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Tissue Plasminogen Activator in Human Aqueous Humor and Its Possible Therapeutic Significance
Tissue Plasminogen Activator in Human Aqueous Humor and Its Possible Therapeutic Significance Ramesh C. Tripathi, M . D . , James K. Park, B . A . , Brenda J. Tripathi, P h . D . , and Charles B. Millard, B.A.
By using an enzyme-linked immunosorbent assay, we detected a significant amount of tissue plasminogen activator (0.8 ± 0.17 ng/ml) in the aqueous humor of ten normal human eyes. It was also identified on Western blot analysis. The ratio of tissue plasminogen activator to total protein in aqueous humor was about 30 times greater than the ratio of tissue plasminogen activator to total protein in plasma. There was evidence that at least some of the tissue plasminogen activator in the aq ueous humor was synthesized locally by the structures bordering the anterior chamber of the eye. Tissue plasminogen activator may be a useful therapeutic modality in clinical condi tions of delayed dissolution of fibrin in the eye. TISSUE-SPECIFIC PLASMINOGEN activator is a
serine protease that plays an important role in the fibrinolytic cascade. 1 Although it is now recognized as an effective therapeutic agent for the dissolution of fibrin clots in the vascular system, 2 precise information concerning its normal level, activity, metabolism, and homeostatic regulation in the human eye is needed before it can be used routinely in the treatment of ocular disorders. We undertook this study to identify and quantitate tissue plasminogen ac tivator circulating in normal human aqueous humor and to provide a rationale for its possi ble therapeutic use in several ocular disorders.
Accepted for publication Sept. 2, 1988. From the Department of Ophthalmology a n d Visual Science, University of Chicago, Chicago, Illinois. This study w a s s u p p o r t e d by United States Public Health Service award EY-03747 from the National Eye Institute. Reprint requests to Ramesh C. Tripathi, M.D., Eye Research Laboratories, University of Chicago, 939 E. 57th St., Chicago, IL 60637.
Material and Methods We obtained samples of aqueous humor by paracentesis from the central pupillary area of the anterior chamber of patients undergoing elective cataract surgery. The detailed method for collection of pure aqueous humor was de scribed previously. 3 Briefly, by using a 30gauge needle for paracentesis, and without touching the corneal endothelium, iris, or lens, we aspirated approximately 100 μΐ of aqueous humor from each subject before the surgical procedure was begun. By using an enzyme-linked immunosorbent assay (ELISA), we measured the amount of tissue plasminogen activator in the ten samples of aqueous humor. To each well of a 96-well microtiter plate we added a coating that con sisted of 200 μΐ of goat anti-human tissue plas minogen activator prepared from a 2-mg/ml 0.1 M sodium bicarbonate stock solution diluted to 1:800. The plates were incubated at 25 C for three hours in a moist chamber. The coating was discarded, and the wells were washed with saline-polyoxyethylene sorbitan monolaurate. Next, 200 μΐ of the aqueous humor samples and human tissue plasminogen activator standards were incubated at 25 C in a moist chamber for 18 hours. The samples were removed, and the wells were washed four times with salinepolyoxyethylene sorbitan monolaurate. To each well, 200 μΐ of peroxidase-conjugated antitissue plasminogen activator IgG (prepared by diluting the stock solution to 1:2,000 with phosphate-buffered saline-polyoxyethylene sorbitan monolaurate) was added and allowed to incubate for three hours at 25 C. We dis carded the conjugate and washed the wells four times with saline-polyoxyethylene sorbitan monolaurate. Finally, 200 μΐ of substrate, con sisting of 0.4 mg/ml ori/zo-phenylenediamine,
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and 0.01% hydrogen peroxide dissolved in citrate-phosphate buffer, pH 5.0, was added to each well, and the plates were incubated for 30 minutes in the dark at 25 C. We terminated the enzymatic reaction by adding 50 μΐ of 4.5 M sulfuric acid per well. Absorbances were read on an ELISA reader at 492 nm. In order to exclude the possibility of falsepositive results, we also identified the tissue plasminogen activator in aqueous humor by Western blot analysis. We brought the samples to approximately 35 times their concentration in vivo on a filter and resuspended them in the solubilizing mix (40% weight/volume urea, 1% weight/volume sodium dodecyl sulfate, and 3% volume/volume 2-mercaptoethanol, pH 7.6). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed with a minor modification 3 of the method of Laemmli, 4 and immunoblotting was carried out according to the technique of Towbin and Gordon. 5 For con trols, 2 ng of human tissue plasminogen activa tor was separated on 10% gels and transferred to nitrocellulose strips. The strips were stained with a mouse anti-human tissue plasminogen activator antiserum using an indirect peroxidase method, 6 8 and the molecular weight stan dards were stained with Ponceau S.
Results As determined by ELISA, the levels of tissue plasminogen activator in the ten samples of human aqueous humor ranged from 0.6 to 1.1 ng/ml (Table). We found no correlation between the level of tissue plasminogen activator and the age, sex, or race of the patients. The mean concentration of tissue plasminogen activator was 0.8 ± 0.17 ng/ml. The Western blot tests stained with the mouse anti-human tissue plas minogen activator antibody showed a band at an approximate molecular weight of 68 kD, consistent with that of tissue plasminogen acti vator (Figure).
Discussion The possible sources of tissue plasminogen activator in the aqueous humor include secre tion by the ciliary processes and synthesis by the cells bordering the anterior chamber. 9 Com pared to the ratio of tissue plasminogen activa-
TABLE LEVELS OF TISSUE PLASMINOGEN ACTIVATOR IN AQUEOUS HUMOR AS DETERMINED BY ELISA PATIENT NO., AGE (YRS),
TISSUE PLASMINOGEN ACTIVATOR*
SEX
RACE
(NG/ML OF AQUEOUS HUMOR)
1, 77, F 2, 61, F 3, 86, F 4, 80, F 5, 79, F 6, 52, M 7, 87, F 8, 76, F 9, 71, M 10, 46, M
W B B B B B W W B W
1.0 0.8 0.7 0.7 0.6 0.7 0.9 0.6 1.1 0.9
*Mean ± S.D., 0.8 ± 0.2 ng/ml.
tor to total protein in plasma (15 ng/ml tissue plasminogen activator:70 mg/ml protein), the ratio in aqueous humor (0.8 ng/ml tissue plas minogen activator:0.12 mg/ml protein) was more than 30 times higher. In view of the known roles of tissue plasminogen activator in the body, we may conjecture that its presence in the aqueous humor serves several functions. For example, tissue plasminogen activator may have a role in modulating the normal facility of aqueous outflow by regulating the amount of extracellular glycoproteins present in the trabecular meshwork. 10 In cases of hyphema, plas minogen activators in the aqueous humor are known to be important in the dissolution of fibrin clots. 11 Patients with traumatic hyphema in whom systemic aminocaproic acid (an inhibi tor of plasminogen activators) is administered show a significant increase in the length of time that clotting persists, 12 thereby lending support to the theory that tissue plasminogen activator plays a role in promoting fibrinolysis in the human anterior chamber. Breakdown of the blood-aqueous barrier in the course of various insults to the eye causes entry of fibrin, other plasma proteins, and inflammatory cells into the aqueous humor. Fibrin provides a structure for cicatrization. The clinical complications that result from the delayed dissolution of fibrinous exudates in the anterior chamber include formation of synechiae, increased intraocular pressure, fail ure of surgical procedures for glaucomas, and compromise in the structural and functional integrity of the cornea, trabecular meshwork,
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hyphema in humans, these agents have failed to gain widespread acceptance mainly because of their toxicity and the resultant complications. The administration of urokinase and streptokinase has been associated with cataract formation, increased intraocular pressure, inflammation, corneal abnormalities, cloudy vitreous, and retinal degeneration. 1921 Furthermore, urokinase does not demonstrate preferential plasminogen activation in the presence of fibrin, which may create indiscriminate plasminogen activation. In contrast, tissue plasminogen activator has minimal toxic effects in experimental animal models. 16 Unlike urokinase and streptokinase, tissue plasminogen activator has the advantages of selective and enhanced plasminogen activation in the presence of fibrin, as well as protection of plasmin from antiplasmin inhibitors until complete clot lysis is achieved. These unique characteristics of tissue plasminogen activator make it the ideal choice for intraocular fibrinolysis while uncontrolled proteolysis is minimized.
■2A
1
2
Figure (Tripathi and associates). Immunoblot of aqueous humor protein (lane 2) stained with mouse anti-human tissue plasminogen activator antibody. Lane 1 contains human tissue plasminogen activator as control. A single specific band at 68 kD (arrow) is present in both lanes.
lens, and uveal tissue. Therefore, rapid dissolution of fibrin in the anterior chamber would reduce the clinical complications that are often associated with hyphema, anterior uveitis, and inflammation after surgery. Recent experimental studies on animals have provided evidence that tissue plasminogen activator has a therapeutic role in the treatment of disorders of both the anterior and posterior segments of the eye. 1318 Although other fibrinolytic agents, such as urokinase and streptokinase, have been used for the resolution of
Cells of the trabecular meshwork that were exposed in vivo to whole blood or to plasma, as well as cultured trabecular cells exposed to thrombin, showed a four- to fivefold increase in the extracellular release of tissue plasminogen activator. 22 · 23 These findings indicate that, given a specific stimulus, the trabecular cells actively synthesize tissue plasminogen activator. Whether other structures bordering the anterior chamber play a similar role remains to be investigated; however, alterations in the levels of tissue plasminogen activator in aqueous humor have been reported in an animal model of uveitis. 24 In view of the normal levels of circulating tissue plasminogen activator in human as well as animal eyes,10,26,26 and of the apparent insufficiency of this naturally occurring protease in various clinical situations, we advocate the therapeutic administration of tissue plasminogen activator. Such therapy could be useful in those cases in which a delay in the dissolution of fibrin in the anterior chamber would otherwise lead to the formation of synechiae, increased intraocular pressure, failure of surgical procedures for glaucoma, and a compromise in the structural and functional integrity of tissues in the anterior segment of the eye. However, because the large size of the tissue plasminogen activator molecule (68 kD) would most likely preclude its passage across the cornea, topical delivery of the drug will be impractical. Direct intracameral or intravitreal
722
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a d m i n i s t r a t i o n , t h e r e f o r e , s h o u l d be the m a i n r o u t e for d e l i v e r y .
References I. Kluft, C : t-PA in fibrin dissolution and hemostasis. In Kluft, C. (ed.): Tissue-type Plasminogen Activator (t-PA). Physiological and Clinical Aspects, vol. 1. Boca Raton, CRC Press, 1988, pp. 47-79. 2. Sobel, B. E.: Thrombolytic therapy with t-PA. In Kluft, C. (ed.): Tissue-type Plasminogen Activator (t-PA). Physiological and Clinical Aspects, vol. 2. Boca Raton, CRC Press, 1988, pp. 109-127. 3. Tripathi, R. C , Millard, C. B., and Tripathi, B. J;: Protein composition of human aqueous humor. SDS-PAGE analysis of surgical and postmortem samples. Exp. Eye Res. In press. 4. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970. 5. Towbin, H., and Gordon, J.: Immunoblotting and dot immunobinding. Current status and outlook. J. Immunol. Methods 72:313, 1984. 6. Stefansson, K., Marton, L. S., Antel, J. P., Wollman, R. P., Roos, R. P., Chejfec, G., and Arnason, B. W.: Neuropathy accompanying IgMgamma-monoclonal gammopathy. Acta Neuropathol. 59:255, 1984. 7. Tripathi, B. J., Tripathi, R. C , Stefansson, K., Havran, W. L., and Fitch, F. W.: Production and characterization of monoclonal antibodies to human trabecular endothelium. Exp. Eye Res. 43:863, 1986. 8. Tripathi, B. J., Marcus, C. H., Millard, C. B., Gulcher, J., Stefansson, K., and Tripathi, R. C : Monoclonal antibodies and lectins as probes for investigation of the cell biology of human trabecular meshwork. A preliminary report. Ophthalmic Res. In press. 9. Tripathi, B. J., Geanon, J. D., and Tripathi, R. C : Distribution of tissue plasminogen activator in human and monkey eyes. An immunohistochemical study. Ophthalmology 94:1434, 1987. 10. Park, J. K., Tripathi, R. C , Tripathi, B. J., and Barlow, G. H.: Tissue plasminogen activator in the trabecular endothelium. Invest. Ophthalmol. Vis. Sci. 28:1341, 1987. I I . Tripathi, R. C : A corneal transfixing irrigation/perfusion device. A new method for evacuation of hyphema. Ophthalmic Surg. 11:569, 1980. 12. Crouch, E. R., Jr., and Frenkel, M.: Aminocaproic acid in the treatment of traumatic hyphema. Am. J. Ophthalmol. 81:355, 1976. 13. Lambrou, F. H., Snyder, R. W., and Williams, G. A.: Use of tissue plasminogen activator in experimental hyphema. Arch. Ophthalmol. 105:995, 1987.
December, 1988
14. Johnson, R. N., Olsen, K., and Hernandez, E.: Tissue plasminogen activator treatment of postoperative intraocular fibrin. Ophthalmology 95:592, 1988. 15. Pandolfi, M.: Intraocular hemorrhages. A hemostatic therapeutic approach. Surv. Ophthalmol. 22:322, 1978. 16. Snyder, R. W., Lambrou, F. H., and Williams, G. A.: Intraocular fibrinolysis with recombinant human tissue plasminogen activator. Arch. Ophthalmol. 105:1277, 1987. 17. O'Rourke, J., Wang, W.-P., Donnelly, L., Wang, E., and Kreutzer, D. L.: Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation. Am. J. Pathol. 126:334, 1987. 18. Jaffe, G. J., Green, G. D. J., McKay, B. S., Hartz, A., and Williams, G. A.: Intravitreal clearance of tissue plasminogen activator in the rabbit. Arch. Ophthalmol. 106:969, 1988. 19. Kozial, J., Peyman, G. A., Sanders, D. L., Vichek, J., and Goldberg, M. S.: Urokinase in experimental vitreous hemorrhage. Ophthalmic Surg. 6:79, 1975. 20. Friedman, M. W.: Streptokinase in ophthalmology. Am. J. Ophthalmol. 35:1184, 1952. 21. Chapman-Smith, J. S., and Crock, G. W.: Urokinase in the management of vitreous hemorrhage. Br. J. Ophthalmol. 61:500, 1977. 22. Park, J. K., Tripathi, R. C , Barlow, G. H., and Tripathi, B. J.: Tissue plasminogen activator in trabecular endothelium and its role in traumatic hyphema. ARVO Abstracts. Supplement to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1986, p. 165. 23. Shuman, M. A., Polansky, J., Merkel, C , Kim, R., and Alvarado, J.: Human trabecular meshwork (HTM) cells express high levels of tissue plasminogen activator. ARVO Abstracts. Supplement to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1986, p. 50. 24. O'Rourke, J., Lindsay, M., Kreutzer, D., Picciano, P., Rowland, F., Joyner, R., and Dobrzanski, L.: Evidence of impaired anterior segment fibrinolytic activity in chronic uveitis. Ophthalmic Res. 14:256, 1982. 25. Geanon, J. D., Tripathi, B. J., Tripathi, R. C , and Barlow, G. H.: Tissue plasminogen activator in avascular tissues of the eye. A quantitative study of its activity in the cornea, lens, and aqueous and vitreous humors of dog, calf, and monkey. Exp. Eye Res. 44:55, 1987. 26. Hayashi, K., Nakashima, Y., Sueishi, K., Tanaka, K., and Inomata, H.: Fibrinolytic activity and localization of plasminogen activator in bovine vitreous fluid and aqueous humor. ARVO Abstracts. Supplment to Invest. Ophthalmol. Vis. Sci. Philadelphia, J. B. Lippincott, 1986, p. 49.
By using an enzyme-linked immunosorbent assay, we detected a significant amount of tissue plasminogen activator (0.8 ± 0.17 ng/ml) in the aqueous humor of ten normal human eyes. It was also identified on Western blot analysis. The ratio of tissue plasminogen activator to total protein in aqueous humor was about 30 times greater than the ratio of tissue plasminogen activator to total protein in plasma. There was evidence that at least some of the tissue plasminogen activator in the aq ueous humor was synthesized locally by the structures bordering the anterior chamber of the eye. Tissue plasminogen activator may be a useful therapeutic modality in clinical condi tions of delayed dissolution of fibrin in the eye. TISSUE-SPECIFIC PLASMINOGEN activator is a
serine protease that plays an important role in the fibrinolytic cascade. 1 Although it is now recognized as an effective therapeutic agent for the dissolution of fibrin clots in the vascular system, 2 precise information concerning its normal level, activity, metabolism, and homeostatic regulation in the human eye is needed before it can be used routinely in the treatment of ocular disorders. We undertook this study to identify and quantitate tissue plasminogen ac tivator circulating in normal human aqueous humor and to provide a rationale for its possi ble therapeutic use in several ocular disorders.
Accepted for publication Sept. 2, 1988. From the Department of Ophthalmology a n d Visual Science, University of Chicago, Chicago, Illinois. This study w a s s u p p o r t e d by United States Public Health Service award EY-03747 from the National Eye Institute. Reprint requests to Ramesh C. Tripathi, M.D., Eye Research Laboratories, University of Chicago, 939 E. 57th St., Chicago, IL 60637.
Material and Methods We obtained samples of aqueous humor by paracentesis from the central pupillary area of the anterior chamber of patients undergoing elective cataract surgery. The detailed method for collection of pure aqueous humor was de scribed previously. 3 Briefly, by using a 30gauge needle for paracentesis, and without touching the corneal endothelium, iris, or lens, we aspirated approximately 100 μΐ of aqueous humor from each subject before the surgical procedure was begun. By using an enzyme-linked immunosorbent assay (ELISA), we measured the amount of tissue plasminogen activator in the ten samples of aqueous humor. To each well of a 96-well microtiter plate we added a coating that con sisted of 200 μΐ of goat anti-human tissue plas minogen activator prepared from a 2-mg/ml 0.1 M sodium bicarbonate stock solution diluted to 1:800. The plates were incubated at 25 C for three hours in a moist chamber. The coating was discarded, and the wells were washed with saline-polyoxyethylene sorbitan monolaurate. Next, 200 μΐ of the aqueous humor samples and human tissue plasminogen activator standards were incubated at 25 C in a moist chamber for 18 hours. The samples were removed, and the wells were washed four times with salinepolyoxyethylene sorbitan monolaurate. To each well, 200 μΐ of peroxidase-conjugated antitissue plasminogen activator IgG (prepared by diluting the stock solution to 1:2,000 with phosphate-buffered saline-polyoxyethylene sorbitan monolaurate) was added and allowed to incubate for three hours at 25 C. We dis carded the conjugate and washed the wells four times with saline-polyoxyethylene sorbitan monolaurate. Finally, 200 μΐ of substrate, con sisting of 0.4 mg/ml ori/zo-phenylenediamine,
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and 0.01% hydrogen peroxide dissolved in citrate-phosphate buffer, pH 5.0, was added to each well, and the plates were incubated for 30 minutes in the dark at 25 C. We terminated the enzymatic reaction by adding 50 μΐ of 4.5 M sulfuric acid per well. Absorbances were read on an ELISA reader at 492 nm. In order to exclude the possibility of falsepositive results, we also identified the tissue plasminogen activator in aqueous humor by Western blot analysis. We brought the samples to approximately 35 times their concentration in vivo on a filter and resuspended them in the solubilizing mix (40% weight/volume urea, 1% weight/volume sodium dodecyl sulfate, and 3% volume/volume 2-mercaptoethanol, pH 7.6). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed with a minor modification 3 of the method of Laemmli, 4 and immunoblotting was carried out according to the technique of Towbin and Gordon. 5 For con trols, 2 ng of human tissue plasminogen activa tor was separated on 10% gels and transferred to nitrocellulose strips. The strips were stained with a mouse anti-human tissue plasminogen activator antiserum using an indirect peroxidase method, 6 8 and the molecular weight stan dards were stained with Ponceau S.
Results As determined by ELISA, the levels of tissue plasminogen activator in the ten samples of human aqueous humor ranged from 0.6 to 1.1 ng/ml (Table). We found no correlation between the level of tissue plasminogen activator and the age, sex, or race of the patients. The mean concentration of tissue plasminogen activator was 0.8 ± 0.17 ng/ml. The Western blot tests stained with the mouse anti-human tissue plas minogen activator antibody showed a band at an approximate molecular weight of 68 kD, consistent with that of tissue plasminogen acti vator (Figure).
Discussion The possible sources of tissue plasminogen activator in the aqueous humor include secre tion by the ciliary processes and synthesis by the cells bordering the anterior chamber. 9 Com pared to the ratio of tissue plasminogen activa-
TABLE LEVELS OF TISSUE PLASMINOGEN ACTIVATOR IN AQUEOUS HUMOR AS DETERMINED BY ELISA PATIENT NO., AGE (YRS),
TISSUE PLASMINOGEN ACTIVATOR*
SEX
RACE
(NG/ML OF AQUEOUS HUMOR)
1, 77, F 2, 61, F 3, 86, F 4, 80, F 5, 79, F 6, 52, M 7, 87, F 8, 76, F 9, 71, M 10, 46, M
W B B B B B W W B W
1.0 0.8 0.7 0.7 0.6 0.7 0.9 0.6 1.1 0.9
*Mean ± S.D., 0.8 ± 0.2 ng/ml.
tor to total protein in plasma (15 ng/ml tissue plasminogen activator:70 mg/ml protein), the ratio in aqueous humor (0.8 ng/ml tissue plas minogen activator:0.12 mg/ml protein) was more than 30 times higher. In view of the known roles of tissue plasminogen activator in the body, we may conjecture that its presence in the aqueous humor serves several functions. For example, tissue plasminogen activator may have a role in modulating the normal facility of aqueous outflow by regulating the amount of extracellular glycoproteins present in the trabecular meshwork. 10 In cases of hyphema, plas minogen activators in the aqueous humor are known to be important in the dissolution of fibrin clots. 11 Patients with traumatic hyphema in whom systemic aminocaproic acid (an inhibi tor of plasminogen activators) is administered show a significant increase in the length of time that clotting persists, 12 thereby lending support to the theory that tissue plasminogen activator plays a role in promoting fibrinolysis in the human anterior chamber. Breakdown of the blood-aqueous barrier in the course of various insults to the eye causes entry of fibrin, other plasma proteins, and inflammatory cells into the aqueous humor. Fibrin provides a structure for cicatrization. The clinical complications that result from the delayed dissolution of fibrinous exudates in the anterior chamber include formation of synechiae, increased intraocular pressure, fail ure of surgical procedures for glaucomas, and compromise in the structural and functional integrity of the cornea, trabecular meshwork,
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hyphema in humans, these agents have failed to gain widespread acceptance mainly because of their toxicity and the resultant complications. The administration of urokinase and streptokinase has been associated with cataract formation, increased intraocular pressure, inflammation, corneal abnormalities, cloudy vitreous, and retinal degeneration. 1921 Furthermore, urokinase does not demonstrate preferential plasminogen activation in the presence of fibrin, which may create indiscriminate plasminogen activation. In contrast, tissue plasminogen activator has minimal toxic effects in experimental animal models. 16 Unlike urokinase and streptokinase, tissue plasminogen activator has the advantages of selective and enhanced plasminogen activation in the presence of fibrin, as well as protection of plasmin from antiplasmin inhibitors until complete clot lysis is achieved. These unique characteristics of tissue plasminogen activator make it the ideal choice for intraocular fibrinolysis while uncontrolled proteolysis is minimized.
■2A
1
2
Figure (Tripathi and associates). Immunoblot of aqueous humor protein (lane 2) stained with mouse anti-human tissue plasminogen activator antibody. Lane 1 contains human tissue plasminogen activator as control. A single specific band at 68 kD (arrow) is present in both lanes.
lens, and uveal tissue. Therefore, rapid dissolution of fibrin in the anterior chamber would reduce the clinical complications that are often associated with hyphema, anterior uveitis, and inflammation after surgery. Recent experimental studies on animals have provided evidence that tissue plasminogen activator has a therapeutic role in the treatment of disorders of both the anterior and posterior segments of the eye. 1318 Although other fibrinolytic agents, such as urokinase and streptokinase, have been used for the resolution of
Cells of the trabecular meshwork that were exposed in vivo to whole blood or to plasma, as well as cultured trabecular cells exposed to thrombin, showed a four- to fivefold increase in the extracellular release of tissue plasminogen activator. 22 · 23 These findings indicate that, given a specific stimulus, the trabecular cells actively synthesize tissue plasminogen activator. Whether other structures bordering the anterior chamber play a similar role remains to be investigated; however, alterations in the levels of tissue plasminogen activator in aqueous humor have been reported in an animal model of uveitis. 24 In view of the normal levels of circulating tissue plasminogen activator in human as well as animal eyes,10,26,26 and of the apparent insufficiency of this naturally occurring protease in various clinical situations, we advocate the therapeutic administration of tissue plasminogen activator. Such therapy could be useful in those cases in which a delay in the dissolution of fibrin in the anterior chamber would otherwise lead to the formation of synechiae, increased intraocular pressure, failure of surgical procedures for glaucoma, and a compromise in the structural and functional integrity of tissues in the anterior segment of the eye. However, because the large size of the tissue plasminogen activator molecule (68 kD) would most likely preclude its passage across the cornea, topical delivery of the drug will be impractical. Direct intracameral or intravitreal
722
AMERICAN JOURNAL OF OPHTHALMOLOGY
a d m i n i s t r a t i o n , t h e r e f o r e , s h o u l d be the m a i n r o u t e for d e l i v e r y .
References I. Kluft, C : t-PA in fibrin dissolution and hemostasis. In Kluft, C. (ed.): Tissue-type Plasminogen Activator (t-PA). Physiological and Clinical Aspects, vol. 1. Boca Raton, CRC Press, 1988, pp. 47-79. 2. Sobel, B. E.: Thrombolytic therapy with t-PA. In Kluft, C. (ed.): Tissue-type Plasminogen Activator (t-PA). Physiological and Clinical Aspects, vol. 2. Boca Raton, CRC Press, 1988, pp. 109-127. 3. Tripathi, R. C , Millard, C. B., and Tripathi, B. J;: Protein composition of human aqueous humor. SDS-PAGE analysis of surgical and postmortem samples. Exp. Eye Res. In press. 4. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970. 5. Towbin, H., and Gordon, J.: Immunoblotting and dot immunobinding. Current status and outlook. J. Immunol. Methods 72:313, 1984. 6. Stefansson, K., Marton, L. S., Antel, J. P., Wollman, R. P., Roos, R. P., Chejfec, G., and Arnason, B. W.: Neuropathy accompanying IgMgamma-monoclonal gammopathy. Acta Neuropathol. 59:255, 1984. 7. Tripathi, B. J., Tripathi, R. C , Stefansson, K., Havran, W. L., and Fitch, F. W.: Production and characterization of monoclonal antibodies to human trabecular endothelium. Exp. Eye Res. 43:863, 1986. 8. Tripathi, B. J., Marcus, C. H., Millard, C. B., Gulcher, J., Stefansson, K., and Tripathi, R. C : Monoclonal antibodies and lectins as probes for investigation of the cell biology of human trabecular meshwork. A preliminary report. Ophthalmic Res. In press. 9. Tripathi, B. J., Geanon, J. D., and Tripathi, R. C : Distribution of tissue plasminogen activator in human and monkey eyes. An immunohistochemical study. Ophthalmology 94:1434, 1987. 10. Park, J. K., Tripathi, R. C , Tripathi, B. J., and Barlow, G. H.: Tissue plasminogen activator in the trabecular endothelium. Invest. Ophthalmol. Vis. Sci. 28:1341, 1987. I I . Tripathi, R. C : A corneal transfixing irrigation/perfusion device. A new method for evacuation of hyphema. Ophthalmic Surg. 11:569, 1980. 12. Crouch, E. R., Jr., and Frenkel, M.: Aminocaproic acid in the treatment of traumatic hyphema. Am. J. Ophthalmol. 81:355, 1976. 13. Lambrou, F. H., Snyder, R. W., and Williams, G. A.: Use of tissue plasminogen activator in experimental hyphema. Arch. Ophthalmol. 105:995, 1987.
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