Kinetics of plasmin inhibition in the presence of a synthetic tripeptide substrate. The reaction with pancreatic trypsin inhibitor and two forms of alpha 2-plasmin inhibitor.

The progressive inhibition of plasmin by pancreatic trypsin inhibitor and by alpha 2-plasmin inhibitor in the presence of D-valyl-L-leucyl-L-lysine 4-nitroanilide was investigated. The kinetics with plasmin were compared with those with miniplasmin. The kinetic properties of two functionally different forms of alpha 2-plasmin inhibitor described by Clemmensen [(1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen. D., Wiman, B & Verstraete, M., eds.), pp 131-136, Elsevier, Amsterdam] were characterized. The two forms differ in their plasminogen-binding capability, and this difference can account for a difference in secondary site interaction suggested from the kinetics. The binding of inhibitor to miniplasmin is a simple pseudo-first-order reaction with both pancreatic trypsin inhibitor and the two alpha 2-plasmin inhibitor forms. Such simple kinetics are also observed for the reaction between plasmin and the non-plasminogen-binding form of alpha 2-plasmin inhibitor. More complicated kinetics are obtained for the reaction between plasmin and the alpha 2-plasmin inhibitor form that binds to plasminogen. With both forms of the alpha 2-plasmin inhibitor, a complex stable to acetic acid/urea and gel electrophoresis is present and fully developed 15 s after initiation of the reaction with plasmin.     

Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α₂-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α₂-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Lys⁷⁶, Glu¹–Arg⁶⁷ or Glu¹–Lys⁷⁷)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α₂-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys. 170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α₂-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Val⁴⁴¹) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α₂-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α₂-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.     

Kringle domains and plasmin denaturation.

The rate of plasmin denaturation was in the order of Lys-plasmin greater than miniplasmin greater than microplasmin. Fibrinogen degradation products (FDP) dose dependently increased the denaturation rate of Lys-plasmin and mini-plasmin with a maximal rate constant at the FDP/plasmin ratio of about 0.5. The denaturation rate constant of microplasmin was not affected. FDP increased the rate of plasmin denaturation was in parallel with its effect on the interaction among kringle domains. Without FDP only trace amounts of plasminogen dimer could be detected by cross-linking with bis-(sulfo-succinimidyl)-suberate followed by SDS gel electrophoresis. In the low concentration of FDP significant amounts of oligomers of Glu-, mini-plasminogens, kringle 1-3 and kringle 1-5 were observed. High concentration of FDP, however, decreased plasminogen oligomer.     

Fibrin digestion by thrombin. Comparison with plasmin-digested fibrinogen.

Solutions of plasminogen-free human fibrinogen alone or (1) treated with sodium p-chloromercuribenzoate in order to inactivate factor XIII, or (2) enriched with factor XIII, cysteine and CaC12, were clotted with plasmin-free human thrombin and incubated under sterile conditions. The clots dissolved gradually within 2 days (fibrin from sodium p-chloromercuribenzoate-treated fibrinogen) to 15 days (fibrin from factor XIII-enriched fibrinogen). This proteolytic process was not affected by soybean trypsin inhibitor but was completely inhibited by hirudin. Gel electrophoresis of the thrombin digests indicated the formation of bands equivalent to bands X, Y, D and E of plasmin digests of fibrinogen. The two latter bands, whose identity was confirmed by immunoelectrophoresis, appeared at a more advanced stage of proteolysis than the corresponding bands of plasmin digests. The number of isopeptide bonds present did not appear to affect the rate of release of acid-soluble peptides. Gel electrophoresis and the rate of release of acid-soluble peptides indicated that fewer bonds are hydrolysed by thrombin at the time of the complete solubilization of the clot than are split by plasmin when fibrinogen becomes unclottable by thrombin.     

Refolding, purification, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies

Two des-kringle derivatives of human plasminogen, microplasminogen and miniplasminogen, have been expressed at high levels as inclusion bodies in Escherichia coli using a T7 expression system. In each case, the isolated inclusion bodies were refolded and purified. A final yield of 10% of total refolded protein was observed in each case. Both refolded molecules were successfully activated to their functional forms, microplasmin and miniplasmin, by the plasminogen activator urokinase. The kinetic properties of the refolded microplasmin and miniplasmin were comparable to full length, native plasmin.     

Structural elements that govern the substrate specificity of the clot-dissolving enzyme plasmin

There is remarkable homology between the core structures of plasmin, a fibrin clot-degrading enzyme, and factor D, a complement-activating enzyme, despite markedly different biological functions. We postulated that sequence divergence in the loop structures between these two enzymes mediated the unique substrate and inhibitor interactions of plasmin. Recombinant microplasminogens chimerized with factor D sequences at loops 3, 5, and 7 were cleaved by the plasminogen activator urokinase and developed titratable active sites. Chimerization abolished functional interactions with the plasminogen activator streptokinase but did not block complex formation. The microplasmin chimeras showed enhanced resistance (k(i) decreased up to two to three times) to inactivation of microplasmin by alpha(2)-antiplasmin. Microplasmin chimerization had minimal ( approximately 2 fold) effects on the catalytic efficiency for cleavage of small substrates and did not alter the cleavage of fibrin. However, microplasmin and the microplasmin chimeras showed enhanced abilities to degrade fibrin in plasma clots suspended in human plasma. These studies indicate that loop regions of the protease domain of plasmin are important for interactions with substrates, regulatory molecules, and inhibitors. Because modification of these regions affected substrate and inhibitor interactions, loop chimerization may hold promise for improving the clot dissolving properties of this enzyme.     

Role of the K4 and K5 plasmin heavy chain kringles in the fibrin clot structure destruction]

A comparative analysis of the rates of polymeric fibrin structure destruction by plasmin (Pm) and its proteolytic derivatives such as Val354-plasmin (c-Pm), Val442-plasmin (m-Pm) and Lys530-plasmin (mu-Pm) has been undertaken. It was shown, that Pm, c-Pm, m-Pm and mu-Pm at equal proteolytic activity, have dissolved fibrin clots with relative rates 40.3:38.0:4.6:1.0 correspondingly. The Pm, m-Pm and mu-Pm relative rates were changed by epsilon-aminocaproic acid to 4.6:1.5:1.0 correspondingly. In this case fibrin clot destruction time was increased for Pm and m-Pm and was not changed for mu-Pm. The rates of fibrinogen hydrolysis were nearly equal for these forms of enzyme. It was suggested, that the specific interactions between plasmin K4 and K5 kringles and solid phase fibrin substrate determine the polymer fibrin structure destruction rate.     

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Isolation and characterization of microplasminogen. A low molecular weight form of plasminogen

A functionally active human microplasminogen without kringle structures was produced by incubation of plasminogen with urokinase-free plasmin at an alkaline pH. The microplasminogen was purified by affinity chromatography on lysine- and soybean trypsin inhibitor-Sepharose and by chromofocusing. Human plasminogen is specifically cleaved at Arg529-Lys530 by plasmin to form microplasminogen, which consists of a single polypeptide of 261 residues from the COOH-terminal portion of native plasminogen. It has an Mr of 28,617, calculated from the sequence, which is consistent with the molecular weight determined by sodium dodecyl sulfate gel electrophoresis. Microplasminogen is a slightly basic protein and is eluted from a chromofocusing column at pH 8.3. It can be activated by urokinase and streptokinase to a catalytically active microplasmin. The specific amidolytic activity of microplasmin is about three times higher than Lys77-plasmin on a weight basis and is about the same on a molar basis. The activation of microplasminogen by streptokinase is slower than that of either Glu-plasminogen or Lys77-plasminogen. On the other hand, the activation of microplasminogen by urokinase is faster than that of either of the latter. The Arg560-Val561 bond is cleaved during activation of both microplasminogen and native plasminogen.     

Regulation of plasmin-dependent fibrin clot lysis by annexin II heterotetramer

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a K(d) of 436 nm and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.     

Fibrinolysis and fibrinogenolysis by Val442-plasmin

Elastase cleavage of Lys77-plasmin results in the formation of Val442-plasmin. This result suggests that small, active plasmin fragments can be produced even under conditions of high plasminogen activator levels such as occur in vivo. We examined the effect of the generation of such fragments by studying the degradation of fibrinogen and fibrin by Val442-plasmin. Val442-plasmin lysis of fibrinogen yielded the same products as obtained with Lys77-plasmin, but at a slightly lower rate. Lysine inhibited fibrinogenolysis by both Lys77-plasmin and Val442-plasmin. The marked inhibition observed at concentrations higher than 10 mM lysine occurred to the same extent for both proteases. In addition, the products and rate of fibrinolysis were the same for both proteases. These results indicate that the lysine binding regions present in Lys77-plasmin but absent in Val442-plasmin do not determine the rate, reaction products, or lysine inhibition of fibrinolysis and fibrinogenolysis by plasmin.     

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.     

Suppressed catalytic efficiency of plasmin in the presence of long-chain fatty acids. Identification of kinetic parameters from continuous enzymatic assay with Monte Carlo simulation

Thrombi, which are dissolved primarily by plasmin (EC 3.4.21.7.), contain up to millimolar concentrations of fatty acids and these are known to affect the action of the protease. In the present study the modulation of plasmin activity was characterized quantitatively in a continuous amidolytic assay based on synthetic plasmin substrate (Spectrozyme-PL). A novel numerical procedure was applied for identification of kinetic parameters and their confidence intervals, with Monte Carlo simulation of the reaction progress curves, providing adequate grounds for discrimination of different models of the enzyme action. All three fatty acids caused a 10-20-fold increase in the Michaelis constant on Spectrozyme-PL (baseline value 5.9 mum). The catalytic constant decreased from 5.8.s(-1) to 2.4-2.8.s(-1) in the presence of arachidonate and oleate, but increased to 14.8.s(-1) in the presence of stearate, implying enhancement of plasmin activity at saturating substrate concentrations. However, based on the ratio of the catalytic and Michaelis constants, all three fatty acids acted as inhibitors of plasmin with various degrees of potency, showing concentration dependence in the range of 10-65 mum for oleate and arachidonate, and 115-230 mum for stearate. The reported effects of the three fatty acids require the presence of kringle 5 in the structure of the protease; miniplasmin (des-kringle 1-4 plasmin) is as sensitive to fatty acids as plasmin, whereas the activity of microplasmin (des-kringle 1-5 plasmin) is not affected.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Glu1-, Lys77-, Val442-, and Val561-plasmin: a comparative study

Thermodynamic and kinetic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human Glu1-, Lys77-, Val442- and Val561-plasmin (EC 3.4.21.7) have been determined between pH 3.0 and 9.5, and from 5.0 to 45.0 degrees C. The inhibitor-binding properties to human Glu1-, Lys77-, Val442- and Val561-plasmin suggest a possible role of BPTI in modulating plasmin activity when the inhibitor is used therapeutically.     

Identification of the plasminogen-binding site of human alpha 2-plasmin inhibitor

A plasminogen-binding site of human alpha 2-plasmin inhibitor was studied. The chromatogram of digest from the amidinated alpha 2-plasmin inhibitor (67K-daltons, plasminogen-binding form) with trypsin was almost identical with that obtained from the 65K-daltons derivative (non-plasminogen-binding form) treated with the same procedure, except for the three tryptic peptides. One of the three peptides, the deamidinated peptide T-11, was found to have a strong ability to inhibit the interaction of alpha 2-plasmin inhibitor with human plasmin. Moreover, the dissociation constant Kd for interaction between the peptide T-11 and plasmin was estimated to be 5.5 microM, indicating that Kd is about 10-fold lower than that of epsilon-aminocaproic acid. The sequence of the peptide T-11 was determined by the Edman method as follows: NH2-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-COOH. alpha 2-Plasmin inhibitor and its 65K-daltons derivative were found to have the same NH2-terminal sequence of Asn(Asp)-Gln-Glu-Gln-. These results indicated that the plasminogen-binding site(s) of alpha 2-plasmin inhibitor could be located in the COOH-terminal region of its molecule and that some of epsilon-NH2-groups in the deamidinated peptide T-11 may be involved in the lysine-binding site(s) of plasmin(ogen).     

Features of the structure of fibrin clots,connected with conditions of gel formation

Fibrinogen to fibrin conversion and then fibrin clot three-dimensional network formation is one of the final steps in the coagulation system activation. Different factors, such as the environment temperature and pH, ions, so on, render an effect on the fibrin gel formation. Recently, the presence or absences of interface between two phases influence on the fibrin gel structure during its formation have been shown. Studies of fibrin gel structure peculiarities formed at different conditions (between two phases and without one phase) are demonstrated in this article. The plasmin enzymatic hydrolysis of fibrin clots both with surface film and without it was investigated. Experimental data allow to make a conclusion that the fibrin clot structure changes depend on its essential influence on the plasmin hydrolysis process of these clots.     

On the interaction of alpha2-plasmin inhibitor and proteases. Evidence for the formation of a covalent crosslinkage and non-covalent weak bondings between the inhibitor and proteases.

alpha2-plasmin inhibitor is a proteinase inhibitor in plasma which efficiently inhibits the lysis of fibrin clots induced by plasminogen activator. The nature of the binding of the inhibitor to trypsin or plasmin was studied by the chemical treatment of the enzyme-inhibitor complex with 7.5 M hydrazine at pH 10.0. With the hydrazine treatment, the complexes were degraded to proteins corresponding to the respective enzyme and inhibitor moieties. These results indicate that the covalent bond between the inhibitor and the enzymes is a carboxylic ester. The binding reaction of the inhibitor to active site-modified trypsin was also studied. The inhibitor formed complexes with anhydrotrypsin and carboxyamidomethylated trypsin. The complexes were dissociated in the presence of 1% sodium dodecyl sulfate, to the individual components: the respective enzyme and inhibitor moieties. The inhibitor, however, did not form a complex with diisopropylphosphorylated trypsin regardless of the presence or absence of the denaturing reagent. These results suggest the contribution of non-covalent interactions to the complex formation between the inhibitor and native enzymes.     

Effects of free fatty acids on fibrinolytic activity.

A novel method for the estimation of fibrinolytic activity is proposed. In this method, a fibrin clot suspension is used as a substrate (fibrin is known to be a physiological substrate of plasmin). The fibrin clot suspension was prepared by homogenization of human fibrin clots. With this method, we found that free fatty acids inhibited the plasmin activity, and long-chain, unsaturated free fatty acids had a particularly strong inhibitory action on plasmin. As regards the mechanism of the inhibitory action, free fatty acids may not inhibit complex formation between plasmin and fibirin, but may make it impossible for plasmin to act on fibrin due to deformation of the surface of the fibrin clot.     

The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis

The effect of heparin on the proteolytic and fibrinolytic activities of plasmin and plasminogen was studied. Heparin at a concentration of 6.3.10(-6) M did not change the caseinolytic activity of plasmin and plasminogen stimulated by streptokinase but suppressed their fibrinolytic activity. At concentrations from 2.10(-8) to 0.5.10(-6) M heparin increased, whereas at 1.10(-6)-4.10(-6) M reduced the time of desAAfibrin clot half-lysis by plasmin. Within the concentration range of 2.10(-8) to 4.10(-6) M heparin did not change the time of the clot half-lysis by glu-plasminogen and slightly decreased the time of fibrin clot half-lysis by lys-plasminogen in the presence of the tissue activator. It was supposed that heparin inhibits the fibrinolytic effect of plasmin by way of formation of complexes with plasmin and reduction of plasmin specificity to the solid phase substrate, i. e., polymeric fibrin.