Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha(2)-antiplasmin

L A Robbie, B Bennett, B A Keyt, N A Booth

Research output: Contribution to journalArticle

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Abstract

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 alpha (2)-antiplasmin (alpha (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 alpha (2)-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha (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 alpha (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 alpha (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 alpha (2)-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha (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 alpha (2)-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha (2)-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha (2)-AP as a regulator of fibrin persistence and thrombus resistance to lysis.

Original languageEnglish
Pages (from-to)517-523
Number of pages7
JournalBritish Journal of Haematology
Volume111
Publication statusPublished - 2000

Keywords

  • alpha(2)-antiplasmin
  • plasminogen activator inhibitor-1
  • tissue-type plasminogen activator
  • fibrin lysis
  • plasmin
  • PLASMINOGEN-ACTIVATOR INHIBITOR-1
  • HUMAN-PLASMA
  • ALPHA-2-PLASMIN INHIBITOR
  • CROSS-LINKING
  • CLOT LYSIS
  • PROTEINASE-INHIBITORS
  • CHANDLER LOOP
  • FIBRINOLYSIS
  • PURIFICATION
  • DEFICIENCY

Cite this

Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha(2)-antiplasmin. / Robbie, L A ; Bennett, B ; Keyt, B A ; Booth, N A .

In: British Journal of Haematology, Vol. 111, 2000, p. 517-523.

Research output: Contribution to journalArticle

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abstract = "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 alpha (2)-antiplasmin (alpha (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 alpha (2)-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha (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 alpha (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 alpha (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 alpha (2)-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha (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 alpha (2)-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha (2)-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha (2)-AP as a regulator of fibrin persistence and thrombus resistance to lysis.",
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TY - JOUR

T1 - Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha(2)-antiplasmin

AU - Robbie, L A

AU - Bennett, B

AU - Keyt, B A

AU - Booth, N A

PY - 2000

Y1 - 2000

N2 - 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 alpha (2)-antiplasmin (alpha (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 alpha (2)-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha (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 alpha (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 alpha (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 alpha (2)-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha (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 alpha (2)-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha (2)-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha (2)-AP as a regulator of fibrin persistence and thrombus resistance to lysis.

AB - 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 alpha (2)-antiplasmin (alpha (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 alpha (2)-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha (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 alpha (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 alpha (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 alpha (2)-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha (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 alpha (2)-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha (2)-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha (2)-AP as a regulator of fibrin persistence and thrombus resistance to lysis.

KW - alpha(2)-antiplasmin

KW - plasminogen activator inhibitor-1

KW - tissue-type plasminogen activator

KW - fibrin lysis

KW - plasmin

KW - PLASMINOGEN-ACTIVATOR INHIBITOR-1

KW - HUMAN-PLASMA

KW - ALPHA-2-PLASMIN INHIBITOR

KW - CROSS-LINKING

KW - CLOT LYSIS

KW - PROTEINASE-INHIBITORS

KW - CHANDLER LOOP

KW - FIBRINOLYSIS

KW - PURIFICATION

KW - DEFICIENCY

M3 - Article

VL - 111

SP - 517

EP - 523

JO - British Journal of Haematology

JF - British Journal of Haematology

SN - 0007-1048

ER -