Type‐2 plasminogen‐activator inhibitor is a substrate for trophoblast transglutaminase and Factor XIII_a

Abstract

Plasminogen‐activator inhibitor type‐2 (PAI‐2), a serine‐proteinase inhibitor, suppresses fibrinolysis by blocking both urokinase and tissue‐type plasminogen activators. The 43‐kDa PAI‐2 molecule is an abundant cytosolic protein in certain cell types, but can upon appropriate stimulation be secreted as an approximately 60–70‐kDa glycoprotein. However, in trophoblast membranes PAI‐2 activity is associated with large covalent complexes (Jensen, P. H., Nykjær, P., Andreasen P. A., Lund, L., Åstedt, B. Lecander, I. & Gliemann, J. (1989) Biochim. Biophys. Acta 986, 135–140). This study shows that PAI‐2 can act as a substrate for both tissue transglutaminase and activated plasma factor XIII. In the presence of Ca2+, either of these will catalyze the incorporation of primary amines, such as putrescine, into PAI‐2. Moreover, in reactions with tissue transglutaminase, PAI‐2 homopolymers and, in conjunction with other biological substrates, heteropolymers were observed. As judged by the test of incorporating 125I‐urokinase into SDS‐resistant 125I‐urokinase/PAI‐2 complexes, polymerized PAI‐2 retained its inhibitory activity. Furthermore, syncytiotrophoblast microvillous membranes and trophoblast detergent extracts incorporated 125I‐PAI‐2 into large structures in a reaction inhibited by putrescine and a synthetic inhibitor of transglutaminase. Trophoblast transglutaminase was identified as a tissue transglutaminase by non‐denaturing gel electrophoresis and dansylcadaverine activity staining, fibronectin binding and Western blotting with a specific antibody. The transglutaminase‐catalyzed and Ca2+‐dependent anchoring of PAI‐2 to extracellular membrane structures might have the purpose of focally regulating fibrinolysis.

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis

“…Transglutaminase activation has indeed be associated with cellular apoptosis (Fesus et al, 1989). and we recently showed PAI-2 to be a substrate for transglutaminase (Jensen et al, 1993(Jensen et al, . 1994 (Jensen et al, 1993(Jensen et al, .…”

“…and we recently showed PAI-2 to be a substrate for transglutaminase (Jensen et al, 1993(Jensen et al, . 1994 (Jensen et al, 1993(Jensen et al, . 1994 First, dye binding to proteins in cells (Bruno et al, 1992) or cell extracts (Kaufmann.…”

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

“…fibronectin) and may provide a means of localising the inhibitor to specific sites (eg. placental microvillous membranes) without affecting inhibitory activity (Jensen et al 1993). Several other serpins, such as protease inhibitor-10 (PI 10) and myeloid/erythroid nuclear termination stage-specific protein (MENT) also contain extended loops between helices C and D which act as nuclear targeting signals , Chuang & Schleef 1999.…”

“…Another structural feature unique to PAI-2 is an extended sequence of amino acids between a-helices C and D (Ala^ to Glu % ) (Figure 1. 7) which in other serpins is much shorter, containing between 6 and 17 residues (Antalis et al 1988;Jensen et al 1993). Three glutamine residues in this sequence (Gln 8 3,84,8 act as sites for transglutaminase mediated cross-linking of PAI-2 to cellular and ECM proteins (eg.…”

“…PAI-2 is expressed by a number of cell types (eg. trophoblasts, keratinocytes, monocytes, endothelial cells, fibroblasts) (as well as in the plasma pregnant women, patients suffering myeloid leukemia and in gingival crevicular flu (2-8 mg/ml) (Jensen et al 1993;Kinnby et al 1994). PAI-2 has been implicated in regulation of a number of physiological processes and pathological conditions involving ECM degradation and tissue remodelling (eg.…”