Tissue Plasminogen Activator Activity in Human Aqueous Humor - IOVS

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further defining the type of PA responsible for the detected activity. t-PA antigen levels showed ..... derestimate i-PA activity because its lysine bonds are cleaved ...
Tissue Plasminogen Activator Activity in Human Aqueous Humor David M. Smalley, John E. Fitzgerald, Daniel M. Taylor, Robert E. Cone, and James O'Rourke Purpose. To determine the levels of free plasminogen activator activity in human aqueous humor and to identify the type of activity (i.e., tissue-type t-PA or urokinase-type u-PA) that is responsible. Methods. Aqueous humor was obtained by a simplified pipette paraccniesis before cataract surgery in 31 subjects, ages 57 to 93 years. Levels of plasminogen activator activity were determined using a modified 1 7-hour specific arnidolyik: assay. The type of plasminogen activator was investigated in selected samples based on its dependence on soluble fibrin, inhibition by amiloride, and specific antibody blocking. Activity-antigen ratios were compared in seven samples. Results. Plasminogen activator (PA) activity was present in all samples tested. PA activity ranged widely between 0.54 and 26.7 inlU/ml, with a mean value of 10.8 ± 8.1 rnlU/ml. Soluble fibrin, a known stimulator of tissue-type plasminogen activator (t-PA), was required in the assay system. Its absence decreased the measured activity by more than 90%. Amiloride, a known inhibitor of urokinase-type PA, had little or no effect in selected samples tested. The activity was blocked by anti-human t-PA antibodies but not by antibodies against human u-PA, further defining the type of PA responsible for the detected activity. t-PA antigen levels showed less variation among individuals than did activity levels. Antigen-activity ratios ranged between 89 and 552. Conclusion. Plasminogen activator activity is present in the human aqueous humor in measurable quantities. The type of PA activity present is almost exclusively t-PA. t-PA activity varies more widely than antigen, as is the case in plasma. Invest Ophthalmol Vis Sci. 1994;35: 48-53

X lasminogen activators (PAs) are serine proteases that cleave plasminogen to form plasmin; thus, they play a key role in the regulation of proteolysis, especially fibrinolysis. The two major types of PA are tissue-type plasminogen activator and urokinase. Tissuetype plasminogen activator (t-PA) is a 68 kd protein whose major source is vascular endothelium. Urokinase (u-PA) is expressed by several cell types, including vascular endothelium.1 t-PA is maximally active when

from the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut. Presented in part at the ARVO Annual Meeting, May 2- 7, 1992, Sarasota, Florida. Supported by NIH grants EY07091 and F.Y00123. Submitted for publication February 25, 1993; revised May 26, 1993; accepted June 22, 1993. Proprietary interest category: N. Reprint requests: Dr. James O'Rourke, Department of Pathology, University of Connecticut School of Medicine, 263 Farmington Avenue, Room LI061, Farmington, CT 06030-3105.

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bound to fibrin but is minimally active in plasma and other fluids where, in the absence of fibrin binding, it is almost entirely bound to an excess of its principal inhibitor, PAI-1.2 In studies of aqueous humor fibrinolysis, discussion for many years has centered on the possibility that aqueous outflow, as well as intracameral clot lysis, may be regulated by a plasminogen activator present in the aqueous. In the evolution of this thinking3"13 (Table 1), a PA released from tissues bordering the anterior chamber has been assumed to cleave plasminogen present in the aqueous to form plasmin, an aggressive and versatile protease that has long been known to accelerate both aqueous outflow and clot lysis.3"3 Although this assumption has undoubtedly provided an attractive rationale for the future use of exogenous PAs in ophthalmic therapy, it has thus far failed ;to explain certain contradictions concerning the capacity

Investigative Ophthalmology & Visual Science, January 1994, Vol. 35, No. 1 Copyright © Association for Research in Vision and Ophthalmology

t-PA in Aqueous Humor

TABLE l.

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Aqueous Humor Plasminogen Activator

Reference

Species

PA Form

Assay

Finding

Francescheiti ei al (1959)20 Saidu/zafar H (1970)9 O'Rourkeeial (1982)IB Mehraeial (1984)9 Hayashieial (1986)22

Human Human/primate Feline Human Bovine

O'Rourkeeial (1985)19

Feline

Activity Activiiy Activity Activity Activiiy Antigen Activiiy

Positive 1:10-1:4 tiler Positive lysis area not given 0.87 ± 0.36 cm'2 lysis 1 10-900 mm2 lysis 0.04 ± 0.09 IU/ml 3.3 ± 0.8 ng/ml .017 ± .009CTA/ml*

23

O'Rourkeeial (J987)

Feline

Activity

Parkcial (1987)°

Canine Canine Primate Bovine/primate Bovine Primate Human Human

Activity Antigen Antigen Activity Antigen Antigen

Hernagglut illation Fibrin plate (0.5% human) Fibrin plate Fibrin plate A m idol y lie RIA Fibrin-agar plaie (human .0125%) I1'25 plasminogen gel SDS zymography I125 fibrin well ELISA ELISA I125 fibrin well ELISA ELISA

Geanonetal(1987) 7 Tripaihi ei al (I988)10 Bernatchezet al (1992)"

Antigen Activity

ELISA SDS gel zymography

.066 ± .019CTA/ml Not detectable Not delectable 0.86 ng/ml Not detectable 1.25 ± 0.19 ng/ml 0.86 ± 0.09 ng/ml 0.8 ± 0.17 ng/ml Not quantified

* 1 CTA L'nii = 1 IU slrcpiokinasc."

of aqueous to produce plasmin from plasminogen. For example, the free, uninhibited PA activity required to cleave plasminogen has been reported to be virtually absent or undeiectable in aqueous,67 even though aqueous has been shown to lyse plasminogcn-rich fibrin plates that plasma cannot lyse89 and to contain far more PA antigen per milligram of total protein than does plasma.10 It is possible that this problem may represent a need for greater assay sensitivity for free PA activity. However, there is also the unresolved disagreement about whether the PA activity present in human aqueous is predominantly tPA,10 which is greatly enhanced by fibrin binding, or u-PA,11 which is not. In this setting, it may be helpful to recall that the regulation of functions such as outflow or clot lysis by an aqueous PA would critically depend on the level of free, uninhibited PA activity as distinguished from the more plentiful PA antigen, which, unless bound to fibrin, is largely inactivated by inhibitors.1 This report deals with one aspect of the complex question of aqueous fibrinolysis, namely, the presence of a free PA activiiy in human aqueous. It is, first, a survey of samples recovered under resting conditions from normal eyes and assayed with enhanced sensitivity and, second, an identification of the type of PA activity found. As such, the work does not investigate activiiy inhibitors, systemic fibrinolysis, tissue sources of aqueous PA, or PA release mechanisms. MATERIALS AND METHODS Human glu-plasminogen, 2 chain human t-PA and u-PA, DESAFIB-X (soluble fibrin 1), spectrozyme

PL (chromogenic substrate H-D-Nle-HHT-LyspNA.2AcOH), goat anti-human t-PA, and goat antihuman u-PA used in PA activiiy assays were obtained from American Diagnostica, Inc. (Greenwich, CT). Vials of lyophyli/ed soluble fibrin 1 were stored at —20°C for up to 1 year; fresh stock dilutions were made at 2-week intervals and stored at —4°C. Human glu-plasminogen, t-PA, and u-PA, (all lyophylized) were stored at -70°C (to offset possible autoactivation by trace plasmin), and fresh stock solutions made at 2-week intervals were stored as aliquots at — 20°C. Ainiloride used to suppress u-PA activiiy was obtained from Boehringer-Mannheim (Indianapolis, IN). Tissue PA antigen was measured using an ELISA kit (American Diagnostica), as previously described by Tripaihi et al.10 An International Standard 83/517, of (500,000 IU/mg, or 600 M IU/ng, (National Institute for Biological Standards and Controls, Minis, Herts, England) was used for activity-antigen ratio computations.12 Aqueous Sample Recovery Aqueous humor specimens were obtained from 31 patients (14 men and 17 women ranging from 57 to 93 years of age) under sterile conditions and local anesthesia immediately before routine cataract surgery. This was the initial surgery done on each eye sampled. Preoperative examination revealed no evidence of glaucoma, uveitis, or medication for either. Twentytwo subjects were taking long-term cardiac or antihyperiensive medication. A 30-gauge, needle-tipped aqueous paracentesis pipette (Visitec Co., Sarasota, FL) was used to minimize the duration (10 seconds), instrumentation, trauma, and resistance to limbal

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puncture.13 The method used to recover aqueous humor during surgery was humane, included proper consent and institutional human experimental committee approval, and followed the tenets of the Declaration of Helsinki. One hundred n\ samples were gently aspirated and promptly transferred to ice cooled mini-centrifuge tubes for transport to the laboratory within 2 hours. Samples were stored at -70°C with no loss of activity compared to fresh samples assayed immediately. Samples were not acidified, as is usually done for plasma to inactivate PA inhibitors, because preliminary trails showed no difference from unacidified samples. Aqueous recovery procedures were done according to approved institutional and ARVO recommended guidelines. Aqueous total protein and plasma PA levels were not measured. PA Activity Assay The functional assay used to quantify free PA activity was a modified amidolytic assay previously described by Karlan et al14 in which incubation times, in the presence of excess plasminogen and soluble fibrin ], were extended to maximize sensitivity. Linearity for u-PA but not for t-PA can be achieved over four orders of magnitude, that is, roughly 0.01 to 100 mlU, using the lysine-containing substrate, spectrozyme PL, appropriate incubation times. An incubation time of 17 hours provided very good sensitivity and linearity for aqueous humor samples and corresponding standards. Samples were diluted 1:4 in buffer Z (50 mM Tris, 0.1 mM NaCl, 1.0 mM EDTA, 0.01% Triton X-100, pH 7.5). The assay was performed in duplicate in 100 fi\ wells of a 96-well microtiter plate by adding 25 ^1 of each of the reactants in the following order: sample (1:4 dilution), DESAFIB (2.5 /zg), Spectrozyme-PL substrate (25 nM), and glu-plasminogen (1.25 /ig)- All solutions were made in buffer Z. A u-PA standard dilution curve and control wells for fibrinogen or plasminogen autoactivation (plasmin trace contamination) and for absent soluble fibrin and plasminogen effects were also prepared for each assay. The plate was incubated at 37°C for 17 hours, and absorbance was measured at 405 nM. For antibody blocking experiments, three randomly selected aqueous samples were preincubated for 2 hours at room temperature with 1:1000 dilution of goat anti-human t-PA or uPA in buffer Z. For suppression of u-PA activity, selected samples were preincubated at room temperature for 2 hours with 2 mg/ml of amiloride in buffer Z. RESULTS PA Activity Levels PA activity was consistently detected in duplicate amidolytic assays of aqueous humor samples recovered from 31 subjects. The activity ranged widely from

(0.54 to 26.7 mlU/ml), as observed in earlier fibrin plate assays8915 (Table 1). The group mean for 31 samples (i.e., duplicate samples means/31) was 10.8 ±8.1 mlU/ml. Differences between duplicate assays (SD, see Figure 1) for each sample were larger for higher levels of activity than for lower levels. Nine samples showed a very low activity (below 5 mlU) and eight were above 20 mlU; however, no correlations to eye or systemic diseases were evident in these subgroups. Free PA activity was thus measurable in human aqueous humor and showed a wide interindividual variation. T-PA Activity Versus Antigen Levels Antigenic levels of t-PA were measured in seven randomly selected samples by an ELISA assay using goal anti-human t-PA as primary antibody (Table 2). As in past studies, antigenic t-PA (i.e. free plus inhibitorcomplexed tPA) was present at much higher levels and showed much smaller differences between samples than did free PA activity measurements (Table 1). Based on the standard equivalent of 600 mlU of free t-PA activity per ng,12 the antigenic levels (2.1 to 5.8 ng/ml) were between 89- and 552-fold higher than the free activity levels (6.3 to 20.6 mlU/inl) measured in the same samples. Thus, unlike unmodified plasma,116 a spontaneous free PA activity is measurable in unmodified human aqueous humor despite its apparent high level of inhibitor complexing. Antigenic levels of u-PA were not tested.

Plasminogen Activator Identification To define the type of activator present, three randomly selected samples were assayed for PA activity in the absence of soluble fibrin 1 (which stimulates t-PA

=

P

20-

•ilii

liti PATIENT

FIGURE l. Plasminogen activator activity levels. Plasminogen activator activity levels were measured in 31 samples of human aqueous humor using an amidolytic assay. All samples were done in duplicate, and the results are shown as mean ± SD.

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t-PA in Aqueous Humor

TABLE

2. Plasminogen Activator Antigen and Activity Levels Antigen Measured (ng/ml)

Patient

3.2

1 2 3 4 5 6 7

3.4 2.1 5.8 4.3 3.0 3.0

Activity Measured (mlU/ml)

Activity (as ng/ml)

.10.5 ±0.2 17.0 ±3.7 14.1 ±0.9 6.3 ± 1.6 17.3 ± 1.0 20.6 ± 0.4 .18.6 ±0.9

0.0175 ±0.0003 0.0283 ± 0.0062 0.0235 ±0.0015 0.0105 ±0.0027 0.0288 ±0.0017 0.0343 ± 0.0007 0.0275 ±0.0015

A n Hgen/Activity Ratio 182 120 89 552 149 87 109

Plasminogen activator antigen activity levels were measured in seven samples of human aqueous humor using an KLISA and an amidolylic assay, respectively. Activity levels were performed in duplicate and are shown as mean ± SD. The amount of activity (mlU/ml) is convened to ng/ml based on the specific activity of t-PA being approximately 600 inIU/ng.17 The antigen/activity ratio is based on the calculated ng/ml of activity and of the measured ng/ml antigen levels.

but not u-PA activity) and in the presence of amiloride (which inhibits u-PA but not t-PA activity). As shown in Figure 2, soluble fibrin 1 was required for the detection of PA activity in human aqueous samples; in its absence, activity was decreased more than 90%. Conversely, amiloride had no significant effect on the PA activity. In another set of three randomly selected samples, preincubation with antibodies against human t-PA and u-PA was done. As shown in Figure 3, activity was effectively blocked by anti-t-PA antibody, whereas antiu-PA antibody had no significant effect. These data indicate that t-PA rather than u-PA was the dominant PA activity type present in the human aqueous samples.

I 0 0 0

i-PA standard UK standard AH#1 AH #2

D

AH #3

DISCUSSION The foregoing results confirm that a measurable presence of basal free t-PA activity exists in unmodified human aqueous humor. Activity levels are low in comparison with antigenic levels and, as in plasma,116"18 show a much wider interindividual variation than do antigenic levels. The various factors that may influence activity in extravascular fluids, such as aqueous humor (e.g., kinins, fibrinogen, thrombin), are complex and not yet fully understood. The detectability of low levels of free t-PA activity in vivo appears to require a high assay sensitivity. In previous studies (Table I), the presence of free activity has been detected in aqueous humor but not in plasma, using semiquantitative fibrin plate as8,9,15,19 with long (17- to 24-hour) incubation times, says and it was earlier found by Franceschetti et al, who

• E3 • E2 •

t-PA Standard UK Standard AH 01 AH 02 AH 03

O

< 3

m —J%%—• PA A c t i v My

-PLASMINOGEN

+AMILORIDE

FIGURE 2. Determination of plasminogen activator type activity. To distinguish t-PA from u-PA plasminogen activator, activity levels were measured in the absence of soluble fibrin (— soluble fibrin) and in the presence of amiloride (+ amiloride) in randomly selected aqueous humor samples (AH # 1 , AH # 2 , AH *3). The measured activity in the absence of plasminogen (— plasminogen) was always minimal. All samples were done in duplicate, and the results are shown as mean ± SD.

No Antibodies

antl-UK

antl-t-PA

FIGURE 3. Antibody blocking plasminogen activator activities. The plasminogen activator activity levels of t-PA, u-PA, and randomly selected aqueous humor samples (AH# 1 , AH # 2 , and AH # 3 ) were measured in the absence and presence of anti-human u-PA and anti-human t-PA antibodies. All samples were done in duplicate, and the results are shown as mean ± SD.

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Investigative Ophthalmology &: Visual Science, January 1994, Vol. 35, No. 1

used hemagglutination assays for fibrinolysis.20 In our hands, the sensitivity of fibrin plates for aqueous humor lysis, but not for plasma, was considerably enhanced when a dilute (0.05%) human fibrinogen preparation was used.15 In the amidolytic assays reported here, a prolonged incubation time was key to detectability. Plausible reasons for the relatively low level of basal aqueous PA activity include the possibility that the lysine-containing substrate used in our assay may underestimate i-PA activity because its lysine bonds are cleaved more efficiently by u-PA used in the standard curves than by t-PA in the aqueous samples. Additional reasons may include the washout effects of aqueous turnover (at approximate hourly intervals) and the probability that the t-PA released to the aqueous pool during that time is from the relatively small tissue volume, represented by the ciliary, iris, and corneal endothelial tissues. t-PA is released from tissue sources in a free active form, unlike other proteolytic components of the coagulation or fibrinolytic systems that are produced as zymogens.1 The brief 3to 4-minute half-life of t-PA in human plasma and the dynamics of its release from tissue sources have suggested to most authors that, under basal conditions, released t-PA activity does not circulate but acts locally before being promptly inhibited.17 It should be noted that repeated assays for t-PA activity or antigen performed in individuals tend to remain stable for long periods of time, whereas interindividual differences for activity are large.21 It is also relevant that the higher levels reported for various tissues often represent a stimulated release or extraction that is artifactually induced, (e.g. fibrin overlay, thrombin stimulation, homogenization, cytolysis, and buffer extractions), whereas aqueous samples reflect basal resting conditions of release from source tissues. The wide variation in aqueous t-PA activity levels observed among individuals in this study is similar to that found with human plasma.16 The broad range for plasma among individuals has been attributed to the fine balance between t-PA and its inhibitors; hence, minor changes in either component will result in large changes in activity and in the antigen-activity ratio.1 The pattern shown in Table 1 using various assay methods also confirms a wider range in aqueous for PA activity than for antigen. Patients studied in this report represent only a cataract age group and were each sampled at the same time of day by one surgeon (DMT) under similar conditions. Variations in exercise, smoking, or alcohol intake, which may alter released t-PA activity in plasma,l() were not controlled in this series. The assay method used is highly sensitive and reproduces well. Differences between duplicate samples (shown as SD in Figure 1) were greater for

higher levels of activity, which have a larger available dynamic range, than for low levels. We have no alternative explanation for the aqueous PA activity variations and suggest that this is the normal basal physiological condition to be expected. The type of PA activity detected was almost exclusively t-PA based on stimulation by soluble fibrin 1, lack of inhibition by amiloride, and differential blocking by anti-t-PA and anti-u-PA antibodies. This is contrary to the findings of Bernatchez et al, who detected u-PA as the predominant PA in human aqueous." One possible explanation for the discrepancy may involve the concentration of soluble fibrin 1 used in assays. Bernatchez et al used casein gels, which may not contain an excess of soluble fibrin and therefore may not fully activate the t-PA. We were also unsuccessful in measuring human aqueous PA activity by zymography using plasminogen-rich fibrinogen gels (unpublished observations, 1992). Based on the measured activity, this technique was not sensitive enough in our hands to measure l-PA in human aqueous humor. The physiological relevance of l-PA activity in aqueous humor lies in the fact that it is a basal level of free, uninhibited activity released from adjacent source tissues under resting conditions. The frequently conjectured involvement of aqueous PA in such functions as outflow and clot lysis regulation6 "•'5.19.20.22,23 WOLl|ci depend on this free activity, as distinguished from inhibitor-complexed antigenic PA. However, it is not yet clear that the activity measured in aqueous humor would play the major role in these two functions. The process of tissue release of tPA in response to various agonist stimulations may play a greater role. For example, it seems evident that the considerable t-PA activity released locally from trabccular meshwork tissues'1 would be carried away from, rather than toward, the anterior chamber. It is also known from clinical observations that anterior chamber clot lysis proceeds most rapidly in areas of tissue contact with the iris or corneal endothelium but slowly in the pupillary space.24 Moreover, recent studies from this laboratory by Ramsby et al have shown that fibrin polymerization, in contact with corneal endothelial monolayers, will induce a more than 20-fold increase in the release of free t-PA activity.25 These observations by others have drawn attention to the significance of the provoked releasability of free t-PA activity from tissues into body fluids, as distinguished from its resting levels in such fluids. The acute release of t-PA activity into human plasma that follows venous occlusion or various agonist stimulations may exceed resting levels more than 50-fold and may thereby negate the dominant influence of inhibitors.1 Whether differences in the acute releasability of t-PA can be linked to specific human diseases remains to be seen.

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t-PA in Aqueous Humor Key Words tissue plasminogen activator, aqueous humor, urokinase, protease

14.

References 1. Prowse CV, MacGregor IR. Regulation of the plasminogen activator level in blood. In: KJuft C, ed. Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects. Boca Raton, FL: CRC Press; 1988;2:49-66. 2. Schleef RR, Loskuioff DL. Fibrinolytic: system of vascular endothelial cells. Haemostasis. 1988; 18:32834 1. 3. Saiduzzafar H. Exp Eye Res. 1970; 10:293-296. 4. Pandolfi M, Astrup T. Effect of plasmin on outflow resistance in the primate eye. Proc Soc Exp Biol and Med. 1966; 121:139-14 1. 5. Tripathi RC, Tripathi BJ, Bornstein S, Gabianelli E, Ernst JT. Use of tissue plasminogen activator for rapid dissolution of fibrin and blood clots in the eye after surgery for glaucoma cataract in humans. Drug Dev Res. I992;27:147-I59. 6. Park JK, Tripaihi RC, Tripaihi BJ, Barlow GH. Tissue plasminogen activator in the irabecular endoiheliuni. Invest Ophthalmol and Vis Sci. 1987;28:1341-1 345. 7. Geanon JD, Tripathi BJ, Tripathi RC, Barlow GH. Tissue plasminogen activator in avascular tissues of the eye. Exp Eye. Res. 1987;44:55-63. 8. Saiduzzafar H. Fibrinolylic activity in the aqueous humor. Exp Eye Res. 1970; I 0:293-296. 9. Mehra KS, Dube B, Mikuni 1, Dube RK. Reduced fibrinolytic activity in aqueous humor in chronic simple glaucoma. Tohai J Exp Ciin Med. 1984;9:33-34. 10. Tripaihi RC, Park JK, Tripaihi BJ, Millard CB. Tissue plasminogen aciivaior in human aqueous humor and its possible significance. Am J Ophthamol 1988; 106: 719-722. 1 1. Bernatchez SF, Tabatabay C, Belin B. Urokinase-type plasminogen activator in human aqueous humor. Invest Ophthalmol and Vis Sci. I 992;33:2687-2692. 12. Dieval J, Nguyen G, Gross S, Delobel J, Kruithof EKO. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type 1. Mood. 1991;77:528-532. 13. O'Rourke J, Taylor DM, McDonald P, Kreulzer DL.

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An aqueous paraceniesis pipel. Ophthalmol Surg. 1991;22:166-167. Karlan BY, Clark AS, Liitlefield BA. A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between urokinase and tissue-type activators. Biochem and Biophys Res Comm. 1987; 142:147-154. O'Rourke J, Lindsay M, Kreulzer DL, el al. Evidence of impaired anterior segment fibrinolylic activity in chronic uveiiis. Ophthalmol Res. 1982;4:256-264. Juhan-Vague I, Aillaud M-F, Allessi M-C. Biological variations in t-PA activator and antigen. In: KJuft C, ed. Tissue-Type Plasminogen Activator (tPA): Physiological and Clinical Aspects. Vol. 2. Boca Raton, FL: CRC Press; 1988:70-87. Kluft C. t-PA in fibrin dissolution and hemostasis. In: KJuft C, ed. Tissue-Type Plasminogen Activator (tPA): Physiological and Clinical Aspects. Vol. 1. Boca Raton, FL: CRC Press; 1988:47-82. Verheisen JH. Purification, assay and standardization of t-PA. In: Kluft C, ed. Tissue-Type Plasminogen Activator (tPA): Physiological and Clinical Aspects. Vol. 1. Boca Raton, FL: CRC Press; 1988:123-144. O'Rourke J, Moore M, Kreulzer DL. Regulation of anterior chamber fibrinolysis. Curr Eye Res. 1985;4: 569-578. Franceschetti A, Eichenberger E. Fibrinolyse im kammerwasser menschlicher und tierischer augen. Experientia. 1 959;4:130-131. Nilsson T, Wallen P, Mellbring G. In vivo metabolism of human tissue-type plasminogen activator. Scand J Haemotol. 1984;33:49-53. Hayashi K, Nakashima Y, Sueishi K, Tanaka K, Inoinata H. Fibrinolytic activity in bovine vitreous fluid and aqueous humor. ARVO Abstracts. Invest Ophthalmol and Vis Sci. 1986;27:49. O'Rourke J, Wang W-P, Donnelly L, Wang E, Kreulzer DL. Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation. Am J Pathol. 1987;28:1341-1345. Pandolfi M. Hemorrhages in Ophthalmology: A Hemostatic Approach. Stuttgart: Georg Thieme;. 1983:3135. Ramsby ML, Kreutzer DL. Fibrin induction of tissue plasminogen activity in corneal endothelial cells in vitro. Invest Ophthalmol Vis Sci. 1993;34:3207-3219.