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.     

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was $\text{40–50}\text{pmol}\text{2.5·10}{}^6$ cells per 24 h. Additions of 20, 60 or 100 μg of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 μg/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.     

Identification of a site in fibrin(ogen) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.     

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.     

Fibronectin decreases the stimulatory effect of fibrin and fibrinogen fragment FCB-2 on plasmin formation by tissue plasminogen activator.

Fibronectin is a dimeric glycoprotein (Mr 440,000) involved in many adhesive processes. During blood coagulation it is bound and cross-linked to fibrin. Fibrin binding is achieved by structures (type I repeats) which are homologous to the "finger" domain of tissue plasminogen activator. Tissue plasminogen activator also binds to fibrin via the finger domain and additionally via the "kringle 2" domain. Fibrin binding of tissue plasminogen activator results in stimulation of its activity and plays a crucial role in fibrinolysis. Since fibronectin might interfere with this binding, we studied the effect of fibronectin on plasmin formation by tissue plasminogen activator. In the absence of fibrin, fibronectin had no effect on plasminogen activation. In the presence of stimulating fibrinogen fragment FCB-2, fibronectin increased the duration of the initial lag phase (= time period until maximally stimulated plasmin formation occurs) and decreased the rate of maximal plasmin formation which occurs after that lag phase mainly by increasing the Michaelis constant (Km). These effects of fibronectin were dose-dependent and were similar with single- and two-chain tissue plasminogen activator. They were also observed with plasmin-pretreated FCB-2. An apparent Ki of 43 micrograms/ml was calculated for the inhibitory effect of fibronectin when plasminogen activation by recombinant single-chain tissue plasminogen activator was studied in the presence of 91 micrograms/ml FCB-2. When a recombinant tissue plasminogen activator mutant lacking the finger domain was used in a system containing FCB-2, no effect of fibronectin was seen, indicating that the inhibitory effect of fibronectin might in fact be due to competition of fibronectin and tissue plasminogen activator for binding to fibrin(ogen) via the finger domain.     

Specific binding of plasminogen to vitronectin. Evidence for a modulatory role of vitronectin on fibrin(ogen)-induced plasmin formation by tissue plasminogen activator.

Vitronectin immobilized onto polystyrene microtiter wells was demonstrated to specifically bind plasminogen in a concentration-dependent manner, yielding an estimated KD = 0.4 microM. Heparin only moderately interfered with the vitronectin-plasminogen interaction, whereas high concentrations of 6-amino-hexanoic acid inhibited binding. Utilizing a ligand-blotting procedure in which plasminogen was reacted with proteolytic fragments of vitronectin, transblotted onto nitrocellulose, the plasminogen-binding site of vitronectin was localized to the heparin-binding domain of the adhesive protein. Moreover, vitronectin was found to inhibit in a dose-dependent fashion the fibrin(ogen)-induced activation of plasminogen by tissue plasminogen activator. These results provide the first evidence for a novel vitronectin-mediated control of plasminogen activation potentially relevant for directional clot-lysis and plasmin-dependent proteolysis in extracellular matrices.     

The role of ligand-ligand interactions in competition by fibrinogen and fibrin degradation products for fibrinogen binding to human platelets

Binding to human platelets of radioiodinated human fibrinogen and fragments X, Y, D, D₁ dimer and E was studied to determine the domain of the fibrinogen molecule responsible for binding to the platelet receptor. Although the fragments did not bind, some wer able to complete for the binding of fibrinogen to platelets. It was postulated that the fragments bound to fibrinogen and subsequently interfered with its binding to the receptor. Two approaches were developed to test this hypothesis. In the first technique, molecular exclusion on Sephacryl S-200 superfine was utilized to examine the interaction of radiolabeled fragments with fibrinogen. In the second seties of studies, fibrinogen-Sepharose was prepared and the binding of degradation products directly determined. A spin dialysis apparatus was employed in each case to achieve rapid separation of bound and free radioligand. These studies demonstrated that fragments D and E bind to fibrinogen. Therefore, the mechanism by which degradation products interfere with fibrinogen binding to the platelet receptor is ligand-ligand interaction rather than binding of the fragments to the receptor. Since none of the radiolabeled degradation products bound to platelets, it appears that receptor recognition requires the intact molecule.     

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)     

Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1

Two acetylation sites on prothrombin fragment 1 (amino-terminal 156 amino acid residues of bovine prothrombin) are essential for the tight calcium and membrane binding functions of the protein; calcium protects both of these sites from acetylation [Welsch, D. J., Pletcher, C. H., & Nelsestuen, G. L. (1988) Biochemistry (first of three papers in this issue)]. The epsilon-amino groups of the lysine residues (positions 3, 11, 44, 57, and 97) were not critical to protein function and were acetylated in the calcium-protected protein. The most reactive of the two essential acetylation sites was identified as amino-terminal alanine. To identify this site, fragment 1 was first acetylated in the presence of calcium to derivatize the nonessential sites. Removal of calcium and partial acetylation with radioactive reagent produced a single major radioactive peptide. Isolation and characterization of this peptide showed that the radioactivity was associated with amino-terminal alanine. In addition, sequence analysis of calcium-protected protein showed the presence of underivatized amino-terminal alanine. Surprisingly, covalent modification with a trinitrophenyl group did not alter membrane binding activity. Thus, the positive charge on the amino terminus did not appear critical to its function. Acetylation of amino-terminal alanine without acetylation of the second essential site produced a fragment 1 derivative which had a high requirement for calcium and which had lost most membrane binding function. However, this protein had only slightly altered affinity for magnesium ion. In agreement with this metal ion selectivity, protection of amino-terminal alanine was calcium specific, and magnesium ion did not protect this site from acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation

Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.     

Inhibition of plasminogen activation by lipoprotein(a): critical domains in apolipoprotein(a) and mechanism of inhibition on fibrin and degraded fibrin surfaces

Similarity between the apolipoprotein(a) (apo(a)) moiety of lipoprotein(a) (Lp(a)) and plasminogen suggests a potentially important link between atherosclerosis and thrombosis. Lp(a) may interfere with tissue plasminogen activator (tPA)-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoagulable state in vivo. A fluorescence-based system was employed to study the effect of apo(a) on plasminogen activation in the presence of native fibrin and degraded fibrin cofactors and in the absence of positive feedback reactions catalyzed by plasmin. Human Lp(a) and a physiologically relevant, 17-kringle recombinant apo(a) species exhibited strong inhibition with both cofactors. A variant lacking the protease domain also exhibited strong inhibition, indicating that the apo(a)-plasminogen binding interaction mediated by the apo(a) protease domain does not ultimately inhibit plasminogen activation. A variant in which the strong lysine-binding site in kringle IV type 10 had been abolished exhibited substantially reduced inhibition whereas another lacking the kringle V domain showed no inhibition. Amino-terminal truncation mutants of apo(a) also revealed that additional sequences within kringle IV types 1-4 are required for maximal inhibition. To investigate the inhibition mechanism, the concentrations of plasminogen, cofactor, and a 12-kringle recombinant apo(a) species were systematically varied. Kinetics for both cofactors conformed to a single, equilibrium template model in which apo(a) can interact with all three fibrinolytic components and predicts the formation of ternary (cofactor, tPA, and plasminogen) and quaternary (cofactor, tPA, plasminogen, and apo(a)) catalytic complexes. The latter complex exhibits a reduced turnover number, thereby accounting for inhibition of plasminogen activation in the presence of apo(a)/Lp(a).     

Toll-like receptor 4 region Glu24-Lys47 is a site for MD-2 binding: importance of CYS29 and CYS40

Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS), but its interaction with MD-2 is required for efficient responses to LPS. Previous studies with deletion mutants indicate a critical role of the amino-terminal TLR4 region in interaction with MD-2. However, it is uncertain which region in the TLR4 molecule directly binds to MD-2. The purpose of this study was to determine a critical stretch of primary sequence in the TLR4 region that directly binds MD-2 and is critical for LPS signaling. The synthetic TLR4 peptide corresponding to the TLR4 region Glu(24)-Lys(47) directly binds to recombinant soluble MD-2 (sMD-2). The TLR4 peptide inhibited the binding of a recombinant soluble form of the extracellular TLR4 domain (sTLR4) to sMD-2 and significantly attenuated LPS-induced NF-kappaB activation and IL-8 secretion in wild type TLR4-transfected cells. Reduction and S-carboxymethylation of sTLR4 abrogated its association with sMD-2. The TLR4 mutants, TLR4(C29A), TLR4(C40A), and TLR4(C29A,C40A), were neither co-precipitated with MD-2 nor expressed on the cell surface and failed to transmit LPS signaling. These results demonstrate that the TLR4 region Glu(24)-Lys(47) is a site for MD-2 binding and that Cys(29) and Cys(40) within this region are critical residues for MD-2 binding and LPS signaling.     

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.     

Plasminogen activation by tissue activator is accelerated in the presence of fibrin(ogen) cyanogen bromide fragment FCB-2

Fibrin, in contrast to fibrinogen, strongly accelerates the plasminogen activation by extrinsic activator (tissue-type plasminogen activator, t-PA). However, when fibrin and fibrinogen are digested with cyanogen bromide, both digests potentiate the t-PA-mediated plasminogen activation equally well. In this report, evidence is presented that this potentiating activity resides in CNBr fragment FCB-2 (= Ho1-DSK) and that a polymeric structure such as fibrin is not a prerequisite for the potentiation.     

Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein

Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K_d value of 1.6 ± 0.4 μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif ₂₆₀VYDLESKAV₂₆₈, similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite.     

N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.