the fibrinolytic system by S. aureus, and thus may promote bacterial spread and invasion.