Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.     

Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator

A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Effects of monoclonal antibodies on tissue-type plasminogen activator (t-PA) binding to lysine, fibrin and heparin and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity

The effects of 4 monoclonal antibodies against human tissue-type plasminogen activator (t-PA) on binding of t-PA to lysine, fibrin, and heparin, and on fibrin-mediated activation of one-chain t-PA-amidolytic activity were investigated. The association constants of the antibodies were determined in a direct assay to be equal to 0.125 l/nmol, 0.225 l/nmol, 0.4 l/nmol, and 0.5 l/nmol for mAB 5, mAB 16, mAB 25, and mAB 31, respectively. All 4 monoclonal antibodies inhibited binding of intact t-PA to lysine-Sepharose and fibrin, and they suppressed fibrin-mediated activation of one-chain t-PA-amidolytic activity. Binding analysis demonstrated that mAB 25 inhibited t-PA binding to lysine-Sepharose and to fibrin as well as fibrin-mediated enhancement of one-chain t-PA-amidolytic activity in a competitive manner with inhibitor constants of 5 nmol/l, 3 nmol/l and 10 nmol/l, respectively. It was also shown that free lysine counteracts the association of t-PA with the antibodies. Binding of t-PA to heparin is only moderately affected by the 4 antibodies. Since t-PA possesses two homologous kringle domains which contain fibrin (lysine) binding sites, the results underline the importance of a lysine binding site for fibrin binding by intact t-PA and show that the binding of the enzyme to fibrin and lysine is mediated by the same binding site of a kringle domain. The parallel effects of antibodies on fibrin binding and on fibrin-mediated enhancement of one-chain t-PA amidolytic activity proves that the site of fibrin binding is identical with the site of fibrin activation. The binding site of heparin apparently differs from lysine and fibrin binding sites.     

Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia

We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.     

Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416

In contrast to most other serine proteases, tissue-type plasminogen activator (t-PA) possesses enzymatic activity as the one-chain zymogen form. The hypothesis that lysine residues 277 or 416 may be involved in stabilization of an active conformation of one-chain t-PA via salt-bridge formation with aspartic acid residue 477 was tested by site-directed mutagenesis. Four recombinant t-PA mutants were constructed. The amidolytic activities of these analogues were compared to that of authentic t-PA. Substitution of arginine-275 provided an analogue [( R275G]t-PA) resistant to plasmin cleavage. The amidolytic activity of [R275G]t-PA was comparable to that of authentic one-chain t-PA, and so was the activity of [R275L,K277L]t-PA, in which additional substitution of lysine residue 277 was carried out. This suggested that its presence was nonessential for obtaining one-chain t-PA activity. In contrast, substitution of lysine residue 416 to obtain [K416S]t-PA and [K416S,H417T]t-PA resulted in substantial quenching of amidolytic one-chain activity. As expected, the amidolytic activities of the two-chain forms were less affected by the substitution. Involvement of lysine residue 416 in one-chain t-PA activity was also indicated by decreased activities of [K416S]t-PA and [K416S,H417T]t-PA with plasminogen as the substrate. The one-chain activity of the lysine residue 416 substitution analogues was partially restored in the presence of fibrin. This could indicate that strong ligands such as fibrin might provide an alternative stabilization of the active conformation of one-chain t-PA.     

Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.     

Activation of fibrin-bound plasminogen by pro-urokinase and its complementariness with that by tissue plasminogen activator

Single chain urokinase (SC-UK) is a precursor of 55 kd two-chain UK (TC-UK). Treatment with catalytic proportions of plasmin or kallikrein converts SC-UK to TC-UK as a consequence of cleavage of its Lys158-Ile159 peptide bond. This plasmin-mediated activation of SC-UK induces a positive feedback secondary reaction and complicates measurement of its activity against its natural substrate, Glu-plasminogen. The fibrin-selective effect of pro-UK-induced clot lysis is not related to fibrin binding. Rather, a conformational change in Glu-plasminogen, conferred when it binds to certain carboxy-terminal lysine residues on fibrin, has been implicated in this mechanism. This is complementary to t-PA. Fibrin-bound t-PA was found to exclusively activate plasminogen bound to certain internal lysine residues. Their complementariness is believed to explain their synergism in fibrinolysis.     

The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.     

A study of the protection of plasmin from antiplasmin inhibition within an intact fibrin clot during the course of clot lysis

Previous work using soluble fibrin surrogates or very dilute fibrin indicate that inhibition of plasmin by antiplasmin is attenuated by fibrin surrogates; however, this phenomenon has not been quantified within intact fibrin clots. Therefore, a novel system was designed to measure plasmin inhibition by antiplasmin in real time within an intact clot during fibrinolysis. This was accomplished by including the plasmin substrate S2251 and a recombinant fluorescent derivative of plasminogen (S741C-fluorescein) into clots formed from purified components. Steady state plasmin levels were estimated from the rates of S2251 hydrolysis, the rates of plasminogen activation were estimated by fluorescence decrease over time, and residual antiplasmin was deduced from residual fluorescence. From these measurements, the second order rate constant could be inferred at any time during fibrinolysis. Immediately after clot formation, the rate constant for inhibition decreased 3-fold from 9.6 x 10(6) m(-1) s(-1) measured in a soluble buffer system to 3.2 x 10(6) m(-1) s(-1) in an intact fibrin clot. As the clot continued to lyse, the rate constant for inhibition continued to decrease by 38-fold at maximum. To determine whether this protection was the result of plasmin exposure of carboxyl-terminal lysine residues, clots were formed in the presence of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). In the presence of TAFIa, the initial protective effect associated with clot formation occurred; however, the secondary protective effect associated with lysine residue exposure was delayed in a TAFIa concentration-dependent manner. This latter effect represents another mechanism whereby TAFIa attenuates fibrinolysis.     

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies

Two murine monoclonal antibodies (MA-2G6 and MA-1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue-type plasminogen activator (t-PA), inhibited the activity of t-PA on fibrin plates. MA-2G6 inhibited the amidolytic activity of t-PA and did not react with t-PA in which the active-site serine was blocked with diisopropylfluorophosphate nor with t-PA in which the active-site histidine was alkylated by reaction with D-Ile-Pro-Arg-CH2Cl. This indicated that MA-2G6 is directed against an epitope covering the active site of t-PA. MA-1C8 did not inhibit the amidolytic activity of t-PA, but abolished both the binding of t-PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t-PA. Thus MA-1C8 is directed against an epitope which covers the fibrin-binding site of t-PA. The A and B chains of partially reduced two-chain t-PA were separated by immunoadsorption on immobilized MA-1C8 and MA-2G6. The purified B chain reacted with MA-2G6 but not with MA-1C8 and activated plasminogen following Michaelis-Menten kinetics with kinetic constants similar to those of intact t-PA (Km = 100 microM and kcat = 0.02 s-1). However, fibrin or CNBr-digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA-1C8 but not with MA-2G6. It bound to fibrin with an affinity similar to that of intact t-PA but did not activate plasminogen. It is concluded that the active center of t-PA is located in the B chain and the fibrin-binding site in the A-chain. Both functional domains are required for the regulation by fibrin of the t-PA-mediated activation of plasminogen.     

The incorporation of a fluorescent probe into the active sites of one- and two-chain tissue-type plasminogen activator

Dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (DEGR-CK) was shown to inactivate both one- and two-chain human, recombinant tissue-type plasminogen activator (t-PA). The interaction of DEGR-CK with both forms of t-PA was accompanied by an identical increase in the fluorescence intensity and a blue shift in the wavelength of maximum emission, which suggests that the environment of the incorporated DEGR is similar in both one- and two-chain t-PA. The kinetics of the interaction of t-PA with DEGR-CK could be followed by both loss of activity and increase in fluorescence. The second order rate constants (k2/Ki) obtained with these two methods agreed quite well. With two-chain t-PA the values were 42 X 10(4) M-1 min-1 and 46 X 10(4) M-1 min-1 by the activity loss and fluorescence methods, respectively. With one-chain t-PA the results were 2.5 X 10(4) M-1 min-1 and 3.1 X 10(4) M-1 min-1. The rate at which one-chain t-PA is inactivated by DEGR-CK is 15 times lower than the rate with two-chain t-PA. The results demonstrated, however, that the cleavage of the one-chain protein to the two-chain form is not required for reactivity with DEGR-CK. This fluorescently labeled t-PA should be useful in probing the interactions of one- and two-chain t-PA with other proteins.     

Tissue-type plasminogen activator binds to and is inhibited by surface-bound lipoprotein(a) and low-density lipoprotein

Elevated levels of lipoprotein(a) [Lp(a)] are associated with an increased risk of atherothrombotic disease, but the mechanism(s) by which Lp(a) potentiates atherogenesis is unknown. The extensive homology of apolipoprotein(a) [apo(a)] to plasminogen has led us and others to postulate that Lp(a) may impair fibrinolysis. We have previously shown that Lp(a) inhibits fibrin stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA); however, we and other investigators have been unable to demonstrate direct inhibition of t-PA by Lp(a) in solution. We now report that t-PA binds reversibly and saturably to surface-bound Lp(a) and to low-density lipoprotein (LDL) and that as a result of this binding activation of plasminogen by t-PA is inhibited. The catalytic efficiency (kcat/Km) of t-PA when bound to polystyrene surface-bound fibrinogen increased 2.9-fold compared to t-PA bound to control wells. When bound to surface-bound Lp(a), however, the catalytic efficiency of t-PA was reduced 9.5-fold compared to t-PA bound to control wells; likewise, by binding to surface-bound LDL, the catalytic efficiency of t-PA was reduced 16-fold compared to the control. Studies with defined monoclonal antibodies suggest that major determinants of t-PA binding are its active site, the LDL receptor binding domain of apolipoprotein B-100 (apoB-100), and apo(a). These data suggest a unique mechanism by which Lp(a) and LDL incorporated in an atheroma can inhibit endogenous fibrinolysis and thereby contribute to the genesis of atherothrombotic disease.     

Trinitrobenzoylated poly(D-lysine) as a stimulator of interactions between plasminogen, plasmin, and tissue-type plasminogen activator

Trinitrobenzyl alkylation of poly(D-lysine) provides a novel powerful stimulator of tissue-type plasminogen activator. Its stimulatory effect on plasminogen activation is far greater than that of the original poly(D-lysine), and even surpasses that of fibrin. Its effect on plasmin-catalysed modification of both tissue-type plasminogen activator (t-PA) and native (Glu-1-) plasminogen are also investigated. Cleavage of one-chain t-PA to its two-chain form is monitored by measuring the increase in amidolytic activity which accompanies this transformation. Presupposing apparent first-order reaction kinetics, a theory is developed by which the rate constant, kcat/Km = 1.0 X 10(6) M-1 X s-1 of plasmin cleavage of one-chain t-PA can be calculated. Plasmin-catalysed transformation of 125I-labelled Glu-1- to Lys-77-plasminogen is quantified following separation by polyacrylamide gel electrophoresis at pH 3.2. A rate constant, kcat/Km = 4.4 X 10(3) M-1 X s-1 is obtained for the reaction between plasmin and Glu-1-plasminogen in the presence of 1 mM trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Both of the above plasmin-catalysed reactions are strongly enhanced by trinitrobenzoylated poly(D-lysine). The mechanism of action of this stimulator is elucidated by studying its binding to both activator and plasmin(ogen), and by direct comparison of the results with measurements of plasminogen activation kinetics in the presence of the stimulator. Binding studies are performed exploiting the observation that an insoluble yellow complex is formed between plasminogen and modified poly(D-lysine). Protein-polymer interactions are also studied with solubilised components in an aqueous two-phase partition system containing dextran and poly(ethylene glycol). The rate enhancement of plasminogen activation is found to be closely correlated to the association of plasminogen to the stimulator. It is proposed that the stimulator effects of this simple polymer on the enzymatic activities of both plasminogen activator and plasmin are brought about by association of the proteinase and its substrate to a common matrix. Similarities between the action of the artificial and the natural stimulator (fibrin) are stressed. These properties of trinitrobenzoylated poly(D-lysine) makes it useful as a model for the study of the regulatory mechanism of the fibrinolytic process at the molecular level.     

Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin.

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.