Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain

• Published in: Archives of biochemistry and biophysics Vol. 431; no. 2; pp. 197 - 206
• Main Authors: Boivin, Dominique, Provençal, Mathieu, Gendron, Sébastien, Ratel, David, Demeule, Michel, Gingras, Denis, Béliveau, Richard
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.11.2004
• Subjects: AE-941; Amino Acid Sequence; Angiogenesis; Angiogenesis Inhibitors - chemistry; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endothelial cells; Fibrin - metabolism; Fibrinolysin - biosynthesis; Immunoglobulin; Immunoglobulin kappa-Chains - chemistry; Immunoglobulin kappa-Chains - isolation & purification; Immunoglobulin kappa-Chains - metabolism; Isoelectric Focusing; Isoelectric Point; Kinetics; Molecular Sequence Data; Molecular Weight; Neovastat; Plasminogen - metabolism; Plasminogen activator; Sequence Analysis, Protein; Shark cartilage; Surface Plasmon Resonance; Tissue Extracts - chemistry; AE-941; Angiogenesis; Plasminogen activator; Immunoglobulin; Neovastat; Endothelial cells; Shark cartilage
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2004.08.022

We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS–PAGE. Two 28-kDa proteins with p Is of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold ( K m decrease from 456 nM to 12–14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen ( K A = 4.0–5.5 × 10 7 M −1; K D = 18–25 nM) and with tPA ( K A = 2.8 × 10 7 M −1; K D = 36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.