Development and clinical application of a new ELISA assay to determine plasmin–α₂‐antiplasmin complexes in plasma

Abstract

Plasmin-2 -antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine-sepharose and elution with " -aminocaproic acid. This material, characterized by SDS-PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme-linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified 2 -antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3 . 5% (intra-assay) and 10 . 6% (interassay). A mean value of 573 . 5 6 131 . 4 ng/ml was obtained from PAP concentration in a healthy population (n 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels.

Endothelial Cell and Hemostatic Activation in Relation to Cytokines in Patients with Sepsis

“…anism, F1ϩ2 and TAT, were significantly elevated Plasmin-␣ 2 -antiplasmin complexes (PAP) were in patients with sepsis as compared with the control measured with an ELISA assay previously degroup (pϽ0.0001), indicating an important degree of prothrombin activation and thrombin generascribed in our laboratory by Montes et al [14]. …”

Hemostatic markers in surgery: a different fibrinolytic activity may be of pathophysiological significance in orthopedic versus abdominal surgery

“…PAP were measured with the ELISA previously described in our laboratory by Montes et al [19]. FDP were measured with the Fibrinostika FDP kit (Organon Teknika, The Nedertands), with a specific anti D-dimer monoclonal antibody [20].…”

Bivalency of plasminogen monoclonal antibodies is required for plasminogen bridging to fibrin and enhanced plasmin formation

“…In the present work, we have evaluated this mechanism using mAbs directed against distinct domain of the plasminogen molecule: A10.2, a mAb that reacts with the LBS of kringle 4 [23], and CPL15, a mAb directed against plasminogen kringle 1 [24]. Affinities of these mAbs for plasminogen and the corresponding fragments were in the low nanomoles per liter range (K d = 10 nmol/l for A10.2 and K d = 32 nmol/l for CPL15).…”

“…MAb A10.2 is an IgG 1 directed against the LBS of plasminogen kringle 4 and plasminogen-like KIV-10 of apo(a) that was raised by immunization of BALB/c mice with recombinant apo(a) [23]. CPL15 is an IgG 1 mAb directed against the kringle 1 of plasminogen as indicated by its reactivity against the plasminogen fragment K1 + 2 + 3 and plasmin -a 2 -antiplasmin complexes [24]. Y18, an IgM mAb that recognises the Aa stretch 1-51 of human fibrinogen [35], was kindly provided by Dr. W. Nieuwenhuizen (Gaubius Institut, Leyden, The Netherlands).…”

“…We hypothesise that mAbs directed against plasminogen may increase its binding to fibrin through a similar mechanism. The availability of the monoclonal antibodies A10.2 that interacts with the LBS of plasminogen kringle 4 [23] and CPL15 that recognises plasminogen kringle 1 [24], prompted us to investigate their effect on plasminogen binding under static and flow conditions using surface plasmon resonance (SPR) analysis. In the present work, we show that these mAbs enhance plasmin formation and fibrinolysis through the formation of bivalent plasminogen -mAb-plasminogen complexes that result in increased plasminogen binding to fibrin surfaces.…”