Effective lysis of model thrombi by a t‐PA mutant (A473S) that is resistant to α₂‐antiplasmin

Abstract

Summary. This study used two mutants of tissue-type plasminogen activator (t-PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)-1 and a 2 -antiplasmin (a 2 -AP) to the control of fibrin lysis. Wild-type t-PA was compared with KHRR296±299AAAA, which is resistant to PAI-1, and with A473S, which is resistant to a 2 -AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of a 2 -AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild-type t-PA or KHRR296±299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of a 2 -AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild-type t-PA; the rate and extent of lysis were enhanced by the addition of antibodies to a 2 -AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to a 2 -AP. This resistance was remarkable, in that no complex formed between A473S t-PA and a 2 -AP, even after extended incubation, when 50% of wild-type t-PA could be converted to complex. Comparison of A473S and KHRR296±299AAAA mutants showed their similar effectiveness in lysis of platelet-rich model thrombi. Thus, PAI-1 and a 2 -AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of a 2 -AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of a 2 -AP as a regulator of fibrin persistence and thrombus resistance to lysis.

Polymorphonuclear leucocytes mediate endogenous thrombus lysis via a u‐PA‐dependent mechanism

“…Model thrombi formed from whole blood lysed without the addition of proteases. This lysis occurred despite the high concentrations of inhibitors present in these thrombi (Robbie et al , 1997, 2000). After the initial observations in model thrombi prepared from whole blood, thrombi prepared from PRP and PMNs were used to allow the cellular contribution to lysis to be analysed more directly.…”

“…Thrombi formed in the Chandler loop system contain all these components and have structures similar to human in vivo thrombi (Robbie et al , 1997). The cellular elements are well placed to modify the lytic process, and platelets have already been identified as modifying lysis via their PAI‐1 content (Robbie et al , 1993, 1997, 2000). This system is more appropriate to the study of thrombus lysis than homogeneous fibrin clots prepared from plasma in the absence of cells, which are often used.…”

“…Chandler model thrombi Thrombi were formed in a Chandler system (Chandler, 1958) as described previously (Robbie et al , 1997, 2000). Citrated whole blood (0·9 ml) or PRP (0·9 ml) and leucocytes (generally 6 × 10 6 ), along with FITC‐labelled fibrinogen (75 µg/ml final concentration), were recalcified to a final concentration of 10 mmol/l CaCl 2 , all in a final volume of 1·15 ml, and placed in the tubing.…”

Congenital α2‐plasmin inhibitor deficiencies: a review

“…In addition to inhibition of plasmin, α 2 ‐PI inhibits plasminogen activators (Moroi & Aoki, 1976; Rijken et al , 1983). The inhibition of fibrin‐bound tPA by fibrin‐bound α 2 ‐PI may significantly contribute to the inhibition of thrombolysis (Robbie et al , 2000).…”