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.

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.

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.

Evidence mounts for a role of the kidney in lipoprotein(a) catabolism.

Numerous studies have suggested a role of the kidney in lipoprotein(a) (Lp(a)) catabolism, but direct evidence is still lacking. Frischmann et al. demonstrate that the marked elevation of Lp(a) observed in hemodialysis patients results from a decrease in Lp(a) clearance rather than an increase in Lp(a) production, consistent with the notion that the kidney degrades Lp(a). More studies are needed to prove the biological relevance.

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.

The relationship between lipoprotein(a) and the complications of diabetes mellitus.

The risk of cardiovascular disease is increased approximately two- to four-fold in patients with diabetes mellitus compared with non-diabetic controls. The nature of this increased risk cannot be completely explained by the contribution of traditional risk factors. As such, there has been a great deal of interest in assessing the role of lipoprotein(a) (Lp(a)), an LDL-like lipoprotein, in the vascular complications of diabetes. Although numerous studies in the non-diabetic population have demonstrated an association between elevated plasma Lp(a) concentration and risk for atherosclerotic disorders, the contribution of Lp(a) to the enhanced risk of vascular disease in the diabetic population is not clearly defined. Herein we review the structure and potential functions of Lp(a), the determination of Lp(a) levels, and the epidemiological evidence supporting its role in coronary heart disease and address the following controversial questions regarding the role of Lp(a) in diabetes mellitus: (1) are plasma Lp(a) levels and phenotype distributions altered in type 1 (insulin-dependent) diabetes mellitus and type 2 (non-insulin-dependent) diabetes mellitus and does the degree of metabolic control influence Lp(a) levels in these patients; (2) what is the relationship between Lp(a) and renal disease in patients with diabetes mellitus; (3) do increased plasma Lp(a) concentrations in patients with diabetes contribute to the vascular complications of this disease; and (4) can the atherogenicity of Lp(a) in diabetes be enhanced in the absence of elevated levels of this lipoprotein due to biochemical modifications.

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate >> arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL. These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.

Effect of low-density lipoprotein buoyant density and cholesterol content on the formation of lipoprotein(a) particles.

Lipoprotein(a) [Lp(a)] is a unique lipoprotein which resembles low-density lipoprotein (LDL) both in lipid composition and the presence of apolipoprotein B-100 (apo B-100). Lp(a) is, however, distinguishable from LDL by the presence of an additional glycoprotein apolipoprotein(a) [apo(a)], which is covalently attached to apo B-100 by a single disulfide bond. It is now generally accepted that Lp(a) assembly is a two-step process in which the initial non-covalent interaction between apo(a) and apo B-100 is mediated by the weak lysine binding sites present in kringle IV types 6, 7 and 8 of apo(a). In the present study, we have investigated the effect of LDL heterogeneity on Lp(a) assembly in a group of 111 individuals. The three parameters of LDL composition assessed in this study were the cholesterol content, the apo B content, and the relative flotation rate (a measure of LDL buoyancy and thus size). We found no correlation between the size of LDL particles and the extent of Lp(a) formation; a weak negative correlation was observed between cholesterol content of LDL and Lp(a) formation (P=0.042). This may suggest a role for free (i.e., surface-associated) cholesterol in the ability of LDL to form Lp(a) particles.

The structure of lipoprotein(a) and ligand-induced conformational changes.

Lipoprotein(a) is composed of low-density lipoprotein linked both covalently and noncovalently to apolipoprotein(a). The structure of lipoprotein(a) and the interactions between low-density lipoprotein and apolipoprotein(a) were investigated by electron microscopy and correlated with analytical ultracentrifugation. Electron microscopy of rotary-shadowed and unidirectionally shadowed lipoprotein(a) prepared without glycerol revealed that it is a nearly spherical particle with no large projections. After extraction of both lipoprotein(a) and low-density lipoprotein with glycerol prior to rotary shadowing, the protein components were observed to consist of a ring of density made up of nodules of different sizes, with apolipoprotein(a) and apolipoprotein B-100 closely associated with each other. However, when lipoprotein(a) was treated with a lysine analogue, 6-aminohexanoic acid, much of the apolipoprotein(a) separated from the apolipoprotein B-100. In 6-aminohexanoic acid-treated preparations without glycerol extraction, lipoprotein(a) particles had an irregular mass of density around the core. In contrast, lipoprotein(a) particles treated with 6-aminohexanoic acid in the presence of glycerol had a long tail, in which individual kringles could be distinguished, extending from the ring of apolipoprotein B-100. The length of the tail was dependent on the particular isoform of apolipoprotein(a). Dissociation of the noncovalent interactions between apolipoprotein(a) and low-density lipoprotein as a result of shear forces or changes in the microenvironment may contribute to selective retention of lipoprotein(a) in the vasculature.

Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding.

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV (2), KIV (7), KIV (9) DeltaCys (which lacks the seventh unpaired cysteine) and KIV (10) which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV (10) to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV (10) is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by approximately 43% at a concentration of 10 microM KIV (10). The ability of the KIV (10) mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp (70) to Arg abolished binding to both lysine-Sepharose and plasmin-modified fibrinogen, while the Trp (70) -->Phe and Arg (35) -->Lys substitutions each resulted in decreased binding to these substrates. None of the KIV (10) mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV (7) contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV (10). However, apo(a) KIV (7) had no effect on fibrinolysis in vitro. Apo(a) KIV (2) and KIV (9) DeltaCys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro.

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a).

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.

Apolipoprotein(a) phenotype, albumin clearance, and plasma levels of lipoprotein(a) in peritoneal dialysis.

This cross-sectional study was undertaken to examine the relationship between plasma lipoprotein(a) [Lp(a)] level and peritoneal dialysis (PD) albumin clearance while controlling for the influence of the apolipoprotein(a) [apo(a)] phenotype. Plasma Lp(a) level, PD albumin clearance, and apo(a) phenotype (high vs. low molecular weight, HMW vs. LMW) were determined in 54 PD patients. Apo(a) phenotypes were 24 LMW and 30 HMW. The plasma Lp(a) level was high (> 65 nmol/l) in 17 of 24 patients with LMW phenotype versus 2 of 30 with HMW phenotype (chi2, p < 0.01). Spearman correlation coefficients of Lp(a) with PD, urine, and total albumin clearances were -0.05 (p = 0.74), -0.04 (p = 0.80), and -0.09 (p = 0.51), respectively. The apo(a) isoform size was the only significant predictor of Lp(a) in multivariate analysis. In this study, there was no association between PD albumin clearance and Lp(a) level. The association between apo(a) phenotype and Lp(a) level is in keeping with studies in the general population. There is a strong genetic influence on Lp(a) level in PD patients.

High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding.

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).

Evaluation of Lp[a] and other independent risk factors for CHD in Asian Indians and their USA counterparts.

Conventional risk factors for coronary heart disease (CHD) do not completely account for the observed increase in premature CHD in people from the Indian subcontinent or for Asian Indians who have immigrated to the USA. The objective of this study was to determine the effect of immigration to the USA on plasma levels of lipoprotein [a] (Lp[a]) and other independent risk factors for CHD in Asian Indians. Three subject groups were studied: group 1, 57 subjects living in India and diagnosed with CHD (CHD patients); group 2, 46 subjects living in India and showing no symptoms of CHD (control subjects); group 3, 206 Asian Indians living in the USA. Fasting blood samples were drawn to determine plasma levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein [LDL cholesterol (LDL-Chol)], high density lipoprotein [HDL cholesterol (HDL-Chol)], apolipoprotein B-100 (apoB-100), and Lp[a]. Apolipoprotein [a] (apo[a]) size polymorphism was determined by immunoblotting. Plasma TG, apoB-100, and Lp[a] concentrations were higher in CHD patients than in control and USA groups. CHD patients had higher levels of TC and LDL-Chol and lower HDL-Chol than control subjects. However, the USA population had higher levels of TC, LDL-Chol, and apoB-100 and lower HDL-Chol than control subjects. Plasma Lp[a] levels were inversely correlated with the relative molecular weight of the more abundant of each subject's two apo[a] isoforms (MAI), and CHD patients showed higher frequencies of lower relative molecular weights among MAI. Our observed changes in lipid profiles suggest that immigrating to the USA may place Asian Indians at increased risk for CHD. This study suggests that elevated plasma Lp[a] confers genetic predisposition to CHD in Asian Indians, and nutritional and environmental factors further increase the risk of CHD. This is the first report implicating MAI size as a predictor for development of premature CHD in Asian Indians. Including plasma Lp[a] concentration and apo[a] phenotype in screening procedures may permit early detection and preventive treatment of CHD in this population.

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.