Development of ELISAs Measuring the Extent of TAFI Activation

Abstract

Objectives-To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. Methods and Results-A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; PϽ0.001) and TAFIa (112.1 versus 103.3; Pϭ0.03), and not of TAFI antigen (92.5 versus 87.9; Pϭ0.07) (results in % of plasma pooled from normolipidemic subjects). Conclusion-ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases. ; 36 kDa). Subsequently, TAFIa is inactivated through a conformational change to an inactive form (TAFIai), followed by proteolytic cleavage resulting in fragments of 25 and 11 kDa. 5 TAFIa exerts an antifibrinolytic effect by removing C-terminal lysine residues from fibrin, resulting in a decreased plasmin formation and a retardation of clot lysis. 6 TAFIa is highly unstable in vitro (half-life at 37°C between 8 to 15 minutes). [7][8][9][10] Different clinical studies have investigated the possible relationship between TAFI and cardiovascular events. A positive correlation was found between TAFI levels and the risk for coronary artery disease, 11 venous thrombosis, 12 angina pectoris, 13 and ischemic stroke. 14 However, another study revealed that elevated TAFI may be protective against myocardial infarction. 15 A possible explanation for these contradictory results was that the different studies used different methods for TAFI determination. 16 Silveira et al used quantitative activation of the zymogen followed by determination of the total enzymatic activity with a chromogenic assay. Van of TAFI antigen levels, as a putative risk marker for cardiovascular events, has been mainly focused on "total" antigen levels (either by ELISA or by activity assays after full activation of TAFI), reporting levels of 4 to 15 g/mL. 16,20 -22 However, it should be stressed that in most of these studies the used immunologic assays were not validated with respect to putative differential reactivities toward different isoforms and fragments...

Thrombin Activatable Fibrinolysis Inhibitor Activation Peptide Shows Association With All Major Subtypes of Ischemic Stroke and With TAFI Gene Variation

“…It is the first study to measure levels of both intact TAFI and released AP, a marker of the extent of TAFI activation. 24 By measuring plasma levels both in the acute phase and at 3-month follow-up, we were able to confirm findings from the work by Leebeek et al that increased TAFI levels in ischemic stroke do not reflect an acute-phase response. 22 Indeed, the present study provides further data to support this hypothesis.…”

“…Plasma was isolated within 2 hours by centrifugation at 4°C and 2000g for 20 minutes. Plasma levels of TAFI were measured by sandwich-type ELISAs using MA-T12D11/MA-T30E5-HRP for intact TAFI 24 and MA-T12D11/ MA-T18A8-HRP for released AP. 24 Values are expressed relative to pooled human plasma.…”

“…Plasma levels of TAFI were measured by sandwich-type ELISAs using MA-T12D11/MA-T30E5-HRP for intact TAFI 24 and MA-T12D11/ MA-T18A8-HRP for released AP. 24 Values are expressed relative to pooled human plasma. Intra-and interassay coefficients of variation were 6.2% and 8.3% for intact TAFI and 3.1% and 7.3% for AP.…”

“…Intra-and interassay coefficients of variation were 6.2% and 8.3% for intact TAFI and 3.1% and 7.3% for AP. 24 High-sensitivity C-reactive protein (CRP) was analyzed as described. 25 …”

“…23 To this end, TAFI was analyzed at the protein level by 2 newly developed immunologic assays with distinct reactivities, recognizing either intact TAFI or the released AP, 24 and, at the genetic level, by a selection of tagging SNPs over the TAFI locus.…”