- Email: [email protected]
Plasma and cerebrospinal fluid activities of tissue plasminogen activator, urokinase and plasminogen activator inhibitor-1 in multiple sclerosis
Fibrinolysis & Proteolysis (1997) 11(2), 109-113 © PearsonProfessionalLtd 1997
Plasma and cerebrospinal fluid activities of tissue plasminogen activator, urokinase and plasminogen a c t i v a t o r inhibitor-1 in multiple sclerosis F. O. 1". AkenamP, M. Koskiniemi 1, S. MustjokP, V. Sirdn 1, M. Fiirkkilii 2, A. VaherP 1Haartman Institute, Department of Virology, FIN-00014 University of Helsinki, Finland. 2Department of Neurology, FIN-00014 University of Helsinki, Finland.
Summary
Objective: To study plasma and cerebrospinal fluid (CSF) levels of tissue plasminogen activator (tPA), urokinase (uPA) and plasminogen activator inhibitor-1 (PAl-l) in multiple sclerosis (MS) patients. Design: Patients diagnosed as, or suspected of having, MS were involved in this study. The reference subjects had lumbar punctures for clinical reasons but were exclusive of having either MS or other types of neurological diseases. The identities of MS and reference samples were unknown to the researcher until laboratory results were ready. Setting: Department of Neurology, University of Helsinki, Finland. Subjects and Methods: tPA, uPA and PAl-1 levels were studied by an immunocapture assay, zymography and enzyme immunoassay in 19 patients with MS and 20 reference subjects. Results: Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. We observed significantly higher mean levels of CSF tPA (MS: 360+_24 mlU/ml, REF: 43.7+_6 mlU/ml; P< 0.001) and PAl-1 (P= 0.048) in patients with MS, in comparison with the reference subjects. Plasma PAl-1 and tPA values were within reference ranges in the patients. Although the CSF tPA activity correlated positively with age in the reference subjects (P= 0.011 ; r= 0.61), no correlation was observed in MS. There was no association between the CSF PAl-1 and age either in the patients or the reference subjects. Nine of 19 CSF samples and 6 of 8 plasma samples of MS patients had quantifiable uPA activity, whereas reference samples did not. Conclusions: CSF tPA activity appears raised in MS patients. The assessment of tPA activity and its specific inhibitor may be useful for understanding the pathogenesis/prognosis of MS and similar neurological diseases.
INTRODUCTION Plasminogen activators (PA), tissue-type (tPA) and urokinase-type (uPA) are serine proteases whose function is the conversion of the inactive zymogen plasminogen into the active protease plasmin. Plasminogen activator inhibitor-1 (PM-1), a 50-54 kDa serpin protease inhibitor, is a major inhibitor of tPA and uPA. 1-3 PAs have been implicated in a number of biological processes including Received 10 December 1996 Accepted after revision 13 March 1997 Correspondence to: Francis Akenami, MSc, Tel: +358-9-434 6480; Fax: +3589-434 6491; E-mail: Francis.Akenami@helsinkLfL
fibrinolysis and thrombolysis, cell migration, tissue invasion by both normal and malignant cells, and tissue remodelling. 4'5 High concentrations of vascular tPA antigen 6-8 and PAI-19'1° are strongly associated with the risk of myocardial infarction and stroke, tPA antigen concentrations have also been found to correlate with the existence of carotid atherosclerosis. 8 Lipoprotein (a) has been reported to exert an atherogenic effect by inhibiting plasminogen activation.11-13 Moreover, roles of PAs and PM-1 in the pathogenesis of neurological diseases and tumour development have been proposed.14-17 tPA gene is expressed in the developing brain, 18-2° during cerebellar motor learning 21 and in h u m a n tumour cells of neuroectodermal origin. 22 109
110
Akenami et al.
In a preliminary study, we observed a spectacular elevation of CSF tPA activity in MS patients in comparison with patients suffering other types of neurological diseases and subjects without neurological disease. 23 Therefore, we conducted this follow-up to include a larger population, and plasma, CSF tPA, uPA and PAId. MATERIALS AND METHODS
The procedures followed were in accord with the Helsinki Declaration of 1975, as revised in 1983, for human experimentation. This study involved 19 MS patients aged 18 to 54 years undergoing spinal taps for clinical reasons, from whom about 1 ml each of CSF was drawn into plain bijou bottles. Twenty subjects without neurological diseases, aged from 1 to 74 years, served as reference. Only samples which were clear and free of blood cells were included for this study. Blood (2.5-3 ml) was collected by venipuncture into EDTA tube. Plasma samples (MS: n=8; reference subjects: n=9) were prepared by centrifuging the blood at about 2 500 x g for 30 min. Samples were assayed immediately for tPA and uPA activities, and were kept frozen at -70°C until they were assayed for PAI-1.
37°C with 50 gl of a solution of rabbit or goat antih u m a n IgG antibodies to either h u m a n uPA (10 gg/ml) or tPA (10gg/ml) (cat. no. 389 and 387, respectively; American Diagnostica). The plates were washed three times with PBS containing 0.05% Tween 20 (PBS/Tween) followed by dispensing 50 gl of CSF or two-fold diluted plasma and were allowed to bind for 2 h at room temperature. After binding, the wells were washed again (three times with PBS/Tween), and h u m a n plasminogen containing traces of plasmin was added to assay the proenzyme activity bound to the wells (2 gg in 50 gl uPA assay buffer consisting of 50 mM glycine pH 7.8, 0.1% Triton X-100, 0.1% gelatin, and 10 mM 6-aminocaproic acid). Plasminogen was purified from fresh h u m a n plasma by affinity chromatography on lysine-Sepharose. 2z The reaction with plasminogen was allowed to proceed for 45 rain at 37°C and the plasmin produced was assayed by its thioesterase activity. 2s To estimate the amount of enzyme produced, high molecular weight two-chain uPA (80 000 IU/mg; American Diagnostica) and twochain tPA (650 000 IU/mg; American Diagnostica) were used as standards. PAl-1 assays
Zymography
Zymography was done as earlier reportedY Briefly, electrophoresis was run in 8% polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS) under non-reducing conditions. 24 The molecular weights of the lysed bands were estimated by making use of prestained low molecular weight marker proteins (Pharmacia, Uppsala, Sweden). Activity standards of uPA (Calbiochem, La Jolla, CA, USA) and tPA (American Diagnostica, Greenwich, CT, USA) were also included. Before zymography, SDS was removed by washing the gel for 6 h with phosphate-buffered saline, pH 7.4 (PBS) containing 2.5% Triton X-100. An agarose gel containing casein and 1.7 gg/ml plasminogen was then placed over the polyacrylamide gel, and the overlay incubated for 24-48 h at 37°C in a humidified chamber. To further characterize the proteinase present, an anticatalytic monoclonal antibody against uPA 10gg/ml (cat. No. 394; American Diagnostica) was added over the polyacrylamide gel before incubation with the caseinagarose gel.
PAI-1 was assayed as previously reported. 29 Briefly, enzyme immunoassay (EL/k) was used as recommended by the manufacturer (Monozyme, Horsholm, Denmark). The assay detected both free (active) and complexed (inactive) PAI-1. The concentration of PAI-1 was determined by comparing the absorbance for each well with a series of absorbance values obtained from known plasma concentrations of PAI-1, plotted as a standard curve. The standard plasma, provided by the manufacturer, with a concentration of 8 ng/ml was diluted with the appropriate buffer to cover the range: 800 pg/ml to 25 pg/ml. Each assay was made in duplicate and the final result was taken as the average of the two determinations. Plasma samples were diluted 1: 20. Statistical analysis
Results are reported as mean _+ standard error of mean (SEM). The student's t-test was used for the comparison of means. Correlation coefficients were evaluated according to Pearson. Results were considered significant when P_<0.05.
Immunocapture assay for tPA and uPA
The uPA and tPA activities of CSF and plasma were measured by an immunocapture assay as previously reported. 23'25'26 Briefly, polystyrene microplate wells (Nunc, Roskilde, Denmark) were coated overnight at Fibrinolysis & Proteolysis (1997) 11(2), 109-113
RESULTS
Samples were qualitatively screened for both uPA and tPA activity by zymography and positive samples were quantitated by the immunocapture assay. Positive samples © Pearson Professional Ltd 1997
Plasminogen activators, PAl- 1 and multiple sclerosis
were seen as lyric bands of tPA at molecular weight of 70 kDa and uPA at molecular weight of 50 kDa (data not shown). We observed significantly higher mean CSF levels of tPA (P< 0.001) and PAI-1 (P= 0.048) in patients with MS, in comparison with the reference subjects (Table). Nine of 19 CSF samples and 6 of 8 plasma samples of MS patients had quantifiable uPA activity, whereas reference samples did not. The CSF tPA activity correlated positively with age in the reference subjects (r=0.61; P=0.011) (Fig. 2), whereas no correlation was observed in MS. This implies a break-down of age-related correlation in the event of disease. There was no association between the CSF PAI-1 and age either in the patients or the reference subjects. We observed a significant negative association between the CSF and plasma (n= 8) PAI-1 in the MS patients (P<0.05; r=-0.64) (Fig. 1). Finally, plasma PAI-1 and tPA values were within reference ranges in the MS patients.
Table CSF and plasma values for tPA, uPA and PAl-1 with their P values in comparison with the reference subjects (mean _+SEM) MS (n = 19)
REF (n = 20)
tPA(mlU/ml) CSF Plasma
360 _+24*** 19.1 _+5.9
43.7 _+6 23.6 _+8
uPA(mlU/ml) CSF Plasma
5.6 -+ 1.6 19.2 -+ 6.7
<0.01 <0.01
PAl-1 (ng/ml) CSF Plasma
0.65 _+0.1"* 11.3 -+ 2.4***
0.31 +_0.1 4.5 -+ 0.7
**P< 0.05 and *** P< 0.001; MS, multiple sclerosis; REF, reference subjects. Plasma : MS : n= 8; REF : n= 9.
© Pearson Professional Ltd 1997
I , . .
I , ,
,
I
.
..
I , , ,
I
,
.
0
1.1 O
~0.9 7 0.7 < 0.5
0.3
O
'
''1
•
"'1
9
'
' ' 1 "
•'
i ' '
'1"
'
' 1 " "
'
i " ' "
i
11 13 15 Plasma PAI-1 (ng/ml)
'
'
•
17
Fig. 1 Regression plot of CSF PAl-1 levels against plasma PAl-1 levels in MS patients (P< 0.05; r=-0.64).
,
100
DISCUSSION
The mean CSF tPA activity in patients with MS was raised and significantly higher than those in the reference subjects. MS is a chronic disease of the central nervous system of largely unknown aefiology affecting young and middle-aged adults. The myelin sheaths surrounding nerves in the brain and spinal cord are damaged, which affects the function of the nerves involved. The disease affects different parts of the brain and spinal cord, resulting in typically scattered symptoms. The involvement of PA induction in the pathogenesis of MS has been proposed in studies involving peripheral blood lymphocytes. 3° The authors also reported the disappearance of PA induction in association with improvement of neuro-
, , ,
1.3
111
E
,
,
i
,
,
t
l
l
l
,
l
,
,
,
I
,
,
,
I
,
,
,
I
,
,
O
80
,
I
,
,
, O
O
o
60 < 40 0
20 0
0
0
1...l ............................... 0
20
40 60 AGE (years)
80
Fig. 2 Regression plot of CSF tPA activity against age in reference subjects (P= 0.011 ; r= 0.61).
logic symptoms. 3° Recemly, tPA has been associated with neuronal cell death and neurodegeneration, 31 which is in agreement with our finding that tPA may be involved in the pathogenesis of MS. Endothelial activation by proteinases and cytokines plays a central role in the pathogenesis of MS. 30'32-36 Activation of endothelial cells in patients with encephalomyelitis by vasoactive amines and proteinases induces vasospasm and breakdown of the blood-brain barrier. 32 Autoimmune encephalomyelitis is t h o u g h t to share the same mechanism of tissue destruction with MS. 32 Endothelial cells are known to produce and secrete tPA, 37 The significance of tPA gene expression in the developing brain is not clearly understood. 18-al In the adult rat brain, transcription of the tPA gene is an immediate early Fibrinolysis & Proteolysis (1997) 1 I(2), 109-113
112
Akenami et al.
r e s p o n s e in t h e h i p p o c a m p u s following t r e a t m e n t s t h a t i n d u c e n e u r o n a l plasticity.18'21 tPA is t h e m a i n PA associa t e d w i t h t h e rat b r a i n g r o w t h cones w h i c h s u p p o r t s t h e view t h a t it is r e q u i r e d for n e u r i t e growth. 19 tPA expression d u r i n g e m b r y o g e n e s i s h a s b e e n d e t e r m i n e d b y in situ h y b r i d i z a t i o n . 2° It s e e m s t h a t tPA is e x p r e s s e d b y a n u m b e r of different cell t y p e s in t h e d e v e l o p i n g n e r v o u s s y s t e m a n d m a y p l a y a role for cell m i g r a t i o n a n d tissue r e m o d e l l i n g in later life as well. Nine of 19 CSF samples a n d 6 of 8 p l a s m a s a m p l e s of MS patients h a d quantifiable uPA activity. Some samples w h i c h h a d d e t e c t a b l e uPA activity b y z y m o g r a p h y , h a d n o m e a s u r a b l e activity b y i m m u n o c a p t u r e assay. Such results can occur if t h e uPA in t h e s a m p l e is n o n c o v a l e n t l y b o u n d either to an i n h i b i t o r or to a n o t h e r p r o t e i n inhibiting t h e b i n d i n g of uPA to t h e i m m u n o c a p t u r e plate. 38 W h i l e t h e m e a n p l a s m a PAI-1 level in MS was n o t different from levels f o u n d in n o r m a l individuals, 3 t h a t of t h e CSF was m o d e r a t e l y e l e v a t e d in c o m p a r i s o n w i t h t h e reference subjects. CSF PAI-1 m a y b e d e r i v e d from t h e plasma, b r a i n or b o t h . Substances are t r a n s p o r t e d across t h e b l o o d - b r a i n barrier b y v a r i o u s active a n d passive m e c h a n i s m s . It h a s b e e n s u g g e s t e d that, in n o r m a l individuals, PAI-1 is s e c r e t e d b y t h e c h o r o i d p l e x u s t o g e t h e r with m i n u t e levels s y n t h e s i z e d b y t h e CNS a n d secreted into t h e CSF. 39 CSF P A I l levels are raised in several n e u rological diseases 16 a n d n e o p l a s t i c astrocytes h a v e b e e n v a r i o u s l y r e p o r t e d to secrete PAI-1.40-42 CSF tPA a c t i v i t y c o r r e l a t e d p o s i t i v e l y w i t h age in t h e r e f e r e n c e s , w h e r e a s n o c o r r e l a t i o n was o b s e r v e d in MS. T h e s i g n i f i c a n t n e g a t i v e a s s o c i a t i o n b e t w e e n t h e CSF a n d p l a s m a PAI-1 in p a t i e n t s w i t h MS ( P < 0 . 0 5 ; r = - 0 . 6 4 ) s u g g e s t s t h a t PAI-1 m a y n o t cross t h e b l o o d b r a i n b a r r i e r b y p a s s i v e t r a n s p o r t only. A s i g n i f i c a n t n e g a t i v e a s s o c i a t i o n b e t w e e n tPA a n d PAI-1 activities h a s b e e n r e p o r t e d . 43 H o w e v e r , p r o b a b l y b e c a u s e w e m e a s u r e d t o t a l PAI-1 ( b o t h free a n d c o m p l e x e d ) , t h e n e g a t i v e a s s o c i a t i o n w h i c h w e o b s e r v e d was n o t significant. 43 This s u g g e s t s t h a t it is t h e active PAI-1 t h a t is i m p o r t a n t in r e g u l a t i n g tPA a c t i v i t y b o t h in t h e p l a s m a a n d CSF. T h e role of t h e e l e v a t e d activity of CSF tPA in t h e p a t h o g e n e s i s of MS is n o t clear. Our l a b o r a t o r y is curr e n t l y s t u d y i n g t h e level of tPA mRNA e x p r e s s i o n in b r a i n sections of patients t h a t d i e d of MS in c o m p a r i s o n w i t h subjects t h a t d i e d of n o n - n e u r o l o g i c a l causes a n d this will p r o b a b l y give us a l e a d to further studies o n MS.
ACKNOWLEDGEMENT This w o r k was s u p p o r t e d in part b y t h e University of Helsinki, Finland; t h e M e d i c a l Research C o u n c i l of t h e A c a d e m y of F i n l a n d a n d t h e Helsinki U n i v e r s i t y Central H o s p i t a l Research Funds, Finland. Fibrinolysis & Proteolysis (1997) 11(2), 109-113
REFERENCES 1. Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381-387. 2. Loskutoff DJ. Type 1 plasminogen activator inhibitor and its potential influence on thrombolytic therapy. Semin Thromb Hemost 1988; 14: 100-109. 3. Kruithof EKO. Plasminogen activator inhibitors - a review. Enzyme 1988; 40:113-121. 4. Dan~ K, Andreasen PA, Grondahl-Hansen J, Kristensen K, Nielsen L, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res 1985; 44: 139-266. 5. P611~nenJ, Stephens RW, Vaheri A. Directed plasminogen activation at the surface of normal and malignant cells. Adv Cancer Res 1991; 57: 273-328. 6. Ridker PM, Vaughan DE, Stampfer MK, Manson JE, Hennekens CH. Endogenous tissue-type plasminogen activator and risk of myocardial infarction. Lancet 1993; 341 : 1165-1168. 7. Ridker PM, Hennekens CH, Stampfer MJ, Manson JE, Vaughan DE. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet 1994; 343: 940-943. 8. Salomaa W , Wu KK, Stinson VL et al. The association of fibrinolytic activity with asymptomatic carotid atherosclerosis: the ARIC study. Circulation 1993; 87: 22. 9. Hamsten A, Wimon B, DeFaire U, Blomback M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1985; 313: 1557-1563. 10. Hamsten A, Walldius G, Szamosi A et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; ii: 3-9. 11. Short CD, Durrington PN, Mallick NP, Bhatnagar D, Hunt NP, Mbewu A. Serum lipoprotein (a) in men with proteinuria due to idiopathic membranous nephropathy. Nephrol Dial Transplant 1992; 7 Suppl 1: 109-113. 12. Bartens W, Wanner C. Lipoprotein (a): new insights into an atherogenic lipoprotein. Clin Investigator 1994; 72: 558-567. 13. Chapman MJ, Huby T, Nigon F, Thillet J. Lipoprotein (a): implication in atherothrombosis. Atherosclerosis 1994; 110 Suppl: S 69-75. 14. Gijtenbeek JM, Emeis JJ, Van-der-Sande JJ, Lulf RE. Different types of plasminogen activator activity in human brain tumours: relation with peritumoral oedema? Clin Neurol Neurosurg 1994; 96: 305-309. 15. Sawaya R, Rayford A, Kono Set al. Plasminogen activator inhibitor-1 in the pathogenesis of delayed radiation damage in rat spinal cord in vivo. J Neurosurg 1994; 81: 381-387. 16. Sutton R, Keohane ME, Van der Berg SR, Gonias SL. Plasminogen activator inhibitor-1 in the cerebrospinal fluid as an index of neurological disease. Blood Coagul Fibrinolysis 1994; 5: 167-171. 17. De Vries TJ, Mooy CM, Van Balken MR et al. Components of the plasminogen activation system in uveal melanoma - a clinico-pathologicalstudy. J Pathol 1995; 175: 59-67. 18. Carroll PM, Tsirka SE, Richards WG, Frohman MA, Strickland S. The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizureenhanced response in the CNS. Development 1994; 120: 3173-3183. 19. Garcia-Rocha M, Avila J, Armas-Porte R. Tissue-type plasminogen activator (tPA) is the main plasminogen activator associated with isolated rat nerve growth cones. Neurosci Lett 1994; 180: 123-126. 20. Friedman GC, Seeds NW. Tissue plasminogen activator expression in the embryonic nervous system. Brain Res Dev Brain Res 1994; 81: 41-49. 21. Seeds NW, Williams BL, Bickford PC. Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning. Science 1995; 270: 1992-1994. 22. Bizik J, Stephens RW, Grofova M, Vaheri A. Binding of tissuetype plasminogen activator to human melanoma cells. J Cell Biochem 1993; 5l: 326-335. © Pearson Professional Ltd 1997
Plasminogen activators, PAl- 1 and multiple sclerosis
23. Akenami FOT, Sir6n V, Koskiniemi M, Siimes MA, Teriiv~iinen H, Vaheri A. Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases. J Clin Pathol 1996; 49: 577-580. 24. LaemmliUK. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 1970; 227: 680-682. 25. Stephens R, Leung KC, P611/inen J, Salonen EM, Vaheri A. Microplate immunocapture assay for plasminogen activators and their specific inhibitors. J Immun Meth 1987; 105: 245-251. 26. Stephens R, Alitalo R, Tapiovaara H, Vaheri A. Production of an active urokinase by leukemia cells: A novel distinction from cell lines of active tumors. Leukemia Res 1988; 12: 419-422. 27. Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1097. 28. Green GDG, Shaw E. Thiobenzyloxycarbonyl-L-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes. Anal Biochem 1979; 93: 223-226. 29. Akenami FOT, Koskiniemi M, F/irkkil/i M, Vaheri A. Cerebrospinal fluid plasminogen activator inhibitor-1 in patients with neurological diseases. J Clin Pathol 1997; 50:157-160. 30. Sugita K, Suzuki N, Shimizu N, Takanashi J, Ishii M, Niimi N. Involvement of cytokines in N-methyl-N'-nitro-Nnitrosoguanidine-induced plasminogen activator activity in acute disseminated encephalomyelitis and multiple sclerosis lymphocytes. Eur Neurol 1993; 33: 358-362. 31. Tsirka SE, Rogove AD, Strickland S. Neuronal cell death and tPA. Nature 1996; 384: 123-124. 32. Norton WT, Brosnan CF, Cammer W, Goldmuntz EA. Mechanisms and suppression of inflammatory demyelination. Acta Neurobiol Exp Warsz 1990; 50: 225-235. 33. Dore-Duffy P, Donovan C, Todd RF 3rd. Expression of monocyte activation antigen Mo3 on the surface of peripheral blood monocytes from patients with multiple sclerosis. Neurology 1992; 42: 1609-1614.
© Pearson Professional Ltd 1997
113
34. Dore-Duffy P, Washington R, Dragovic L. Expression of endothelial cell activation antigens in microvessels from patients with multiple sclerosis. Adv Exp Med Biol 1993; 331: 243-248. 35. Washington R, Burton J, Todd RF 3rd, Newman W, Dragovic L, Dore-Duffy P. The expression of immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis. Ann Neurol 1994; 35: 89-97. 36. Romanic AM, Madri JA. The extraceflular matrix-degrading proteinases in the nervous system. Brain Pathol 1994; 4: 145-156. 37. Niedbala MJ. Cytokine regulation of endothelial cell extracellular proteolysis. Agents Actions Suppl 1993; 42: 179-193. 38. Stephens RW, Tapiovaara H, Reisberg T, Bizik J, Vaheri A. Mpha-a-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells. Cell Regul 1991; 2: 1057-1065. 39. Rao JS, Chen M, Festoff BW. Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in the normal human cerebrospinal fluid. J Neurosci Res 1993; 34: 340-345. 40. Andreasen PA, Christensen TH, Huang J-Y, Nielsen IS, Wilson EL, Dano K. Hormonal regulation of extracellular plasminogen activators and Mr-54,000 plasminogen activator in human neoplastic cell lines, studied with monoclonal antibodies. Mol Cell Endocrinol 1986; 45: 137-147. 41. Keohanne ME, Hall SW, Vandenberg SR, Gonias SL. Secretion of ~2-macroglobulin, ~2-antiplasmin and plasminogen activator inhibitor 1 by glioblastoma multiforme in primary organ in culture. J Neurosurg 1990; 73: 234-241. 42. Murphy P, Heart DA. Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators. Exp Cell Res 1992; 198: 93-100. 43. R&nby M, Sundell IB, Nflsson TK. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal LPA activity. Thromb Haemost 1989; 62:917-922.
Fibrinolysis & Proteolysis (1997) 11(2), 109-113
Plasma and cerebrospinal fluid activities of tissue plasminogen activator, urokinase and plasminogen a c t i v a t o r inhibitor-1 in multiple sclerosis F. O. 1". AkenamP, M. Koskiniemi 1, S. MustjokP, V. Sirdn 1, M. Fiirkkilii 2, A. VaherP 1Haartman Institute, Department of Virology, FIN-00014 University of Helsinki, Finland. 2Department of Neurology, FIN-00014 University of Helsinki, Finland.
Summary
Objective: To study plasma and cerebrospinal fluid (CSF) levels of tissue plasminogen activator (tPA), urokinase (uPA) and plasminogen activator inhibitor-1 (PAl-l) in multiple sclerosis (MS) patients. Design: Patients diagnosed as, or suspected of having, MS were involved in this study. The reference subjects had lumbar punctures for clinical reasons but were exclusive of having either MS or other types of neurological diseases. The identities of MS and reference samples were unknown to the researcher until laboratory results were ready. Setting: Department of Neurology, University of Helsinki, Finland. Subjects and Methods: tPA, uPA and PAl-1 levels were studied by an immunocapture assay, zymography and enzyme immunoassay in 19 patients with MS and 20 reference subjects. Results: Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. We observed significantly higher mean levels of CSF tPA (MS: 360+_24 mlU/ml, REF: 43.7+_6 mlU/ml; P< 0.001) and PAl-1 (P= 0.048) in patients with MS, in comparison with the reference subjects. Plasma PAl-1 and tPA values were within reference ranges in the patients. Although the CSF tPA activity correlated positively with age in the reference subjects (P= 0.011 ; r= 0.61), no correlation was observed in MS. There was no association between the CSF PAl-1 and age either in the patients or the reference subjects. Nine of 19 CSF samples and 6 of 8 plasma samples of MS patients had quantifiable uPA activity, whereas reference samples did not. Conclusions: CSF tPA activity appears raised in MS patients. The assessment of tPA activity and its specific inhibitor may be useful for understanding the pathogenesis/prognosis of MS and similar neurological diseases.
INTRODUCTION Plasminogen activators (PA), tissue-type (tPA) and urokinase-type (uPA) are serine proteases whose function is the conversion of the inactive zymogen plasminogen into the active protease plasmin. Plasminogen activator inhibitor-1 (PM-1), a 50-54 kDa serpin protease inhibitor, is a major inhibitor of tPA and uPA. 1-3 PAs have been implicated in a number of biological processes including Received 10 December 1996 Accepted after revision 13 March 1997 Correspondence to: Francis Akenami, MSc, Tel: +358-9-434 6480; Fax: +3589-434 6491; E-mail: Francis.Akenami@helsinkLfL
fibrinolysis and thrombolysis, cell migration, tissue invasion by both normal and malignant cells, and tissue remodelling. 4'5 High concentrations of vascular tPA antigen 6-8 and PAI-19'1° are strongly associated with the risk of myocardial infarction and stroke, tPA antigen concentrations have also been found to correlate with the existence of carotid atherosclerosis. 8 Lipoprotein (a) has been reported to exert an atherogenic effect by inhibiting plasminogen activation.11-13 Moreover, roles of PAs and PM-1 in the pathogenesis of neurological diseases and tumour development have been proposed.14-17 tPA gene is expressed in the developing brain, 18-2° during cerebellar motor learning 21 and in h u m a n tumour cells of neuroectodermal origin. 22 109
110
Akenami et al.
In a preliminary study, we observed a spectacular elevation of CSF tPA activity in MS patients in comparison with patients suffering other types of neurological diseases and subjects without neurological disease. 23 Therefore, we conducted this follow-up to include a larger population, and plasma, CSF tPA, uPA and PAId. MATERIALS AND METHODS
The procedures followed were in accord with the Helsinki Declaration of 1975, as revised in 1983, for human experimentation. This study involved 19 MS patients aged 18 to 54 years undergoing spinal taps for clinical reasons, from whom about 1 ml each of CSF was drawn into plain bijou bottles. Twenty subjects without neurological diseases, aged from 1 to 74 years, served as reference. Only samples which were clear and free of blood cells were included for this study. Blood (2.5-3 ml) was collected by venipuncture into EDTA tube. Plasma samples (MS: n=8; reference subjects: n=9) were prepared by centrifuging the blood at about 2 500 x g for 30 min. Samples were assayed immediately for tPA and uPA activities, and were kept frozen at -70°C until they were assayed for PAI-1.
37°C with 50 gl of a solution of rabbit or goat antih u m a n IgG antibodies to either h u m a n uPA (10 gg/ml) or tPA (10gg/ml) (cat. no. 389 and 387, respectively; American Diagnostica). The plates were washed three times with PBS containing 0.05% Tween 20 (PBS/Tween) followed by dispensing 50 gl of CSF or two-fold diluted plasma and were allowed to bind for 2 h at room temperature. After binding, the wells were washed again (three times with PBS/Tween), and h u m a n plasminogen containing traces of plasmin was added to assay the proenzyme activity bound to the wells (2 gg in 50 gl uPA assay buffer consisting of 50 mM glycine pH 7.8, 0.1% Triton X-100, 0.1% gelatin, and 10 mM 6-aminocaproic acid). Plasminogen was purified from fresh h u m a n plasma by affinity chromatography on lysine-Sepharose. 2z The reaction with plasminogen was allowed to proceed for 45 rain at 37°C and the plasmin produced was assayed by its thioesterase activity. 2s To estimate the amount of enzyme produced, high molecular weight two-chain uPA (80 000 IU/mg; American Diagnostica) and twochain tPA (650 000 IU/mg; American Diagnostica) were used as standards. PAl-1 assays
Zymography
Zymography was done as earlier reportedY Briefly, electrophoresis was run in 8% polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS) under non-reducing conditions. 24 The molecular weights of the lysed bands were estimated by making use of prestained low molecular weight marker proteins (Pharmacia, Uppsala, Sweden). Activity standards of uPA (Calbiochem, La Jolla, CA, USA) and tPA (American Diagnostica, Greenwich, CT, USA) were also included. Before zymography, SDS was removed by washing the gel for 6 h with phosphate-buffered saline, pH 7.4 (PBS) containing 2.5% Triton X-100. An agarose gel containing casein and 1.7 gg/ml plasminogen was then placed over the polyacrylamide gel, and the overlay incubated for 24-48 h at 37°C in a humidified chamber. To further characterize the proteinase present, an anticatalytic monoclonal antibody against uPA 10gg/ml (cat. No. 394; American Diagnostica) was added over the polyacrylamide gel before incubation with the caseinagarose gel.
PAI-1 was assayed as previously reported. 29 Briefly, enzyme immunoassay (EL/k) was used as recommended by the manufacturer (Monozyme, Horsholm, Denmark). The assay detected both free (active) and complexed (inactive) PAI-1. The concentration of PAI-1 was determined by comparing the absorbance for each well with a series of absorbance values obtained from known plasma concentrations of PAI-1, plotted as a standard curve. The standard plasma, provided by the manufacturer, with a concentration of 8 ng/ml was diluted with the appropriate buffer to cover the range: 800 pg/ml to 25 pg/ml. Each assay was made in duplicate and the final result was taken as the average of the two determinations. Plasma samples were diluted 1: 20. Statistical analysis
Results are reported as mean _+ standard error of mean (SEM). The student's t-test was used for the comparison of means. Correlation coefficients were evaluated according to Pearson. Results were considered significant when P_<0.05.
Immunocapture assay for tPA and uPA
The uPA and tPA activities of CSF and plasma were measured by an immunocapture assay as previously reported. 23'25'26 Briefly, polystyrene microplate wells (Nunc, Roskilde, Denmark) were coated overnight at Fibrinolysis & Proteolysis (1997) 11(2), 109-113
RESULTS
Samples were qualitatively screened for both uPA and tPA activity by zymography and positive samples were quantitated by the immunocapture assay. Positive samples © Pearson Professional Ltd 1997
Plasminogen activators, PAl- 1 and multiple sclerosis
were seen as lyric bands of tPA at molecular weight of 70 kDa and uPA at molecular weight of 50 kDa (data not shown). We observed significantly higher mean CSF levels of tPA (P< 0.001) and PAI-1 (P= 0.048) in patients with MS, in comparison with the reference subjects (Table). Nine of 19 CSF samples and 6 of 8 plasma samples of MS patients had quantifiable uPA activity, whereas reference samples did not. The CSF tPA activity correlated positively with age in the reference subjects (r=0.61; P=0.011) (Fig. 2), whereas no correlation was observed in MS. This implies a break-down of age-related correlation in the event of disease. There was no association between the CSF PAI-1 and age either in the patients or the reference subjects. We observed a significant negative association between the CSF and plasma (n= 8) PAI-1 in the MS patients (P<0.05; r=-0.64) (Fig. 1). Finally, plasma PAI-1 and tPA values were within reference ranges in the MS patients.
Table CSF and plasma values for tPA, uPA and PAl-1 with their P values in comparison with the reference subjects (mean _+SEM) MS (n = 19)
REF (n = 20)
tPA(mlU/ml) CSF Plasma
360 _+24*** 19.1 _+5.9
43.7 _+6 23.6 _+8
uPA(mlU/ml) CSF Plasma
5.6 -+ 1.6 19.2 -+ 6.7
<0.01 <0.01
PAl-1 (ng/ml) CSF Plasma
0.65 _+0.1"* 11.3 -+ 2.4***
0.31 +_0.1 4.5 -+ 0.7
**P< 0.05 and *** P< 0.001; MS, multiple sclerosis; REF, reference subjects. Plasma : MS : n= 8; REF : n= 9.
© Pearson Professional Ltd 1997
I , . .
I , ,
,
I
.
..
I , , ,
I
,
.
0
1.1 O
~0.9 7 0.7 < 0.5
0.3
O
'
''1
•
"'1
9
'
' ' 1 "
•'
i ' '
'1"
'
' 1 " "
'
i " ' "
i
11 13 15 Plasma PAI-1 (ng/ml)
'
'
•
17
Fig. 1 Regression plot of CSF PAl-1 levels against plasma PAl-1 levels in MS patients (P< 0.05; r=-0.64).
,
100
DISCUSSION
The mean CSF tPA activity in patients with MS was raised and significantly higher than those in the reference subjects. MS is a chronic disease of the central nervous system of largely unknown aefiology affecting young and middle-aged adults. The myelin sheaths surrounding nerves in the brain and spinal cord are damaged, which affects the function of the nerves involved. The disease affects different parts of the brain and spinal cord, resulting in typically scattered symptoms. The involvement of PA induction in the pathogenesis of MS has been proposed in studies involving peripheral blood lymphocytes. 3° The authors also reported the disappearance of PA induction in association with improvement of neuro-
, , ,
1.3
111
E
,
,
i
,
,
t
l
l
l
,
l
,
,
,
I
,
,
,
I
,
,
,
I
,
,
O
80
,
I
,
,
, O
O
o
60 < 40 0
20 0
0
0
1...l ............................... 0
20
40 60 AGE (years)
80
Fig. 2 Regression plot of CSF tPA activity against age in reference subjects (P= 0.011 ; r= 0.61).
logic symptoms. 3° Recemly, tPA has been associated with neuronal cell death and neurodegeneration, 31 which is in agreement with our finding that tPA may be involved in the pathogenesis of MS. Endothelial activation by proteinases and cytokines plays a central role in the pathogenesis of MS. 30'32-36 Activation of endothelial cells in patients with encephalomyelitis by vasoactive amines and proteinases induces vasospasm and breakdown of the blood-brain barrier. 32 Autoimmune encephalomyelitis is t h o u g h t to share the same mechanism of tissue destruction with MS. 32 Endothelial cells are known to produce and secrete tPA, 37 The significance of tPA gene expression in the developing brain is not clearly understood. 18-al In the adult rat brain, transcription of the tPA gene is an immediate early Fibrinolysis & Proteolysis (1997) 1 I(2), 109-113
112
Akenami et al.
r e s p o n s e in t h e h i p p o c a m p u s following t r e a t m e n t s t h a t i n d u c e n e u r o n a l plasticity.18'21 tPA is t h e m a i n PA associa t e d w i t h t h e rat b r a i n g r o w t h cones w h i c h s u p p o r t s t h e view t h a t it is r e q u i r e d for n e u r i t e growth. 19 tPA expression d u r i n g e m b r y o g e n e s i s h a s b e e n d e t e r m i n e d b y in situ h y b r i d i z a t i o n . 2° It s e e m s t h a t tPA is e x p r e s s e d b y a n u m b e r of different cell t y p e s in t h e d e v e l o p i n g n e r v o u s s y s t e m a n d m a y p l a y a role for cell m i g r a t i o n a n d tissue r e m o d e l l i n g in later life as well. Nine of 19 CSF samples a n d 6 of 8 p l a s m a s a m p l e s of MS patients h a d quantifiable uPA activity. Some samples w h i c h h a d d e t e c t a b l e uPA activity b y z y m o g r a p h y , h a d n o m e a s u r a b l e activity b y i m m u n o c a p t u r e assay. Such results can occur if t h e uPA in t h e s a m p l e is n o n c o v a l e n t l y b o u n d either to an i n h i b i t o r or to a n o t h e r p r o t e i n inhibiting t h e b i n d i n g of uPA to t h e i m m u n o c a p t u r e plate. 38 W h i l e t h e m e a n p l a s m a PAI-1 level in MS was n o t different from levels f o u n d in n o r m a l individuals, 3 t h a t of t h e CSF was m o d e r a t e l y e l e v a t e d in c o m p a r i s o n w i t h t h e reference subjects. CSF PAI-1 m a y b e d e r i v e d from t h e plasma, b r a i n or b o t h . Substances are t r a n s p o r t e d across t h e b l o o d - b r a i n barrier b y v a r i o u s active a n d passive m e c h a n i s m s . It h a s b e e n s u g g e s t e d that, in n o r m a l individuals, PAI-1 is s e c r e t e d b y t h e c h o r o i d p l e x u s t o g e t h e r with m i n u t e levels s y n t h e s i z e d b y t h e CNS a n d secreted into t h e CSF. 39 CSF P A I l levels are raised in several n e u rological diseases 16 a n d n e o p l a s t i c astrocytes h a v e b e e n v a r i o u s l y r e p o r t e d to secrete PAI-1.40-42 CSF tPA a c t i v i t y c o r r e l a t e d p o s i t i v e l y w i t h age in t h e r e f e r e n c e s , w h e r e a s n o c o r r e l a t i o n was o b s e r v e d in MS. T h e s i g n i f i c a n t n e g a t i v e a s s o c i a t i o n b e t w e e n t h e CSF a n d p l a s m a PAI-1 in p a t i e n t s w i t h MS ( P < 0 . 0 5 ; r = - 0 . 6 4 ) s u g g e s t s t h a t PAI-1 m a y n o t cross t h e b l o o d b r a i n b a r r i e r b y p a s s i v e t r a n s p o r t only. A s i g n i f i c a n t n e g a t i v e a s s o c i a t i o n b e t w e e n tPA a n d PAI-1 activities h a s b e e n r e p o r t e d . 43 H o w e v e r , p r o b a b l y b e c a u s e w e m e a s u r e d t o t a l PAI-1 ( b o t h free a n d c o m p l e x e d ) , t h e n e g a t i v e a s s o c i a t i o n w h i c h w e o b s e r v e d was n o t significant. 43 This s u g g e s t s t h a t it is t h e active PAI-1 t h a t is i m p o r t a n t in r e g u l a t i n g tPA a c t i v i t y b o t h in t h e p l a s m a a n d CSF. T h e role of t h e e l e v a t e d activity of CSF tPA in t h e p a t h o g e n e s i s of MS is n o t clear. Our l a b o r a t o r y is curr e n t l y s t u d y i n g t h e level of tPA mRNA e x p r e s s i o n in b r a i n sections of patients t h a t d i e d of MS in c o m p a r i s o n w i t h subjects t h a t d i e d of n o n - n e u r o l o g i c a l causes a n d this will p r o b a b l y give us a l e a d to further studies o n MS.
ACKNOWLEDGEMENT This w o r k was s u p p o r t e d in part b y t h e University of Helsinki, Finland; t h e M e d i c a l Research C o u n c i l of t h e A c a d e m y of F i n l a n d a n d t h e Helsinki U n i v e r s i t y Central H o s p i t a l Research Funds, Finland. Fibrinolysis & Proteolysis (1997) 11(2), 109-113
REFERENCES 1. Sprengers ED, Kluft C. Plasminogen activator inhibitors. Blood 1987; 69: 381-387. 2. Loskutoff DJ. Type 1 plasminogen activator inhibitor and its potential influence on thrombolytic therapy. Semin Thromb Hemost 1988; 14: 100-109. 3. Kruithof EKO. Plasminogen activator inhibitors - a review. Enzyme 1988; 40:113-121. 4. Dan~ K, Andreasen PA, Grondahl-Hansen J, Kristensen K, Nielsen L, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res 1985; 44: 139-266. 5. P611~nenJ, Stephens RW, Vaheri A. Directed plasminogen activation at the surface of normal and malignant cells. Adv Cancer Res 1991; 57: 273-328. 6. Ridker PM, Vaughan DE, Stampfer MK, Manson JE, Hennekens CH. Endogenous tissue-type plasminogen activator and risk of myocardial infarction. Lancet 1993; 341 : 1165-1168. 7. Ridker PM, Hennekens CH, Stampfer MJ, Manson JE, Vaughan DE. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet 1994; 343: 940-943. 8. Salomaa W , Wu KK, Stinson VL et al. The association of fibrinolytic activity with asymptomatic carotid atherosclerosis: the ARIC study. Circulation 1993; 87: 22. 9. Hamsten A, Wimon B, DeFaire U, Blomback M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med 1985; 313: 1557-1563. 10. Hamsten A, Walldius G, Szamosi A et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; ii: 3-9. 11. Short CD, Durrington PN, Mallick NP, Bhatnagar D, Hunt NP, Mbewu A. Serum lipoprotein (a) in men with proteinuria due to idiopathic membranous nephropathy. Nephrol Dial Transplant 1992; 7 Suppl 1: 109-113. 12. Bartens W, Wanner C. Lipoprotein (a): new insights into an atherogenic lipoprotein. Clin Investigator 1994; 72: 558-567. 13. Chapman MJ, Huby T, Nigon F, Thillet J. Lipoprotein (a): implication in atherothrombosis. Atherosclerosis 1994; 110 Suppl: S 69-75. 14. Gijtenbeek JM, Emeis JJ, Van-der-Sande JJ, Lulf RE. Different types of plasminogen activator activity in human brain tumours: relation with peritumoral oedema? Clin Neurol Neurosurg 1994; 96: 305-309. 15. Sawaya R, Rayford A, Kono Set al. Plasminogen activator inhibitor-1 in the pathogenesis of delayed radiation damage in rat spinal cord in vivo. J Neurosurg 1994; 81: 381-387. 16. Sutton R, Keohane ME, Van der Berg SR, Gonias SL. Plasminogen activator inhibitor-1 in the cerebrospinal fluid as an index of neurological disease. Blood Coagul Fibrinolysis 1994; 5: 167-171. 17. De Vries TJ, Mooy CM, Van Balken MR et al. Components of the plasminogen activation system in uveal melanoma - a clinico-pathologicalstudy. J Pathol 1995; 175: 59-67. 18. Carroll PM, Tsirka SE, Richards WG, Frohman MA, Strickland S. The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizureenhanced response in the CNS. Development 1994; 120: 3173-3183. 19. Garcia-Rocha M, Avila J, Armas-Porte R. Tissue-type plasminogen activator (tPA) is the main plasminogen activator associated with isolated rat nerve growth cones. Neurosci Lett 1994; 180: 123-126. 20. Friedman GC, Seeds NW. Tissue plasminogen activator expression in the embryonic nervous system. Brain Res Dev Brain Res 1994; 81: 41-49. 21. Seeds NW, Williams BL, Bickford PC. Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning. Science 1995; 270: 1992-1994. 22. Bizik J, Stephens RW, Grofova M, Vaheri A. Binding of tissuetype plasminogen activator to human melanoma cells. J Cell Biochem 1993; 5l: 326-335. © Pearson Professional Ltd 1997
Plasminogen activators, PAl- 1 and multiple sclerosis
23. Akenami FOT, Sir6n V, Koskiniemi M, Siimes MA, Teriiv~iinen H, Vaheri A. Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases. J Clin Pathol 1996; 49: 577-580. 24. LaemmliUK. Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 1970; 227: 680-682. 25. Stephens R, Leung KC, P611/inen J, Salonen EM, Vaheri A. Microplate immunocapture assay for plasminogen activators and their specific inhibitors. J Immun Meth 1987; 105: 245-251. 26. Stephens R, Alitalo R, Tapiovaara H, Vaheri A. Production of an active urokinase by leukemia cells: A novel distinction from cell lines of active tumors. Leukemia Res 1988; 12: 419-422. 27. Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1097. 28. Green GDG, Shaw E. Thiobenzyloxycarbonyl-L-lysinate, substrate for a sensitive colorimetric assay for trypsin-like enzymes. Anal Biochem 1979; 93: 223-226. 29. Akenami FOT, Koskiniemi M, F/irkkil/i M, Vaheri A. Cerebrospinal fluid plasminogen activator inhibitor-1 in patients with neurological diseases. J Clin Pathol 1997; 50:157-160. 30. Sugita K, Suzuki N, Shimizu N, Takanashi J, Ishii M, Niimi N. Involvement of cytokines in N-methyl-N'-nitro-Nnitrosoguanidine-induced plasminogen activator activity in acute disseminated encephalomyelitis and multiple sclerosis lymphocytes. Eur Neurol 1993; 33: 358-362. 31. Tsirka SE, Rogove AD, Strickland S. Neuronal cell death and tPA. Nature 1996; 384: 123-124. 32. Norton WT, Brosnan CF, Cammer W, Goldmuntz EA. Mechanisms and suppression of inflammatory demyelination. Acta Neurobiol Exp Warsz 1990; 50: 225-235. 33. Dore-Duffy P, Donovan C, Todd RF 3rd. Expression of monocyte activation antigen Mo3 on the surface of peripheral blood monocytes from patients with multiple sclerosis. Neurology 1992; 42: 1609-1614.
© Pearson Professional Ltd 1997
113
34. Dore-Duffy P, Washington R, Dragovic L. Expression of endothelial cell activation antigens in microvessels from patients with multiple sclerosis. Adv Exp Med Biol 1993; 331: 243-248. 35. Washington R, Burton J, Todd RF 3rd, Newman W, Dragovic L, Dore-Duffy P. The expression of immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis. Ann Neurol 1994; 35: 89-97. 36. Romanic AM, Madri JA. The extraceflular matrix-degrading proteinases in the nervous system. Brain Pathol 1994; 4: 145-156. 37. Niedbala MJ. Cytokine regulation of endothelial cell extracellular proteolysis. Agents Actions Suppl 1993; 42: 179-193. 38. Stephens RW, Tapiovaara H, Reisberg T, Bizik J, Vaheri A. Mpha-a-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells. Cell Regul 1991; 2: 1057-1065. 39. Rao JS, Chen M, Festoff BW. Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in the normal human cerebrospinal fluid. J Neurosci Res 1993; 34: 340-345. 40. Andreasen PA, Christensen TH, Huang J-Y, Nielsen IS, Wilson EL, Dano K. Hormonal regulation of extracellular plasminogen activators and Mr-54,000 plasminogen activator in human neoplastic cell lines, studied with monoclonal antibodies. Mol Cell Endocrinol 1986; 45: 137-147. 41. Keohanne ME, Hall SW, Vandenberg SR, Gonias SL. Secretion of ~2-macroglobulin, ~2-antiplasmin and plasminogen activator inhibitor 1 by glioblastoma multiforme in primary organ in culture. J Neurosurg 1990; 73: 234-241. 42. Murphy P, Heart DA. Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators. Exp Cell Res 1992; 198: 93-100. 43. R&nby M, Sundell IB, Nflsson TK. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal LPA activity. Thromb Haemost 1989; 62:917-922.
Fibrinolysis & Proteolysis (1997) 11(2), 109-113