Role of the lysine binding regions in the kinetic properties of human plasmin

Morris, Joseph P.; Blatt, Stephen P.; Powell, J.R.; Strickland, Dudley K.; Castellino, Francis J.

doi:10.1021/bi00520a001

Cited by 46 publications

(18 citation statements)

References 34 publications

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“…Similarly to fibrinogen as a substrate (21,22), the kinetic parameters of plasmin and miniplasmin on non-cross-linked fibrin are essentially identical. The only exception is k -1 , which is markedly higher for miniplasmin (Table I; our model discriminates the association and dissociation constants for the formation of enzyme-substrate complex based on the different order of the two reactions).…”

mentioning

confidence: 98%

“…According to this concept, in addition to plasmin and PMN-proteases, a fibrinolytic role is attributed to miniplasmin, 2 which is generated after activation of the elastase-degraded form of plasminogen (M r 38,000) lacking four of the five kringle domains that represent the N-terminal Glu 1 -Val 441 sequence of plasminogen (20). The catalytic properties of some of these proteases have been evaluated in homogeneous systems for fibrinogen as a substrate (21,22) http://www.jbc.org/ Downloaded from min and PMN-elastase are the most efficient fibrinolytic enzymes, and a conclusion is drawn that the high affinity lysine binding site in the N-terminal kringle domains of plasmin is involved in the interactions with the native polymerized fibrin, whereas the fifth kringle found in both enzymes participates in binding to lysine residues newly exposed in the course of fibrin degradation (23). Two possible access routes to the fibrin substrate exist for an enzyme (24), an intrinsic one (when the protease is entrapped within the fibrin network) and an extrinsic one (when the fluid phase-borne enzyme attacks the surface of the clot).…”

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confidence: 99%

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Functional Evaluation of the Structural Features of Proteases and Their Substrate in Fibrin Surface Degradation

Kolev

Tenekedjiev²,

Komorowicz

et al. 1997

Journal of Biological Chemistry

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A new model has been introduced to characterize the action of a fluid phase enzyme on a solid phase substrate. This approach is applied to evaluate the kinetics of fibrin dissolution with several proteases. The model predicts the rate constants for the formation and dissociation of the protease-fibrin complex, the apparent order of the association reaction between the enzyme and the substrate, as well as a global catalytic constant (k cat ) for the dissolution process. These kinetic parameters show a strong dependence on the nature of the applied protease and on the structure of the polymerized substrate. The kinetic data for trypsin, PMN-elastase, and three plasminogen-derived proteases with identical catalytic domain, but with a varied N-terminal structure, are compared. The absence of kringle 5 in des-kringle 1-5 -plasmin (microplasmin) is related to a markedly lower k cat (0.008 s ؊1) compared with plasmin and des-kringle 1-4 -plasmin (miniplasmin) (0.039 s ؊1 ). The essentially identical kinetic parameters for miniplasmin and plasmin with the exception of k diss , which is higher for miniplasmin (81.8 s ؊1 versus 57.6 s ؊1 ), suggest that the first four kringle domains are needed to retain the enzyme in the enzyme-fibrin complex. Trypsin, a protease of similar primary specificity to plasmin, but with a different catalytic domain, shows basically the same k cat as plasmin, but its affinity to fibrin is markedly lower compared with plasmin and even microplasmin. The latter suggests that in addition to the kringle domains, the structure of the catalytic domain in plasmin also contributes to its specificity for fibrin. The thinner and extensively branched fibers of fibrin are more efficiently dissolved than the fibers with greater diameter and lower number of branching points. When the polymer is stabilized through covalent cross-linking, the k cat for plasmin and miniplasmin is 2-4-fold higher than on non-cross-linked fibrin, but the decrease in the association rate constant for the formation of enzyme-substrate complex explains the relative proteolytic resistance of the cross-linked fibrin. Thus, the functional evaluation of the discrete steps of the fibrinolytic process reveals new aspects of the interactions between proteases and their polymer substrate.The complex network of blood coagulation culminates in the conversion of fibrinogen to the rod-shaped fibrin monomers (for review, see Ref. 1). The non-covalent interactions among the fibrin monomers result in the formation of long doublestranded polymers in which the protomeric units are positioned in a half-staggered manner. Factors that influence the rate of the conversion of fibrinogen to fibrin and the strength of the polymerization interactions determine the final fiber diameter and clot structure. Increase in the NaCl concentration or in the pH reduces the number of protofibrils in a single fiber and the porosity of the fibrin network (2, 3); e.g. as the NaCl concentration is changed from 50 to 400 mM, the fiber diameter decreases from 180 to 39 nm, a...

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confidence: 98%

mentioning

confidence: 99%

Functional Evaluation of the Structural Features of Proteases and Their Substrate in Fibrin Surface Degradation

Kolev

Tenekedjiev²,

Komorowicz

et al. 1997

Journal of Biological Chemistry

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“…Neither agent affected the rate of substrate hydrolysis by more than 7% in the specified concentration range (data not shown), consistent with the results of previous investigations. 29 ' 40 -62 - 63 Equivalent results were obtained with miniplasmin and streptokinase-plasmin complex. NALME markedly inhibited the hydrolysis of S-2251 by plasmin, miniplasmin, and streptokinase-plasmin complex ( Table 1).…”

Section: Plasmin Amidase Activitymentioning

confidence: 98%

Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition.

Anonick

Vasudevan

Gonias

1992

Arterioscler Thromb

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methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents e-amino-n-caproic acid (eACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting er-thrombin or tissue plasminogen activator. eACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Leu-L-Lys-p-nitroanilide HC1 (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The K, for the plasmin-NALME interaction was 0.4 mM. eACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that eACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and eACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with o^-antiplasmin and a 2 -macroglobulin, eACA increased the fraction of plasmin reacting with o^-macroglobulin; NALME had no effect on the plasmin distribution. eACA, TEA, and NALME increased the euglobulin clot lysis time of normal plasma. NALME did not prolong the prothrombin time or activated partial thromboplastin time. These studies demonstrate that the antifibrinolytic activity of NALME is based on inhibition of the plasmin active site, whereas eACA and TEA are active due to kringle domain interactions. ( I n the circulation, fibrinolysis and fibrinogenolysis are mediated primarily by plasmin, the active serine proteinase counterpart of the zymogen plasminogen. 1 ' 2 The intact structure of plasminogen includes 791 amino acids with an Af-terminal glutamic acid 2 " 5 and either one or two oligosaccharide chains. 6 -8 Plasminogen activation results from the cleavage of the Arg^-Val 56 ' peptide bond. 9 Low concentrations of plasmin cleave [Glu'J-plasminogen to form [Lys 78 ]-plasminogen, a second more readily activatable form of the zymogen. 10 Plasminogen activation and the subsequent function of plasmin are modulated by noncovalent binding inter-

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“…18 The kinetics of Glu 1 -plasmin, Lys 77 -plasmin, and Val442-plasmin toward a variety of substitutes have been examined. 28 ' 62 Subtle differences do exist in these parameters. Interestingly, Lys77-plasmin and Val 442 -plasmin show virtually identical reactiv ities toward human fibrinogen, in terms of reaction rates, reaction products, and effect of є-ACA.…”

Section: Studies With Plasminogen Fragmentsmentioning

confidence: 99%

Biochemistry of Human Plasminogen

Castellino¹

1984

Semin Thromb Hemost

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Role of the lysine binding regions in the kinetic properties of human plasmin

Cited by 46 publications

References 34 publications

Functional Evaluation of the Structural Features of Proteases and Their Substrate in Fibrin Surface Degradation

Functional Evaluation of the Structural Features of Proteases and Their Substrate in Fibrin Surface Degradation

Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition.

Biochemistry of Human Plasminogen

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