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 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.