Monoclonal Antibodies Against Four Different Epitopes of Plasminogen Exhibit Different Effects on Plasminogen Activation Kinetics

Abstract

Monoclonal antibodies against four different epitopes of plasminogen (PG) were obtained and characterized for their binding towards PG using immunoblotting of native, degraded, elastase cleaved PG and alpha-2-antiplasmin complexed plasmin. One antibody (MPW1PG) recognized exclusively Glu-forms of PG and plasmin, a second antibody (MPW2PG) was bound to the kringle 1-3 fragment, one antibody (MPW7PG) recognized the kringle 4 fragment and the fourth antibody (MPW4PG) miniplasminogen. All antibodies exhibited affinities towards PG in the same order of magnitude. From these antibodies only MPW4PG inhibited plasmin action (competitive inhibitor, Ki = 4nM) and after complex formation of plasmin with alpha-2-antiplasmin the complex was not recognized. All other antibodies exhibited binding also to the respective alpha-2-antiplasmin complexed forms. The three antibodies directed against the different epitopes of the heavy chain of plasmin stimulated activation of Glu-PG by urokinase (UK), thereby mimicking the effect of lysine (KM unchanged, kcat increased) but not activation of Lys-PG. When single chain tPA was used instead of UK and PG activation was studied in the absence and presence of CNBr fragments of fibrinogen none of the three antibodies (MPW1PG, MPW2PG, MPW7PG) interfered significantly with the stimulating effect of fibrin, but the lag phase, until maximal fibrin stimulated plasmin formation occured, was reduced. In the absence of fibrin all three antibodies increased the rate of Glu-PG activation by tPA also increasing kcat values while addition of lysine rather inhibited PG activation by tPA and completely abolished the fibrin effect. These results are consistent with the plasmin induced decrease in lag time of the fibrin effect found for these antibodies. From these data it can be concluded that binding of an antibody to one of the three described domains in the heavy chain induces conformational changes in PG leading to an increased rate of activation by both activators. Interestingly, binding of these antibodies did not interfere with the fibrin effect and is different from the effect of lysine.

Soluble M6P/IGF2R Released by TACE Controls Angiogenesis via Blocking Plasminogen Activation

“…MEM-240 recognizes an epitope within the extracellular repeat domains 6 to 9 of M6P/IGF2R 7 ; 4Pg recognizes an epitope within the catalytic part of Plg. 20 In contrast, the mAbs MEM-238 and 7Pg, recognizing an epitope within the amino-terminal region of M6P/IGF2R 7 and the kringle 4 of Plg 20 respectively, were less efficient; however, both recognized the precipitated material on the immunoblot indicating that the corresponding epitopes were hidden within the complex and not released or destroyed, which was in line with a recently published study. 21 We scrutinized the Plg-sM6P/IGF2R complex in serum by BN-PAGE, which allowed a complex dissection by the separation of native protein complexes in the first dimension followed by SDS-PAGE in the second dimension.…”

The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis

“…B). The mAb MEM‐240 recognizes an epitope within the extracellular repeat domains 6 to 9 of M6P/IGF2R and mAb 4Pg an epitope within the catalytic part of Plg . We were able previously to coprecipitate the Plg–M6P/IGF2R complex from human serum with these two mAbs, suggesting that they do not interfere with the Plg–M6P/IGF2R binding but are able, maybe due to steric hindrance, to inhibit the efferocytosis process.…”