Biological Effects of Combined Inactivation of Plasminogen Activator and Plasminogen Activator Inhibitor-1 Gene Function in Mice

Summary

Mice with combined homozygous deficiency of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (T‾U‾), of t-PA and plasminogen activator inhibitor-1 (PAI-1) (T‾P‾), of u-PA and PAI-1 (U‾P‾) or of t-PA, u-PA, and PAI-1 (T‾U‾P‾) were generated by inbreeding of mice with the respective deficiencies. Homologous recombination at the t-PA, u-PA and PAI-1 locus was verified by Southern blot analysis of genomic tail tip DNA, and confirmed by measurement of antigen levels in plasma or urine.

T‾P‾ and U‾P‾ mice were apparently healthy and fertile. T‾U‾ mice showed extensive fibrin deposition with calcification in the liver, whereas T‾U‾P‾ mice were significantly (p <0.001) less affected. Spontaneous in vivo clot lysis measured 4 h after injection of a ¹²⁵I-fibrin-labeled clot prepared from plasma of wild-type (WT) mice into the jugular vein, was (mean ± SEM of n experiments) 2 ± 1% (n = 8) for T P , 49 ± 6% (n = 9) for U‾P‾, 1 ± 1% (n = 4) for T‾U‾ and 3 ± 3% (n = 3) for TU‾P‾ mice, as compared to 32 ± 4% (n = 10) for WT, 1 ± 0% (n = 7) for T‾, 30 ± 5% (n = 5) for U‾ and 58 ± 10% (n = 6) for P‾ mice. Plasminogen-dependent lysis of ¹²⁵I-fibrin-labeled matrix and of ³H-proline-labeled subendothelial matrix (mean ± SEM; n = 4 to 6) was lower with thioglycollate-stimulated macrophages obtained from U‾P‾ mice (22 ± 7% and 5 ± 1%, respectively), as compared to WT mice (57 ± 14% and 18 ± 5%, respectively) and T‾P‾ mice (87 ± 6% and 27 ± 4%, respectively). A similar decrease was previously observed with U‾ mice, but not with T‾ or P‾ mice. Thus, the phenotype of mice with combined deficiency of t-PA and PAI-1 or of u-PA and PAI-1 is similar to the phenotype observed in mice with single deficiency of the plasminogen activator. Additional deletion of PAI-1 does not affect viability, fertility, macrophage function or thrombolytic potential of the single deficient mice. Additional deletion of PAI in mice with combined deficiency of t-PA and u-PA does not restore the deficient in vivo fibrinolytic capacity, but significantly reduces the thrombotic phenotype, as revealed by fewer, smaller and less calcified fibrin deposits in the liver.

• References

• 1 Astrup T. Fibrinolysis: an overview. In: Progress in Chemical Fibrinolysis and Thrombolysis, Vol 3. Davidson JF, Rowan RM, Samama MM, Desnoyers PC. eds Raven Press; New York: 1978. pp 1-57
• 2 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 1991; 78: 3114-3124
• 3 Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest 1991; 88: 1067-1072
• 4 Ohlsson M, Peng XR, Liu YX, Jia XC, Hsueh AJ W, Ny T. Hormone regulation of tissue-type plasminogen activator gene expression and plasminogen activator-mediated proteolysis. Semin Thromb Haemost 1991; 17: 286-290
• 5 Tsafriri A, Bicsak TA, Cajander SB, Ny T, Hsueh AJ W. Suppression of ovulation rate by antibodies to tissue-type plasminogen activator and α₂-antiplasmin. Endocrinology 1989; 124: 415-421
• 6 Strickland S, Reich E, Sherman MI. Plasminogen activator in early embryogenesis: enzyme production by trophoblast and parietal endoderm. Cell 1976; 9: 231-240
• 7 Qian Z, Gilbert ME, Colicos MA, Kandel ER, Kuhl D. Tissue-plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation. Nature 1993; 361: 453-457
• 8 Lulu PK, Graham GH. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev 1990; 9: 369-379
• 9 Rifkin DB, Moscatclli D, Bizik J, Quarto N, Blei F, Dennis P, Flaumenhaft R, Mignatti P. Growth factor control of extracellular proteolysis. Cell Differ Develop 1990; 32: 313-318
• 10 Blasi F, Verde P. Urokinase-dependent cell surface proteolysis and cancer. Semin Cancer Biol 1990; 1: 117-126
• 11 Cannelid P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, van den oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature 1994; 368: 419-424
• 12 Carmeliet P, Kieckens L, Schoonjans L, Ream B, Van NulTelen A, Prcndergast 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: 2746-2755
• 13 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: 2756-2760
• 14 Carmeliet P, Bouchd A, De Clercq C, Janssen S, Pollefeyt S, Wyns S, Mulligan RC, Collen D. Biological effects of disruption of the tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1 genes in mice. Ann NY Acad Sei 1995; 748: 367
• 15 Deelerck PJ, Carmeliet P, Vcrstreken M, De Cock F, Collen D. Generation of monoclonal antibodies against autologous proteins in gene-inactivated mice. J Biol Cheni 1995; 270: 8397-8400
• 16 Lijnen HR, Van Hoef B, Beclen V, Collen D. Characterization of the murine plasma fibrinolytic system. Eur J Biochem 1994; 224: 863-871
• 17 Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost 1987; 58: 1078-1084
• 18 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: 1747-1752
• 19 Dièval J, Nguyen G, Gross S, Delobel J, Kruithof EK O. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type I. Blood 1989; 77: 528-532
• 20 Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Brief report: complete deficiency of plasminogen-activator inhibitor type I due to a frame-shift mutation. N Engl J Med 1992; 327: 1729-1733