The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of α2-antiplasmin

Abstract

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.

Plasmin on adherent cells: from microvesiculation to apoptosis

“…The calculated Km value is comparable to values obtained for tPA expressed by other cell types including endothelial cells [35] and neurons [16]. It is also comparable to values determined for the activation of plasminogen by tPA on fibrin (40 to 160 nM) [36,37], suggesting that the cell surface provides a similar plasminogen activation enhancement. Lysine-dependant specific plasminogen binding was indicated by inhibition of plasmin formation both with the lysine analogue ε-ACA that blocks the LBS of plasminogen, and with CpB that cleaves C-ter lysine residues from the cell surface.…”

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

“…The dissociation constants calculated for plasminogen, K d = 1.16 F 0.22 Amol/l, and for apo(a), K d = 54 F 5 nmol/l, are in agreement with previously published data [12,27,29] in which fibrin was immobilised on multi-well plates using polymerised glutaraldehyde as a linker. This procedure has been previously proven to result in a fibrin monolayer that mimics a fibrin clot surface with regard to plasminogen binding and activation in a static system [27,40,41]. The finding of similar results in polyglutaraldehyde-activated C1 sensor chips indicates that binding of plasminogen preceding its activation on fibrin in a static system and binding of plasminogen observed in real time under flow conditions (see below) obeys a similar mechanism.…”

“…This procedure is similar to the one used for the preparation of fibrin surfaces on C1 sensor chips except for the use of multi-well polyvynil chloride plates as solid support. The activation of plasminogen by t-PA on these fibrin plates was performed as indicated elsewhere [40], with minor modifications. Briefly, a solid-phase fibrin plate was washed three times with binding buffer, and 50 Al per well of the same buffer containing 100 IU/ml t-PA was incubated for 1 h at 37 jC.…”

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

“…In contrast, when natural elastase inhibitors were added after the preincubation of fibrin with PMN-conditioned media, they were unable to prevent mesenchymal cell anoikis, suggesting that fibrin-bound elastase is not sensitive to circulating inhibitors. Such a phenomenom has already been reported for other serine proteases such as plasmin and plasminogen activators 74 and for elastase bound to elastin. 75 In conclusion, our data first demonstrate that PMNs entrapped in the luminal pole of the mural thrombus of abdominal aortic aneurysms release proteinases such as MMP-9 and MMP-8 and elastase; and secondly, provide evidence that PMN elastase adsorbed in the fibrin matrix can prevent re-colonization of the thrombus by both SMCs and BMSCs.…”