Enhanced Fibrinolytic Potential in Mice with Combined Homozygous Deficiency of α2-antiplasmin and PAI-1

M. Dewerchin; D. Collen; H. R. Lijnen

doi:10.1055/s-0037-1616099

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 86(02): 640-646
DOI: 10.1055/s-0037-1616099

Review Article

Schattauer GmbH

Enhanced Fibrinolytic Potential in Mice with Combined Homozygous Deficiency of α₂-antiplasmin and PAI-1

M. Dewerchin

¹Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

,

D. Collen

¹Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

²Center for Molecular and Vascular Biology, University of Leuven, Belgium

,

H. R. Lijnen

²Center for Molecular and Vascular Biology, University of Leuven, Belgium

› Author Affiliations

Abstract

Summary

α₂-antiplasmin (α₂-AP) and plasminogen activator inhibitor-1 (PAI-1) are the main physiological inhibitors of the plasminogen/ plasmin system in mammalian plasma. In the present study, the relative importance of both inhibitors was evaluated with the use of mice with single or combined deficiency of α₂-AP and PAI-1 in the same genetic background. Mice with combined deficiency (α₂-AP^–/–:PAI-1^–/–) are viable, develop normally and are fertile. After amputation of the tail, bleeding times are prolonged (>15 min) in α₂-AP^–/–:PAI-1^–/– mice, as compared to double wild-type or single deficient mice (4.6 to 10 min). Spontaneous lysis after 4 h of intravenously injected ¹²⁵I-fibrin labeled plasma clots is significantly higher in mice with α₂-AP deficiency both in the PAI-1^+/+ background (89 ± 2% versus 42 ± 3%; p = 0.002) and in the PAI-1^–/– background (83 ± 4% versus 53 ± 5%; p = 0.002). PAI-1 deletion in the α₂-AP^+/+ or α₂-AP^–/– background, however, has no significant effect (p = 0.13 or 0.18, respectively). Four hours after endotoxin injection, fibrin deposition in the kidneys is not significantly affected by PAI-1 deletion in mice with α₂-AP^+/+ or α₂-AP^–/– background (p = 0.07 and 0.19, respectively). In contrast, α₂-AP deletion causes significantly reduced fibrin deposition in the PAI-1^+/+ background (p = 0.01). Endotoxin injection causes a dramatic increase in PAI-1 antigen levels in kidney extracts of PAI-1^+/+ animals, without effect on α₂-AP levels.

Taken together, these data indicate that the higher endogenous fibrinolytic capacity observed in mice with combined deficiency is mainly due to the lack of α₂-AP and suggest a less important role for PAI-1.

Keywords

α₂-antiplasmin - plasminogen activator inhibitor-1 - gene targeting - fibrinolysis

Full Text

References

References
1 Lijnen HR, Bachmann F, Collen D, Ellis V, Pannekoek H, Rijken DC, Thorsen S. Mechanisms of plasminogen activation. J Intern Med 1994; 236: 415-24.
2 Collen D. Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. Eur J Biochem 1976; 69: 209-16.
3 Moroi M, Aoki N. Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J Biol Chem 1976; 251: 5956-65.
4 Müllertz S, Clemmensen I. The primary inhibitor of plasmin in human plasma. Biochem J 1976; 159: 545-53.
5 Menoud PA, Sappino N, Boucal-Koshbeen M, Vassalli JD, Sappino AP. The kidney is a major site of α₂-antiplasmin production. J Clin Invest 1996; 97: 2478-84.
6 Lijnen HR, Van Hoef B, Beelen V, Collen D. Characterization of the murine plasma fibrinolytic system. Eur J Biochem 1994; 224: 863-71.
7 Wiman B, Collen D. On the mechanism of the reactions between human α₂-antiplasmin and plasmin. J Biol Chem 1979; 254: 9291-7.
8 Kruithof EKO, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-13.
9 Wiman B, Hamsten A. The fibrinolytic enzyme system and its role in the etiology of thromboembolic disease. Sem Thromb Hemost 1990; 16: 207-16.
10 Kruithof EKO. Plasminogen activator inhibitors – a review. Enzyme 1988; 40: 113-21.
11 Carmeliet P, Collen D. Targeted gene manipulation and transfer of the plasminogen and coagulation systems in mice. Fibrinolysis 1996; 10: 195-213.
12 Lijnen HR, Okada K, Matsuo O, Collen D, Dewerchin M. α₂-Antiplasmin gene-deficiency in mice is associated with enhanced fibrinolytic potential without overt bleeding phenotype. Blood 1999; 93: 2274-81.
13 Carmeliet P, Kieckens L, Schoonjans L, Ream B, van Nuffelen A, Prendergast G, Cole M, Bronson R, Collen D, Mulligan RC. Plasminogen activator inhibitor-1 gene-deficient mice. I. Generation by homologous recombination and characterization. J Clin Invest 1993; 92: 2746-55.
14 Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 gene-deficient mice. II. Effects on hemostasis, thrombosis and thrombolysis. J Clin Invest 1993; 92: 2756-60.
15 Kawasaki T, Dewerchin M, Lijnen HR, Vermylen J, Hoylaerts M. Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice. Blood 2000; 96: 153-60.
16 Declerck PJ, Verstreken M, Collen D. Immunoassay of murine t-PA, u-PA and PAI-1 using monoclonal antibodies raised in gene-inactivated mice. Thromb Haemost 1995; 74: 1305-9.
17 Lijnen HR, Van Hoef B, Lupu F, Moons L, Carmeliet P, Collen D. Function of the plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes. Arterioscler Thromb Vasc Biol 1998; 18: 1035-45.
18 Lijnen HR, Carmeliet P, Bouché A, Moons L, Ploplis VA, Plow EF, Collen D. Restoration of thrombolytic potential in plasminogen-deficient mice by bolus administration of plasminogen. Blood 1996; 88: 870-6.
19 Declerck PJ, Juhan-Vague I, Felez J, Wiman B. Pathophysiology of fibrinolysis. J Int Med 1994; 236: 425-32.
20 Schleef RR, Higgins DL, Pillemer E, Levitt LJ. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. J Clin Invest 1989; 83: 1747-52.
21 Diéval J, Nguyen G, Gross S, Delobel J, Kruithof EK. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type 1. Blood 1991; 77: 528-32.
22 Takahashi Y, Tanaka T, Minowa H, Ookubo Y, Sugimoto M, Nakajima M, Miyauchi Y, Yoshioka A. Hereditary partial deficiency of plasminogen activator inhibitor-1 associated with a lifelong bleeding tendency. Int J Hematol 1996; 64: 61-8.
23 Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Brief report: complete deficiency of plasminogen-activator inhibitor type 1 due to a frameshift mutation. N Engl J Med 1992; 327: 1729-33.
24 Lind B, Thorsen S. A novel missense mutation in the human plasmin inhibitor (alpha2-antiplasmin) gene associated with a bleeding tendency. Br J Haematol 1999; 107: 317-22.
25 Kluft C, Nieuwenhuis HK, Rijken DC, Groeneveld E, Wijngaards G, van Berkel W, Dooijewaard G, Sixma JJ. Alpha2-antiplasmin Enschede: dys-functional alpha2-antiplasmin molecule associated with an autosomal recessive hemorrhagic disorder. J Clin Invest 1987; 80: 1391-400.
26 Yamamoto K, Loskutoff DJ. Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator. J Clin Invest 1996; 97: 2440-51.
27 Lijnen HR, Van Hoef B, Collen D. Characterization of the murine plasminogen/urokinase-type plasminogen activator system. Eur J Biochem 1996; 241: 840-8.