Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.     

The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Kinetic parameters (k(Pg) and K(Pg)) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin-staphylokinase (Pm-STA) complex. The K(Pg) values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 microM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 microM, respectively), while only minor differences in the k(Pg) values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k(Pg)/K(Pg) values for activation of all four plasminogens due to a decrease in the K(Pg) values but did not alter the k(Pg) values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm-STA-Pg ternary enzyme-substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.     

Regulation of plasminogen activation by components of the extracellular matrix

The kinetics of activation of Glu-plasminogen (Glu-Pg) and Lys77-Pg by two-chain recombinant tissue plasminogen activator (t-PA) were determined in the presence of isolated protein components of the extracellular matrix (ECM) and compared to activation in the presence of fibrinogen and fibrinogen fragments and in the absence of added protein. Several ECM protein components were as effective as fibrinogen fragments at stimulating Pg activation. Stimulation of Glu-Pg activation resulted from both a decrease in Km and an increase in Vmax, whereas stimulation of Lys77-Pg was due primarily to increases in Vmax. The most effective stimulators of activation were basement membrane type IV collagen and gelatin which resulted in a 21- and 55-fold increase, respectively, in the kcat/Km of Glu-Pg (relative to a 10-fold increase observed with fibrinogen fragments). Amidolytic activity of t-PA was also enhanced up to 12-fold by ECM proteins. However, plasmin amidolytic activity was unaffected by the presence of added proteins. These data suggest that several ECM-associated proteins can enhanced the activation of Pg in the absence of fibrin.     

Features of plasminogen activation in a complex with antiplasminogen monoclonal antibody IV-1C

Antiplasminogen monoclonal antibody IV-1c (IV-1c) with antigenic determinant in V709-G718 site of plasminogen (Pg) protease domain (Druzhina N.N. et al. 1996.) can induce catalytic activity in Pg moiety of the complex. Catalytic activity appeared in Pg-IV-1c complex after approximately 2 h lag-period. Rate of Lys-Pg activation was higher then that of Glu-Pg. Amidolytic activity of Pg-SK equimolar complex was completely inhibited by IV-1c at 2:1 = Pg:IV-1c molar ratio. At constant Glu-Pg concentration increasing of the IV-1c concentration to equimolar of Pg accelerated Pg activation. Subsequent increase of IV-1c concentration inhibited the Pg activation sharply. Increasing of Glu-Pg concentration at constant IV-1c one did not inhibit Glu-Pg activation in Pg-IV-1c complex. The rate dependence of Pg activation from Glu-Pg-IV-1c complex concentration curve had bell-shaped form with maximum at 500 nM. Electrophoretic analysis of components of Glu-Pg-IV-1c complex showed that Lys-Pg and Lys-Pm were not observed at 100 nM complex concentration for 6 h period of reaction. At 680 nM concentration Glu-Pg-IV-1c complex these forms appeared in initial moments of reaction activation after lag-period. Kinetic scheme and peculiarities of Pg activation reaction in Pg-IV-1c complex are discussed.     

Effect of Specific Cleavage of Immunoglobulin G by Plasmin on the Binding and Activation of Plasminogen

A method of ELISA for measuring the binding of different samples of immunoglobulin (IgG) and its fragments to human plasminogen (Pg) has been developed. Instead of plasminogen, the heavy chain of plasminogen (Pg-H) containing five ligand-binding kringle domains, immobilized on the surface of the plate, was used in this method as a detector. It was found that IgG treated with plasmin (IgG_Pm-t) binds to the immobilized Pg-H 2.84 times more strongly than intact IgG. Both IgG samples showed a weak nonspecific binding to the immobilized light chain of plasminogen (Pg-L). It was shown that 0.2 M L-lysine inhibits the binding of IgG_Pm-t and does not affect the nonspecific binding of intact IgG to the immobilized Pg-H, indicating the involvement of lysine-binding regions of Pg-H in binding to IgG_Pm-t. A preliminary treatment of IgG samples with carboxypeptidase В (CPB) inhibited the binding of IgG_Pm-t and did not affect the nonspecific binding of intact IgG to the immobilized Pg-H, which indicates a key role of the С-terminal lysine of IgG_Pm-t in the specific binding to the lysine-binding sites of Pg. The study of the effects of intact IgG and IgG_Pm-t on the rate of activation of Glu- and Lys-forms of Pg (Glu-Pg and Lys-Pg) by a tissue activator of Pg (tPA) and urokinase (uPA) in buffer showed that intact IgG completely inhibited the activation of Glu-Pg and Lys-Pg with both tPA and uPA. Presumably, the inhibitory effect of intact IgG is due to steric hindrances that it creates for protein–protein interactions of the activators with the zymogen. IgG_Pm-t accelerated the generation of plasmin from Pg. In this case, the stimulatory effect of IgG_Pm-t on the activation of Glu-Pg under the action of tPA was ～25% higher than on the activation of Lys-Pg, which is explained by more significant conformational changes in the Glu-Pg molecule compared with the Lys-Pg molecule after their binding to IgG_Pm-t. The results suggest that the specific cleavage of IgG by plasmin may be one of the ways by which the plasminogen/plasmin system is involved in various physiological and pathological processes.     

Plasmin binding to the plasminogen receptor enhances catalytic efficiency and activates the receptor for subsequent ligand binding.

Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts plasmin (Pm) activity to specific sites and serves to promote fibrinolysis and local Pg activation. These studies show that both Pg and Pm bind to cellular receptors on monocytoid U937 cells. Limited Pm pretreatment of the cells enhances total Pg binding and alters the kinetics of Pm binding. Furthermore, surface-bound Pg is converted to Pm in the absence of exogenous activators. Cell-bound Pm exhibits a 12-fold increase in catalytic efficiency (kcat/Km) relative to Pm free in solution. These studies demonstrate that Pg/Pm receptor occupancy can be regulated by Pm in the microenvironment and may play a significant regulatory role in fibrinolysis and extravascular proteolysis.     

Role of lysine binding sites in activation of plasminogen by streptokinase

The function of lysine-binding sites in kringle domains K1-4 and K5 of plasminogen (Pg) during its activation by streptokinase (SK) was studied. Activation rates of Glu- and Lys-Pg exceed activation rate of mini- and micro-Pg 26 and 40 times, respectively. 6-Animohexanoic acid (6-AHA) in concentrations from 10(-5) to 10(-2) M inhibits activation of Glu-, Lys- and mini-Pg and does not impact the activation of micro-Pg. Complete inhibition of Lys-Pg activation occurs with presence of 10(-3) M 6-AHA while 90% inhibition of mini-Pg activation and 70% inhibition of Glu-Pg activation occur with 10(-2) M 6-AHA. Isolated kringles K1-3 and K4 of Pg inhibit activation of Glu-Pg by SK and concentrations [I]50 are 4.0 and 8.1 x 10(-6) M, respectively. Catalytic activity of Glu-Pg-SK, Lys-Pg-SK and Pm-SK complexes with respect to S 2251 is not inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M. Activation of substrate Pg by Pm-SK complex is also inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M; however, this effect of inhibition is significantly weaker than that with activation by SK. Cleavage of C-terminal Lys or chemical modification of NH2-groups of amino acid residues in SK molecule also results in the decrease of the Glu-Pg activation rate. Lysin-binding sites in K1-4 and K5 of Pg molecule are important at different steps of Pg activation process which includes formation of equimolar complex; structural reorganizations resulted in formation of active center in Pg; and binding of substrate Pg with Pg-SK complex. Lysin-binding sites in K1-4 of Pg are necessary for maintenance of high rate of Pg activation by SK.     

A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator

Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.     

Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions

Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.     

Coupling of conformational and proteolytic activation in the kinetic mechanism of plasminogen activation by streptokinase

Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v(1)) representing conformational activation and subsequent rates of activity increase (v(2)) corresponding to the rate of Pm generation determined by a specific discontinuous assay. The v(2) dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of approximately 2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an approximately 12-fold lower affinity for SK and an approximately 20-fold lower k(cat)/K(m). Blocking lysine-binding sites on Pg inhibited SK.Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu]Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK.Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK.Pg* and subsequent cleavage of Pg by SK.Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK.Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK.Pm, which then converts the remaining free Pg to Pm.     

Binding of the COOH-terminal lysine residue of streptokinase to plasmin(ogen) kringles enhances formation of the streptokinase.plasmin(ogen) catalytic complexes

Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK.Pg* catalytic complex. SK.Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKDeltaK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKDeltaK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKDeltaK414.[Lys]Pg* complex. SKDeltaK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK.[Lys]Pg* and SK.[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.     

Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells

Adenosine deaminase (ADA) is expressed intracellularly by all cells, but in some tissues, it is also associated with the cell surface multifunctional glycoprotein CD26/dipeptidyl peptidase IV. By modulating extracellular adenosine, this "ecto-ADA" may regulate adenosine receptor signaling implicated in various cellular functions. CD26 is expressed on the surface of human prostate cancer 1-LN cells acting as a receptor for plasminogen (Pg). Since ADA and Pg bind to CD26 at distinct but nearby sites, we investigated a possible interaction between these two proteins on the surface of 1-LN cells. Human ADA binds to CD26 on the surface 1-LN cells and immobilized CD26 isolated from the same cells with similar affinity. In both cases, ADA binding is diminished by mutation of ADA residues known to interact with CD26. ADA was also found to bind Pg 2 in the absence of CD26 via the Pg kringle 4 (K4) domain. In the presence of 1-LN cells or immobilized CD26, exogenous ADA enhances conversion of Pg 2 to plasmin by 1-LN endogenous urinary plasminogen activator (u-PA), as well as by added tissue Pg Activator (t-PA), suggesting that ADA and Pg bind simultaneously to CD26 in a ternary complex that stimulates the Pg activation by its physiologic activators. Consistent with this, in melanoma A375 cells that bind Pg, but do not express CD26, the rate of Pg activation was not affected by ADA. Thus, ADA may be a factor regulating events in prostate cancer cells that occur when Pg binds to the cell surface and is activated.     

Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen

Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 +/- 2 pm and 12 +/- 4 pm, which represented a 1,000-10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK(55-414) species had a 120-360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK.Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.     

Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins

We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.     

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.     

Resolution of conformational activation in the kinetic mechanism of plasminogen activation by streptokinase

Streptokinase (SK) activates plasminogen (Pg) by specific binding and nonproteolytic expression of the Pg catalytic site, initiating Pg proteolysis to form the fibrinolytic proteinase, plasmin (Pm). The SK-induced conformational activation mechanism was investigated in quantitative kinetic and equilibrium binding studies. Progress curves of Pg activation by SK monitored by chromogenic substrate hydrolysis were parabolic, with initial rates (v(1)) that indicated no transient species and subsequent rate increases (v(2)). The v(1) dependence on SK concentration for [Glu]Pg and [Lys]Pg was hyperbolic with dissociation constants corresponding to those determined in fluorescence-based binding studies for the native Pg species, identifying v(1) as rapid SK binding and conformational activation. Comparison of [Glu]Pg and [Lys]Pg activation showed an approximately 12-fold higher affinity of SK for [Lys]Pg that was lysine-binding site dependent and no such dependence for [Glu]Pg. Stopped-flow kinetics of SK binding to fluorescently labeled Pg demonstrated at least two fast steps in the conformational activation pathway. Characterization of the specificity of the conformationally activated SK.[Lys]Pg* complex for tripeptide-p-nitroanilide substrates demonstrated 5-18- and 10-130-fold reduced specificity (k(cat)/K(m)) compared with SK.Pm and Pm, respectively, with differences in K(m) and k(cat) dependent on the P1 residue. The results support a kinetic mechanism in which SK binding and reversible conformational activation occur in a rapid equilibrium, multistep process.