Characterization of the I4399M variant of apolipoprotein(a): implications for altered prothrombotic properties of lipoprotein(a).

Essentials Elevated lipoproteinp(a) is an independent and causal risk factor for atherothrombotic diseases. rs3798220 (Ile/Met substitution in apo(a) protease-like domain) is associated with disease risk. Recombinant I4399M apo(a) altered clot structure to accelerate coagulation/delay fibrinolysis. Evidence was found for increased solvent exposure and oxidation of Met residue. SUMMARY: Background Lipoprotein(a) (Lp[a]) is a causal risk factor for a variety of cardiovascular diseases. Apolipoprotein(a) (apo[a]), the distinguishing component of Lp(a), is homologous with plasminogen, suggesting that Lp(a) can interfere with the normal fibrinolytic functions of plasminogen. This has implications for the persistence of fibrin clots in the vasculature and hence for atherothrombotic diseases. A single-nucleotide polymorphism (SNP) (rs3798220) in the gene encoding apo(a) has been reported that results in an Ile→Met substitution in the protease-like domain (I4399M variant). In population studies, the I4399M variant has been correlated with elevated plasma Lp(a) levels and higher coronary heart disease risk, and carriers of the SNP had increased cardiovascular benefit from aspirin therapy. In vitro studies suggested an antifibrinolytic role for Lp(a) containing this variant. Objectives We performed a series of experiments to assess the effect of the Ile→Met substitution on fibrin clot formation and lysis, and on the architecture of the clots. Results We found that the Met variant decreased coagulation time and increased fibrin clot lysis time as compared with wild-type apo(a). Furthermore, we observed that the presence of the Met variant significantly increased fibrin fiber width in plasma clots formed ex vivo, while having no effect on fiber density. Mass spectrometry analysis of a recombinant apo(a) species containing the Met variant revealed sulfoxide modification of the Met residue. Conclusions Our data suggest that the I4399M variant differs structurally from wild-type apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.

Identification of a thrombomodulin interaction site on thrombin-activatable fibrinolysis inhibitor that mediates accelerated activation by thrombin.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM) or plasmin. Evidence from several studies suggests that TM and TAFI make direct contact at sites remote from the activating cleavage site to facilitate acceleration of thrombin-mediated TAFI activation. The elements of TAFI structure that allow accelerated activation of thrombin by TM are incompletely defined. OBJECTIVES: To identify TM interaction regions on TAFI that mediate acceleration of activation by thrombin and therefore indicate TM binding sites on TAFI. METHODS: We mutated selected surface-exposed charged residues on TAFI to alanine in order to identify sites that mediate acceleration of activation by TM. The kinetics of activation of the mutants by thrombin in the presence or absence of TM, as well as their thermal stabilities and antifibrinolytic potentials, were determined. RESULTS: TAFI variants R15A, E28A, K59A, D75A/E77A/D78A, E99A and E106A all exhibited moderately reduced catalytic efficiencies of activation by thrombin-TM. TAFI variants R377A and, particularly, R12A and R12A/R15A exhibited severely reduced activation by thrombin-TM that was not explained by differences in activation by thrombin alone. CONCLUSIONS: We have identified R12 as a critical residue for the activation of TAFI by thrombin-TM, extending a previous report that identified a role for this residue. R12 is likely to directly bind to TM while another key residue, R377, may affect the thrombin-TAFI interaction specifically in the presence of TM.

Pro-inflammatory cytokines reduce human TAFI expression via tristetraprolin-mediated mRNA destabilisation and decreased binding of HuR.

Thrombin activatable fibrinolysis inhibitor (TAFI) is the zymogen form of a basic carboxypeptidase (TAFIa) with both anti-fibrinolytic and anti-inflammatory properties. The role of TAFI in inflammatory disease is multifaceted and involves modulation both of specific inflammatory mediators as well as of the behaviour of inflammatory cells. Moreover, as suggested by in vitro studies, inflammatory mediators are capable of regulating the expression of CPB2, the gene encoding TAFI. In this study we addressed the hypothesis that decreased TAFI levels observed in inflammation are due to post-transcriptional mechanisms. Treatment of human HepG2 cells with pro-inflammatory cytokines TNFα, IL-6 in combination with IL-1ß, or with bacterial lipopolysaccharide (LPS) decreased TAFI protein levels by approximately two-fold over 24 to 48 hours of treatment. Conversely, treatment of HepG2 cells with the anti-inflammatory cytokine IL-10 increased TAFI protein levels by two-fold at both time points. We found that the mechanistic basis for this modulation of TAFI levels involves binding of tristetraprolin (TTP) to the CPB2 3'-UTR, which mediates CPB2 mRNA destabilisation. In this report we also identified that HuR, another ARE-binding protein but one that stabilises transcripts, is capable of binding the CBP2 3'UTR. We found that pro-inflammatory mediators reduce the occupancy of HuR on the CPB2 3'-UTR and that the mutation of the TTP binding site in this context abolishes this effect, although TTP and HuR appear to contact discrete binding sites. Interestingly, all of the mediators tested appear to increase TAFI protein expression in THP-1 macrophages, likewise through effects on CPB2 mRNA stability.

Identification of tristetraprolin as a factor that modulates the stability of the TAFI transcript through binding to the 3'-untranslated region.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen encoded by the human gene CPB2. TAFI constitutes a molecular link between coagulation and fibrinolysis, and between coagulation and inflammation. The 3'-untranslated region (UTR) of the human CPB2 mRNA plays a key role in regulating CPB2 mRNA abundance, but the exact mechanisms that mediate this regulation are largely unexplored. OBJECTIVES: To pinpoint cis-acting elements in the CPB2 3'-UTR that act as stability determinants and to identify protein factors binding to these sites. METHODS: We constructed a series of plasmids encoding mRNAs containing rabbit ß-globin sequences (as a reporter) fused to sequences of the CPB2 3'-UTR (encompassing 5' and internal deletions). These plasmids were transfected into HepG2 (human hepatoma) cells and the stability of the fusion transcripts measured. We performed a series of gel mobility shift analyses using RNA probes encompassing putative (in)stability elements. RESULTS: We identified one element conferring stability and three elements conferring instability. Supershift assays identified the protein bound to the site between the second and third polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP site abolished TTP binding in gel mobility shift assays and also stabilized ß-globin/CPB2 fusion transcripts. TTP knockdown stabilized the fusion transcript containing the TTP site, but not a fusion transcript in which this site was mutated. CONCLUSIONS: Our findings are indicative of a role for TTP in constitutive, and perhaps regulated, control of CPB2 mRNA stability and hence abundance.

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells.

TAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

Secretion and antifibrinolytic function of thrombin-activatable fibrinolysis inhibitor from human platelets.

BACKGROUND: The thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen first characterized in human plasma that is activated through proteolytic cleavage by thrombin, thrombin in complex with thrombomodulin, or plasmin. Active TAFI attenuates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin, thereby inhibiting a potent positive feedback loop in the fibrinolytic cascade. The existence of a separate pool of TAFI within platelets has been described. OBJECTIVES AND METHODS: We aimed to confirm the presence of TAFI in the medium of washed, thrombin-stimulated platelets and to evaluate the characteristics of platelet TAFI by western blot analysis and with a quantitative assay for activated TAFI. We also assessed the ability of platelet TAFI to inhibit fibrinolysis in vitro, using a platelet-rich thrombus lysis assay. RESULTS: Our data are consistent with the presence of TAFI in the α-granules of resting platelets. In contrast to previous reports, platelet TAFI is very similar in electrophoretic mobility to plasma-derived TAFI. We also show, for the first time, that platelet-derived TAFI is capable of attenuating platelet-rich thrombus lysis in vitro independently of plasma TAFI. Moreover, we demonstrate additive effects on thrombolysis of platelet-derived TAFI and TAFI present in plasma. CONCLUSIONS: Taken together, these observations indicate that the secretion of platelet-derived TAFI can augment the concentrations of TAFI already present in plasma to enhance attenuation of the fibrinolytic cascade. This could be significant in regions of vascular damage or pathologic thrombosis, where activated platelets are known to accumulate.

Functional analysis of mutant variants of thrombin-activatable fibrinolysis inhibitor resistant to activation by thrombin or plasmin.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) defines a pathway that functionally links the coagulation and fibrinolytic cascades. TAFI is activated by proteolytic cleavage, a reaction that can be performed by thrombin and plasmin, but most efficiently by thrombin in complex with the endothelial cofactor thrombomodulin (TM). The respective roles of these activators in regulating the TAFI pathway are largely unknown. OBJECTIVE AND METHODS: In the present study, we constructed and expressed mutant variants of TAFI that have key substitutions in the amino acids surrounding the scissile Arg92-Ala93 bond. RESULTS AND CONCLUSIONS: We identified variants that showed patterns of resistance to specific activators. For example, the P91S, R92K and S90P variants exhibited specific impairment of activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The variants that we tested also showed antifibrinolytic potentials that can be rationalized in terms of which enzymes are capable of activating them. On the other hand, certain predictions from peptide studies of mutations that would be expected to interfere with plasmin cleavage were not satisfied by our data, indicating that protein context, as well as the identity of amino acids at protease cleavage sites, dictates protease specificity.

Apolipoprotein(a) inhibits the conversion of Glu-plasminogen to Lys-plasminogen: a novel mechanism for lipoprotein(a)-mediated inhibition of plasminogen activation.

BACKGROUND: Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for thrombotic disorders. Lp(a) is a unique lipoprotein consisting of a low-density lipoprotein-like moiety covalently linked to apolipoprotein(a) [apo(a)], a homologue of the fibrinolytic proenzyme plasminogen. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu-plasminogen to Lys-plasminogen. OBJECTIVE: Using acid-urea gel electrophoresis to resolve the two forms of radiolabeled plasminogen, we determined whether apo(a) is able to inhibit Glu-plasminogen to Lys-plasminogen conversion. METHODS: The assays were performed in the absence or presence of different recombinant apo(a) species, including point mutants, deletion mutants and variants that represent greater than 90% of the known apo(a) isoform sizes. RESULTS: Apo(a) substantially suppressed Glu-plasminogen conversion. Critical roles were identified for the kringle IV types 5-9 and kringle V; contributory roles for sequences within the amino-terminal half of the molecule were also observed. Additionally, with the exception of the smallest naturally-occurring isoform of apo(a), isoform size was found not to contribute to the inhibitory capacity of apo(a). CONCLUSION: These findings underscore a novel contribution to the understanding of Lp(a)/apo(a)-mediated inhibition of plasminogen activation: the ability of the apo(a) component of Lp(a) to inhibit the key positive feedback step of plasmin-mediated Glu-plasminogen to Lys-plasminogen conversion.

Role of mRNA transcript stability in modulation of expression of the gene encoding thrombin activable fibrinolysis inhibitor.

Regulation of mRNA stability has emerged as a major control point in eukaryotic gene expression. The abundance of a particular mRNA can be rapidly regulated in response to a stimulus by altering the stability of existing translatable transcripts rather than by altering the rate of transcription initiation. Alternative polyadenylation of transcripts during mRNA processing can be important in determining transcript abundance if the different forms of mRNA possess different stabilities or translatability. The mRNA transcript encoding thrombin activable fibrinolysis inhibitor (TAFI) is an attractive candidate for regulation of mRNA stability because of the relatively long length of its 3'-untranslated region and because the transcript can be polyadenylated at three different sites. As well, we have previously reported that treatment of HepG2 cells with interleukins (IL) - 1beta and - 6 destabilizes the endogenous TAFI mRNA expressed in this cell line. In the current study, we report that the TAFI 3'-untranslated region contains cis-acting instability element(s) and that these elements in fact determine the intrinsic stability of the TAFI transcript. Moreover, we found that the three different polyadenylated mRNA forms have different intrinsic stabilities, with the mRNA half-life increasing from the longest to the shortest transcript. Interestingly, treatment with IL-1beta plus IL-6 not only resulted in a 2-fold decrease in stability of the transcript produced using the 3'-most polyadenylation site but also resulted in profound shifts in the relative abundances of the respective polyadenylated forms through changes in the frequency of utilization of the three polyadenylation sites. As such, in the presence of IL-1beta and IL-6, the longest transcript is over a thousand times more abundant than the two shorter transcripts whereas in the absence of the stimulus it comprises only 1% of the total TAFI transcripts.

Association of a single nucleotide polymorphism in CPB2 encoding the thrombin-activable fibrinolysis inhibitor (TAF1) with blood pressure.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a hepatically secreted zymogen, whose substrates include bradykinin. The CPB2 gene encoding TAFI is a candidate gene for blood pressure. A recently identified single nucleotide polymorphism (SNP) in the CPB2 coding region, designated as 1057C > T, results in an amino acid change at TAFI residue 325 (Ile > Thr325). We found that the genotype based on this SNP was significantly associated with blood pressure in aboriginal Canadians. Specifically, analysis of variance showed that homozygotes for CPB2 1057T had significantly lower diastolic blood pressure than subjects with other CPB2 genotypes. CPB2 genotype accounted for approximately 3% of the total variation in diastolic blood pressure. consistent with the expected magnitude of a modest genetic effect in a complex trait such as blood pressure. Although the mechanism underlying the association is unclear, the findings are of interest because TAFI may provide a link between coagulation and blood pressure regulation.

Thrombin activable fibrinolysis inhibitor (TAFI): molecular genetics of an emerging potential risk factor for thrombotic disorders.

The balance between the activities of the coagulation and fibrinolytic cascades is crucial for normal hemostasis. However, imbalances can lead to pathological thrombotic events, as is observed in heart attacks and strokes, as well as excessive bleeding, as in hemophilia. Recent investigations have uncovered a novel molecular connection between the two cascades that has been termed thrombin-activable fibrinolysis inhibitor (TAFI) as well as procarboxypeptidase U, procarboxypeptidase R or plasma procarboxypeptidase B. TAFI is the precursor of an enzyme (TAFIa) with basic carboxypeptidase activity that attenuates the lysis of fibrin clots by removal of the carboxyl-terminal lysine residues from partially-degraded fibrin that mediate positive feedback in the fibrinolytic cascade. The plasma concentration of TAFI varies substantially (up to approximately 10-fold) in the human population and may constitute a novel risk factor for thrombotic disorders. Sixteen single nucleotide polymorphisms have been identified in the 5'-flanking, protein coding, and 3'-untranslated regions of the TAFI gene. The polymorphisms all have been shown to be associated with variations in plasma TAFI concentrations. One amino acid substitution has been found to directly alter the properties of the TAFIa enzyme. This review provides a general overview of the TAFI pathway, including a discussion of the spectrum of inhibitors of TAFIa that have been described, and summarizes the recent advances in the molecular genetics of the TAFI gene as well as the results of studies that may implicate the TAFI pathway in risk for arterial and venous thrombotic disorders.

Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.

We have used site-directed mutagenesis and a recombinant expression system for thrombin-activable fibrinolysis inhibitor (TAFI) in order to identify the thrombin cleavage site in activated TAFI (TAFIa) and to determine the relative contribution of proteolytic cleavage and thermal instability in regulation of TAFIa activity in clots. Arg-330 of TAFIa had been proposed to be the thrombin cleavage site based on studies with trypsin, but mutation of this residue to Gln did not prevent thrombin-mediated cleavage nor did mutation to Gln of the nearby Arg-320 residue. However, mutation of Arg-302 to Gln abolished thrombin-mediated cleavage of TAFIa. All TAFIa variants were susceptible to plasmin cleavage. Interestingly, all Arg to Gln substitutions decreased the thermal stability of TAFIa. The antifibrinolytic potential of the TAFI mutants in vitro correlates with the thermal stability of their respective TAFIa species, indicating that this property plays a key role in regulating the activity if TAFIa. Incubation of TAFIa under conditions that result in complete thermal inactivation of the enzyme accelerates subsequent thrombin- and plasmin-mediated cleavage of TAFIa. Moreover, the extent of cleavage of TAFIa by thrombin does not affect the rate of decay of TAFIa activity. Collectively, these studies point to a role for the thermal instability, but not for proteolytic cleavage, of TAFIa in regulation of its activity and, thus, of its antifibrinolytic potential. Finally, we propose a model for the thermal instability of TAFIa.

Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B).

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.

A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor.

TAFI (thrombin-activable fibrinolysis inhibitor) is a recently described plasma zymogen that, when exposed to the thrombin-thrombomodulin complex, is converted by proteolysis at Arg92 to a basic carboxypeptidase that inhibits fibrinolysis (TAFIa). The studies described here were undertaken to elucidate the molecular basis for the inhibition of fibrinolysis. When TAFIa is included in a clot undergoing fibrinolysis induced by tissue plasminogen activator and plasminogen, the time to achieve lysis is prolonged, and free arginine and lysine are released over time. In addition, TAFIa prevents a 2.5-fold increase in the rate constant for plasminogen activation which occurs when fibrin is modified by plasmin in the early course of fibrin degradation. The effect is specific for the Glu- form of plasminogen. TAFIa prevents or at least attenuates positive feedback expressed through Lys-plasminogen formation during the process of fibrinolysis initiated by tissue plasminogen activator and plasminogen. TAFIa also inhibits plasmin activity in a clot and prolongs fibrinolysis initiated with plasmin. We conclude that TAFIa suppresses fibrinolysis by removing COOH-terminal lysine and arginine residues from fibrin, thereby reducing its cofactor functions in both plasminogen activation and the positive feedback conversion of Glu-plasminogen to Lys-plasminogen. At relatively elevated concentrations, it also directly inhibits plasmin.

Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B. Cleavage of the zymogen by thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo. In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with kcat = 0.55 s-1, K(m) = 0.54 microM, and Kd = 6.0 nM for rTAFI and kcat = 0.61 s-1, K(m) = 0.55 microM, and Kd = 6.6 nM for pTAFI. The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis. The half-life of both enzymes at 37 degrees C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to approximately 62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme. Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (delta Ho++ approximately equal to 45 kcal/mol and delta So++ approximately equal to 80 cal/mol/K). Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and epsilon-aminocaproic acid. rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis. These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.

The solution phase interaction between apolipoprotein(a) and plasminogen inhibits the binding of plasminogen to a plasmin-modified fibrinogen surface.

In the present study, we assessed the binding of recombinant forms of apolipoprotein(a) [r-apo(a)] to plasminogen. Apo(a)-plasminogen interactions were demonstrated to be lysine-dependent, as they were abolished by the addition of epsilon-aminocaproic acid. Binding of r-apo(a) and plasma-derived Lp(a) to Glu-plasminogen was assessed in solution using a mutant form of recombinant plasminogen [Plg(S741C)] labeled at the active site with 5'-(iodoacetamido)fluorescein. High-affinity binding of apo(a) to plasminogen was observed with the 17-kringle r-apo(a) (Kd = 20.1 +/- 3.3 nM) as well as with plasma-derived Lp(a) (Kd = 5.58 +/- 0.08 nM). Binding studies using various truncated and mutant forms of r-apo(a) demonstrated that sequences within apo(a) kringle IV types 2-9 and the strong lysine binding site (LBS) in apo(a) kringle IV type 10 are not required for high-affinity binding to plasminogen. In all cases, the binding stoichiometry for the apo(a)-plasminogen interaction was determined to be 1:1. Binding data obtained using a 17-kringle r-apo(a) derivative lacking the protease-like domain (17KDeltaP; Kd = 3158 +/- 138 nM) indicate that sequences within the protease-like domain of apo(a) mediate its interaction with LBS in plasminogen. We determined that r-apo(a) and plasminogen bind to distinct sites on plasmin-modified fibrinogen with the concentration of plasminogen binding sites exceeding the concentration of r-apo(a) sites by a factor of 10. Furthermore, r-apo(a) is capable of inhibiting the binding of plasminogen to plasmin-modified fibrinogen surfaces, an effect which we show is attributable to the formation of a solution phase apo(a)/plasminogen complex which exhibits a greatly reduced affinity for plasminogen binding sites on plasmin-modified fibrinogen. The results of this study provide new insights into the mechanism by which apo(a) and Lp(a) may inhibit fibrinolysis, thus contributing to the atherothrombotic risk associated with this lipoprotein.

New retinoid X receptor subtypes in zebra fish (Danio rerio) differentially modulate transcription and do not bind 9-cis retinoic acid.

Retinoid X receptors (RXRs), along with retinoic acid (RA) receptors (RARs), mediate the effects of RA on gene expression. Three subtypes of RXRs (alpha, beta, and gamma) which bind to and are activated by the 9-cis stereoisomer of RA have been characterized. They activate gene transcription by binding to specific sites on DNA as homodimers or as heterodimers with RARs and other related nuclear receptors, including the vitamin D receptor, thyroid hormone receptors (TRs), and peroxisome proliferator-activated receptors. Two additional RXR subtypes (delta and epsilon) isolated from zebra fish cDNA libraries are described here; although both subtypes form DNA-binding heterodimers with RARs and TR, neither binds 9-cis RA, and both are transcriptionally inactive on RXR response elements. In cotransfection studies with TR, the delta subtype was found to function in a dominant negative manner, while the epsilon subtype had a slight stimulatory effect on thyroid hormone (T3)-dependent transcriptional activity. The discovery of these two novel receptors in zebra fish expands the functional repertoire of RXRs to include ligand-independent and dominant negative modulation of type II receptor function.

A zebrafish retinoic acid receptor expressed in the regenerating caudal fin.

Retinoic acid (RA) is an important signalling molecule in vertebrate pattern formation both in developing and regenerating tissues. The effects of RA are due largely to regulation of gene transcription, mediated by retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma) and retinoid X receptors (RXR-alpha, RXR-beta, RXR-gamma). We have been using zebrafish as a model of regeneration to study the role of retinoic acid and its receptors in vertebrate pattern formation. In this report, we describe the molecular cloning and characterization of one of the zebrafish RARs that is the predominant receptor in the regenerating caudal fin and corresponds most closely to the RAR-gamma subtype isolated from mouse and human and to RAR-delta from newt. Zebrafish RAR-gamma (zfRAR-gamma) exhibits both structural and functional conservation with its mammalian counterparts. Studies utilizing both normal and regenerating caudal fins of the zebrafish have indicated that it is the RAR-gamma subtype, compared to RAR-alpha or RAR-beta, which is expressed at the highest levels in the tail fin. To localize the expression pattern of RAR-gamma during fin regeneration, we have carried out whole-mount in situ hybridization. ZfRAR-gamma transcripts, during fin regeneration, are localized in the blastemal tissue formed at the distal ends of the bony rays following amputation. Treatment of fish with RA during fin regeneration induces a number of striking morphological effects on the regenerate. When amputations are performed distal to the branch points or dichotomies, where a single ray bifurcates to extend two individual 'daughter' rays, RA treatment causes a dichotomy reduction where the two 'daughter' rays fuse to once again form a single ray. The single ray subsequently bifurcates in a comparatively normal manner. Our data suggest that exogenous RA can respecify pattern in the regenerating caudal fin and identifies the blastemae as possible RA target tissues.