- Email: [email protected]
Route of activation, in vitro, of the factor XII-dependent pathway of fibrinolysis
Fibrinolysis(1994) 8, 172-176 9
Longman Group Ltd
Route of Activation, In Vitro, of the Factor XII-dependent Pathway of Fibrinolysis
C. Kluft, A. E H. Jie
SUMMARY Plasma has been shown to contribute to fibrinolysis in vitro by plasminogen activator activities
from tissue-type plasminogen activator, from urokinase-type plasminogen activator and from a factor XII-dependent pathway. The aim of the present study was to further outline the activation route for factor XII-dependent fibrinolysis. To this end effects of purified active enzymes added to dextran sulphate euglobulin fractions of plasma's deficient in factor XII (Hageman trait) or prekallikrein (Fletcher trait) were studied. In Hageman and Fletcher trait plasma, the activation of factor XII-dependent activator activity could be corrected by the addition of the activated factor, kailikrein. Activated, ~-factor XIIa was only effective in Hageman trait plasma. Additions of plasmin, tissue-type plasminogen activator or urokinase-type plasminogen activator were not effective in Hageman trait plasma. It is concluded that factor XII-dependent plasminogen activator activity is generated predominatly via kallikrein.
was present, which could be restored after addition of the missing component. 2'3 This circumstance and the availability of completely deficient plasma for factor XII (Hageman trait) and prekallikrein (Fletcher trait) gives rise to the possibility of studying the route of activation of the factor XII-dependent contribution to plasminogen activation.
In the process of fibrinolysis, the conversion of plasminogen to plasmin is achieved by limited proteolysis of a specific Arg-Val peptide bond in the plasminogen molecule. Two enzymes, the plasminogen activators tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) catalyze the above cleavage and show a high specific activity. 1 Another, third contribution to plasminogen activation comes from factor XII-dependent processes. In these processes, kallikrein, factor XIIa and factor XIa are also capable of plasminogen activation with, however, a significantly lower specific activity. 2 Additionally, a factor XII-dependent proactivator has been postulated to contribute with the quantitative major factor XII-related activity. 3 In the present study, we studied plasminogen activator activity, in vitro, in euglobulin fractions made with the addition of dextran sulphate, a potent activator of factor XII. 4 The so-called dextran sulphate euglobulin fraction (DEF) has previously been shown to be activated optimally, showing a maximal plasminogen activator activity of 100 BAU (blood activator units)/ml. 3'4 However, in plasma of patients deficient in factor XII or prekallikrein, a lower activity of around 50 BAU/ml
MATERIALS AND M E T H O D S Unless otherwise specified, reagents were of analytical grade and purchased from E. Merck, Darmstadt, Germany. Dextran sulphate, sodium salt, MW 500 000 was from Pharmacia Ltd., Uppsala, Sweden. Flufenamic acid was from Aldrich Europe, Beerse, Belgium. Human plasmin labelled 15 casein units/mg protein was from Kabi Diagnostica, Amsterdam, The Netherlands. Purified IgG against t-PA or u-PA were from Organon Teknika, Turnhout, Belgium. Bz-ProPhe-Arg-pNA.HC1 was from Pentapharm AG, Basel, Switzerland. Plasminogen-rich bovine fibrinogen was prepared according to Brakman. 5 Human two chain tPA was prepared from conditioned media of melanoma Bowes cells as described before. 6 High molecular weight urokinase (Mr = 54 000) was from Choay, Paris, France, and labelled in Ploug units (P1).
C. Kluft, A. F. H. Jie, Gaubius Laboratory, IVVO-TNO, Leiden, The Netherlands. 172
Fibrinolysis
Platelet-poor citrated plasma and pooled samples were prepared as described previously. 7 Factor XIIdeficient plasmas were obtained from the following sources: No. 1 in Table 1 from the Department of Haematology, University Hospital, Leiden, The Netherlands; No. 2 and 5 from George King Biomedical Inc., Overland Park, Kansas, USA; No. 3 through the courtesy of Dr H.L. Haak, the Hague, The Netherlands, No. 4 through the courtesy of Drs M.M. Samama and F. Perenet, Paris, France. Fletcher trait plasma (case 3 as described by Hattersley and Hayse 8 was from George King Biomedical Inc., Overland Park, Kansas, USA, lot 06-1701. The [~-factor XIIa (Mr 30000)95% pure, was a gift from Dr N. Heimburger, Behringwerke, Marburg, Germany. Plasma kallikrein was used as its active complex with kinin-free high molecular weight kininogen (gel filtration, Mr 300000), purified as previously described. 9 The DEF was prepared from a mixture of 1 ml plasma, 8 ml distilled water and 1 ml dextran sulphate (100 gg/ml in distilled water). Titration at 0~ with acetic acid to pH 5.9 was started immediately after mixing. After centrifugation in the cold, the precipitate was redissolved in 0.5 ml EDTA buffer (0.05 sodium diethylbarbiturate, 0.10 M NaCI, 0.25% (w/v) gelatin and 2.7 nM ethylene diamine tetraacetate (EDTA) adjusted to pH 7.8 with a HC1 solution). 4 Before assay, the DEF was diluted to a final volume of 1 ml, in some experiments with supplements (factor XIIa, kallikrein, t-PA, u-PA or plasmin) in EDTA buffer. Amounts for additions of IgG against uPA or t-PA were selected for optimal effect on DEF activity, as described. 9, io Plasminogen activator activity of the DEF was assayed on plasminogen-rich bovine fibrin plates, l J This activity is fully dependent on plasminogen. 4 Drops of 30 gl, supplemented with 5 lal of a 14 mM solution of flufenamate, were placed on the fibrin plates, which were incubated for 18 h at 37~ Diameters of lysed zones were intrapolated on a reference curve and activity was expressed in arbitrary BAU. 4 Kallikrein activity was measured on the synthetic tripeptide Bz-Pro-Phe-ArgpNA.HC1 as described. 3
Table 1. Factor Xll-dependent activator activity (BAU/ml)
Volunteers*
Mean SD:
49 40 35 51 40 39 31 41+7
Hageman trait** + [3-factor Xila 36 26 27 44 29 32+8
* Residual activity after addition of excesses of antibodies against t-PA and u-PA in the DEF of plasma of seven volunteers. ** Response to addition of 100% [3-factor Xlla (see Fig. I ) in the presence of excesses of antibodies against u-PA and t-PA. in the DEF of plasma of 5 different patients.
173
RESULTS Factor XII-dependent Activator Portion
The addition of both an excess of neutralizing antibodies against u-PA (see ref. 9) and against t-PA results in inhibition of the plasminogen activator activity of the DEF from 100 BAU/ml to a residual activity of around 40 BAU/ml. As shown in Table 1, 7 healthy volunteers show a residual (= factor XII-dependent) activity of 41+7 (SD) BAU/ml. In the DEF of 5 Hageman trait patients, activator activity was reduced, compared to normal (9+1 (SD) BAU/ml) in the presence of excesses of IgG against t-PA and u-PA. However, the addition of an excess of ~-factor XIIa induced the appearance of 32+8 (SD) BAU/ml of activator activity (Table 1). This activity appeared despite the presence of excesses of both neutralizing antibodies against t-PA and u-PA. The activator activity portions given in Table 1 are apparently unrelated to t-PA and u-PA and are designated as factor XII-dependent.
~-Factor XIIa Added to Contact Factor Deficient Plasmas
Addition of 13-factor XIIa to the DEF of Hageman trait plasma induced appearance of plasminogen activator activity, which was already maximal with the lowest concentration used (30%) (Fig. 1). 13-factor XIIa added to the DEF of normal plasma and Fletcher trait plasma does not elicit much plasminogen activator activity (Fig. 1). Only at very high concentrations, some effects of 13-factor XIIa become apparent (Fig. 1, panel B).
Kallikrein Added to Contact Factor Deficient Plasmas
Addition of kallikrein to the DEF of normal plasma showed a linear increase in plasminogen activator activity amounting to 10 to 15 BAU/ml/100% kallikrein (Fig.2). In the case of Hageman and Fletcher trait plasma, kallikrein induced in a similar experient more plasminogen activator activity than expected from its intrinsic activity revealed when added to normal plasma (Fig. 2).
Plasmin, u-PA or t-PA Added to Contact Factor Deficient Plasma
Results of additions of plasmin, u-PA or t-PA to DEFs of Hageman trait plasma were compared to results of these additions to DEFs of normal plasma (Fig. 3). Recovery of added activity in both situations was similar - no evidence of restoration of factor XII-dependent activity in Hageman trait plasma was apparent.
174
Factor XII-dependent Fibrinolysis
|
ACTIVATOR ACTIVITY
ACTIVATOR ACTIVITY (BAU/ml)
(BAU/ml NORMAL PLASMA
100,
f x
80
x
x
x
100
|
NORMAL PLASMA
HAGEMAN TRAIT
CHER TRAIT 60
.....----.0 FLETCHER TRAIT
50-
40
20
o
0 0
50
100 150 2 0 0 2 5 0 ~-FACTOR
Xlla (%)
0
5()0 1000 1500 13-FACTOR Xlla (%)
Fig. 1 Plasminogen activator activity changes induced by additions of [3-factor Xlla (abscissa) to the DEF of normal plasma ( 0 ) , Hageman trait plasma (x) or Fletcher trait plasma (O). Concentrations of added [3-factor Xlla are expressed in percentage relative to the normal plasma level of factor XII. Panel B shows the effect of additions of large amounts of [3-factor Xlla to normal (O) and Fletcher trait (O)-
DISCUSSION In accordance with previous report, 3'9'~2 the plasminogen activator activity of the DEF of plasma is shown to comprise a factor XII-dependent activator activity which is unrelated to u-PA and t-PA. The factor XIIdependent activator activity can be assayed in the DEF by using quenching antibodies against t-PA and u-PA, and amounts to about 40 BAU/ml of a total activity in a normal DEF of 100 BAU/ml. ACTIVATOR A C T I V I T Y (BAU/ml) 120
m•••O
NORMAL P L A S M A
~
100
80
60,
40
FLETCHER TRAIT EMAN TRAIT
20
0 0
50
100
150
K A L L I K R E I N (%)
Fig. 2 Plasminogen activator activity changes induced by additions of plasma kallikrein to the DEF of normal (O), Hageman trait plasma (x) or Fletcher trait plasma (C)). Concentrations of added kallikrein are expressed in percentage relative to the normal plasma level of prekallikrein.
The activation of the factor XII-dependent activator activity can be studied using congenitally deficient plasmas. Both Hageman and Fletcher traits show absence of the factor XII-dependent plasminogen activator activity. The restoration of activity in the these plasma can be elecited by addition of factor XIIa (Fig. 1) and kallikrein (Fig. 2), respectively. In both cases, the activator activity that appears is larger than the intrinsic plasminogen activating activity of the added factors. This phenomenon has formed the basis for postulation of the presence of an entity (proactivator) that needs activation 2'3 and is different from factor XII and prekallikrein. Evidence for a molecular entity that might represent this component has been reported. ~2.~3In the present report, we further defined the route of activation of this putative plasminogen proactivator. To circumvent the complication of the mutual dependence of factor XII and prekallikrein for their activation, we used purified activated components to reconstitute the deficient plasmas. The reconstitution could be done in the DEE but not in plasma (results not shown) because of the presence of protease inhibitors for the active enzymes in the plasma. In the DEE the reconstitution was followed by immediate addition of flufenamate, which further eliminates inhibitors (notably, C 1inactivator) in euglobulin fractions which are already poor in inhibitors. 14 Our results show that the factor XII-dependent activator activity can be restored in both Hageman and Fletcher trait plasma by addition of active kallikrein. This indicates firstly that active factor XII is not required, and secondly that kallikrein directly or indirectly activates the putative proactivator. It is further shown that the activation of factor XII-dependent activity cannot very effectively be achieved by p-factor XIIa when prekallikrein is absent (Fig. 1).
Fibrinolysis
ACTIVATOR ACTIVITY (BAU/rnl)
ACTIVATOR ACTIVITY (BAU/ml)
ACTIVATOR ACTIVITY (BAUIml)
200"
200'
200"
100
100
100
0
0
o
;
175
0
o
o.s
PLASMIN (CU/ml)
1.o
is
o
u-PA (PL/ml)
15
3o
I-PA (IUIrnl)
Fig. 3 Changes in fibrinolytic activity induced by additions of plasmin, u-PA or t-PA to the DEF of normal ( 0 ) or Hageman trait (C)) plasma. The effects of the additions are all converted to apparent activator activity expressed in BAU/ml using the dose-response curve for the DEF (see ref. 4). CU=Casein units; Pl=Plough units; IU=lnternational units.
Comparing the activation of the factor Xll-dependent fibrinolytic pathway, subject of the present study, and the activation of pro-u-PA, similarities and differences can be noted. Similarities concern the activation of both routes/components by kallikrein and weak activation by activated factor XII. 15 A clear difference is the ability of plasmin to activate pro-u-PA, while plasmin is ineffective in activating the factor XII-dependent process directly (in the absence of factor XII). Thus, although both mentioned pathways can be activated by kallikrein and might be called factor XII-related processes, pro-u-PA activation can proceed independently via plasmin, in contrast to the factor XII-dependent activation we studied, which is fully dependent upon factor XII/prekallikrein.
The addition of plasmin, t-PA or u-PA to Hageman trait plasma showed only recovery of added activity, similar to that of normal plasma. This indicates that neither plasmin, t-PA nor u-PA can replace kallikrein. Further, they cannot directly activate prekallikrein. It is therefore concluded that the factor XII-dependent plasminogen activator activity is generated predominantly by the route 'factor Xlla - kallikrein - fibrinolysis', as indicate in Figure 4. Also indicated in Figure 4 is that factor XII activation can elicit fibrinolysis activation by more than one route. In addition to the process studied in the present manuscript, generation of bradykinin in vivo can result in release of t-PA, 2 and kallikrein can also activate pro-u-PA. 15
FACTOR X,, FAOTi X"a KALLIKRE!N ~
BRADYKININ
PROACTIVATOR~
PREKALLIKREIN
ACTIVATOR
FIBRINOLYSIS
Fig. 4 Factor Xll-dependent fibrinolytic mechanisms.
PRO-u-PA ~
u-PA
176
Factor XII-dependent Fibrinolysis
T h u s o n l y the p a t h w a y s t u d i e d in the p r e s e n t i n v e s t i g a tion is really f a c t o r X I I - ' d e p e n d e n t ' .
REFERENCES I. Bachmann F. Fibrinolysis. In: Verstraete M, Vermylen J, Lijnen H R, Amour J, eds. Thrombosis and haemostasis. Leuven: International Society on Thrombosis and Haemostasis and Leuven University Press, 1987: 227-265. 2. Kluft C, Dooijewaard G, Emeis J J. Role of the contact system in fibrinolysis. Semin Thromb Haemostas 1987; 13: 50-68. 3. Kluft C, Trumpi-Kalshoven M M, Jie A F H, Veldhuyzen-Stolk E C. Factor XII-dependent fibrinolysis: A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII- and kallikrein-dependent plasminogen proactivator. Thromb Haemostas 1979; 41 : 756-773. 4. Kluft C. Studies on the fibrinolytic system in human plasma: Quantitative determination of plasminogen activators and proactivators. Thromb Haemostas 1979; 41: 365-383. 5. Brakman P. Fibrinolysis, a standardized fibrin plate method and a fibrinolytic assay of plasminogen [Thesis] Amsterdam: Scheltema & Holkema, 1967. 6. Kluft C, Van Wezel A L, Van der Velden C A M, Emeis J J, Verheijen J H, Wijngaards G. Large-scale production of extrinsic (tissue-type) plasminogen activator from human melanoma cells. In: Mizrahi A, Van Wezel A L, eds. Advances in biotechnological processes, Vol 2. New York: Alan R Liss, 1983: 97.
Received: I I January 1993 Accepted after revision: 7 December 1993 Offprint orders to" Dr C. Kluft, Gaubius Laboratory IVVO-TNO, PO Box 430, 2300 AK Leiden, The Netherlands.
7. Kluft C, Brakman P, Veldhuyzen-Stolk E C. Screening of fibrinolytic activity in plasma euglobulin fractions on the fibrin plate. In: Davidson J F, Samama M M, Desnoyers P C, eds. Progress in chemical fibrinolysis and thrombolysis, Vol 2. New York: Raven Press, 1976: 57. 8. Hattersley P G, Hayse D. Fletcher factor deficiency: A report of three unrelated cases. BrJ Haematol 1970; 18:411-416. 9. Kluft C, Wijngaards G, Jie A F H. Intrinsic plasma fibrinolysis: Involvement of urokinase-related activity in the factor XIIindependent plasminogen proactivator pathway. J Lab Clin Med 1984; 103: 408418. 10. Rijken D C, Wijngaards G, Welbergen J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb Res 1980; 18:815-830. I 1. Haverkate F, Brakman P. Fibrin plate assay. In: Davidson J F, Samama M M, Desnoyers P C, eds. Progress in chemical fibrinolysis and thrombolysis, Vol 1. New York: Raven Press, 1975: 151. 12. Binnema D J, Dooijewaard G, Van lersel J J L. Identification of two types of protein immunochemically related to urinary urokinase occurring in human plasma. Biochem Biophys Res Commun 1987; 142: 162-168. 13. Binnema D J, Dooijewaard G, Van Iersel J J L, Turion P N C, Kluft C. The contact-system dependent plasminogen activator from human plasma: Identification and characterization. Thromb Haemos 1990; 63: 390-397. 14. Kluft C. Elimination of inhibition in euglobulin fibrinolysis by use of flufenamate: Involvement of C 1-inactivator. Haemostasis 1977; 6:351-369. 15. lchinose A, Fujikawa K, Suyama T. The activation of prourokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 3486-3489.
Longman Group Ltd
Route of Activation, In Vitro, of the Factor XII-dependent Pathway of Fibrinolysis
C. Kluft, A. E H. Jie
SUMMARY Plasma has been shown to contribute to fibrinolysis in vitro by plasminogen activator activities
from tissue-type plasminogen activator, from urokinase-type plasminogen activator and from a factor XII-dependent pathway. The aim of the present study was to further outline the activation route for factor XII-dependent fibrinolysis. To this end effects of purified active enzymes added to dextran sulphate euglobulin fractions of plasma's deficient in factor XII (Hageman trait) or prekallikrein (Fletcher trait) were studied. In Hageman and Fletcher trait plasma, the activation of factor XII-dependent activator activity could be corrected by the addition of the activated factor, kailikrein. Activated, ~-factor XIIa was only effective in Hageman trait plasma. Additions of plasmin, tissue-type plasminogen activator or urokinase-type plasminogen activator were not effective in Hageman trait plasma. It is concluded that factor XII-dependent plasminogen activator activity is generated predominatly via kallikrein.
was present, which could be restored after addition of the missing component. 2'3 This circumstance and the availability of completely deficient plasma for factor XII (Hageman trait) and prekallikrein (Fletcher trait) gives rise to the possibility of studying the route of activation of the factor XII-dependent contribution to plasminogen activation.
In the process of fibrinolysis, the conversion of plasminogen to plasmin is achieved by limited proteolysis of a specific Arg-Val peptide bond in the plasminogen molecule. Two enzymes, the plasminogen activators tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) catalyze the above cleavage and show a high specific activity. 1 Another, third contribution to plasminogen activation comes from factor XII-dependent processes. In these processes, kallikrein, factor XIIa and factor XIa are also capable of plasminogen activation with, however, a significantly lower specific activity. 2 Additionally, a factor XII-dependent proactivator has been postulated to contribute with the quantitative major factor XII-related activity. 3 In the present study, we studied plasminogen activator activity, in vitro, in euglobulin fractions made with the addition of dextran sulphate, a potent activator of factor XII. 4 The so-called dextran sulphate euglobulin fraction (DEF) has previously been shown to be activated optimally, showing a maximal plasminogen activator activity of 100 BAU (blood activator units)/ml. 3'4 However, in plasma of patients deficient in factor XII or prekallikrein, a lower activity of around 50 BAU/ml
MATERIALS AND M E T H O D S Unless otherwise specified, reagents were of analytical grade and purchased from E. Merck, Darmstadt, Germany. Dextran sulphate, sodium salt, MW 500 000 was from Pharmacia Ltd., Uppsala, Sweden. Flufenamic acid was from Aldrich Europe, Beerse, Belgium. Human plasmin labelled 15 casein units/mg protein was from Kabi Diagnostica, Amsterdam, The Netherlands. Purified IgG against t-PA or u-PA were from Organon Teknika, Turnhout, Belgium. Bz-ProPhe-Arg-pNA.HC1 was from Pentapharm AG, Basel, Switzerland. Plasminogen-rich bovine fibrinogen was prepared according to Brakman. 5 Human two chain tPA was prepared from conditioned media of melanoma Bowes cells as described before. 6 High molecular weight urokinase (Mr = 54 000) was from Choay, Paris, France, and labelled in Ploug units (P1).
C. Kluft, A. F. H. Jie, Gaubius Laboratory, IVVO-TNO, Leiden, The Netherlands. 172
Fibrinolysis
Platelet-poor citrated plasma and pooled samples were prepared as described previously. 7 Factor XIIdeficient plasmas were obtained from the following sources: No. 1 in Table 1 from the Department of Haematology, University Hospital, Leiden, The Netherlands; No. 2 and 5 from George King Biomedical Inc., Overland Park, Kansas, USA; No. 3 through the courtesy of Dr H.L. Haak, the Hague, The Netherlands, No. 4 through the courtesy of Drs M.M. Samama and F. Perenet, Paris, France. Fletcher trait plasma (case 3 as described by Hattersley and Hayse 8 was from George King Biomedical Inc., Overland Park, Kansas, USA, lot 06-1701. The [~-factor XIIa (Mr 30000)95% pure, was a gift from Dr N. Heimburger, Behringwerke, Marburg, Germany. Plasma kallikrein was used as its active complex with kinin-free high molecular weight kininogen (gel filtration, Mr 300000), purified as previously described. 9 The DEF was prepared from a mixture of 1 ml plasma, 8 ml distilled water and 1 ml dextran sulphate (100 gg/ml in distilled water). Titration at 0~ with acetic acid to pH 5.9 was started immediately after mixing. After centrifugation in the cold, the precipitate was redissolved in 0.5 ml EDTA buffer (0.05 sodium diethylbarbiturate, 0.10 M NaCI, 0.25% (w/v) gelatin and 2.7 nM ethylene diamine tetraacetate (EDTA) adjusted to pH 7.8 with a HC1 solution). 4 Before assay, the DEF was diluted to a final volume of 1 ml, in some experiments with supplements (factor XIIa, kallikrein, t-PA, u-PA or plasmin) in EDTA buffer. Amounts for additions of IgG against uPA or t-PA were selected for optimal effect on DEF activity, as described. 9, io Plasminogen activator activity of the DEF was assayed on plasminogen-rich bovine fibrin plates, l J This activity is fully dependent on plasminogen. 4 Drops of 30 gl, supplemented with 5 lal of a 14 mM solution of flufenamate, were placed on the fibrin plates, which were incubated for 18 h at 37~ Diameters of lysed zones were intrapolated on a reference curve and activity was expressed in arbitrary BAU. 4 Kallikrein activity was measured on the synthetic tripeptide Bz-Pro-Phe-ArgpNA.HC1 as described. 3
Table 1. Factor Xll-dependent activator activity (BAU/ml)
Volunteers*
Mean SD:
49 40 35 51 40 39 31 41+7
Hageman trait** + [3-factor Xila 36 26 27 44 29 32+8
* Residual activity after addition of excesses of antibodies against t-PA and u-PA in the DEF of plasma of seven volunteers. ** Response to addition of 100% [3-factor Xlla (see Fig. I ) in the presence of excesses of antibodies against u-PA and t-PA. in the DEF of plasma of 5 different patients.
173
RESULTS Factor XII-dependent Activator Portion
The addition of both an excess of neutralizing antibodies against u-PA (see ref. 9) and against t-PA results in inhibition of the plasminogen activator activity of the DEF from 100 BAU/ml to a residual activity of around 40 BAU/ml. As shown in Table 1, 7 healthy volunteers show a residual (= factor XII-dependent) activity of 41+7 (SD) BAU/ml. In the DEF of 5 Hageman trait patients, activator activity was reduced, compared to normal (9+1 (SD) BAU/ml) in the presence of excesses of IgG against t-PA and u-PA. However, the addition of an excess of ~-factor XIIa induced the appearance of 32+8 (SD) BAU/ml of activator activity (Table 1). This activity appeared despite the presence of excesses of both neutralizing antibodies against t-PA and u-PA. The activator activity portions given in Table 1 are apparently unrelated to t-PA and u-PA and are designated as factor XII-dependent.
~-Factor XIIa Added to Contact Factor Deficient Plasmas
Addition of 13-factor XIIa to the DEF of Hageman trait plasma induced appearance of plasminogen activator activity, which was already maximal with the lowest concentration used (30%) (Fig. 1). 13-factor XIIa added to the DEF of normal plasma and Fletcher trait plasma does not elicit much plasminogen activator activity (Fig. 1). Only at very high concentrations, some effects of 13-factor XIIa become apparent (Fig. 1, panel B).
Kallikrein Added to Contact Factor Deficient Plasmas
Addition of kallikrein to the DEF of normal plasma showed a linear increase in plasminogen activator activity amounting to 10 to 15 BAU/ml/100% kallikrein (Fig.2). In the case of Hageman and Fletcher trait plasma, kallikrein induced in a similar experient more plasminogen activator activity than expected from its intrinsic activity revealed when added to normal plasma (Fig. 2).
Plasmin, u-PA or t-PA Added to Contact Factor Deficient Plasma
Results of additions of plasmin, u-PA or t-PA to DEFs of Hageman trait plasma were compared to results of these additions to DEFs of normal plasma (Fig. 3). Recovery of added activity in both situations was similar - no evidence of restoration of factor XII-dependent activity in Hageman trait plasma was apparent.
174
Factor XII-dependent Fibrinolysis
|
ACTIVATOR ACTIVITY
ACTIVATOR ACTIVITY (BAU/ml)
(BAU/ml NORMAL PLASMA
100,
f x
80
x
x
x
100
|
NORMAL PLASMA
HAGEMAN TRAIT
CHER TRAIT 60
.....----.0 FLETCHER TRAIT
50-
40
20
o
0 0
50
100 150 2 0 0 2 5 0 ~-FACTOR
Xlla (%)
0
5()0 1000 1500 13-FACTOR Xlla (%)
Fig. 1 Plasminogen activator activity changes induced by additions of [3-factor Xlla (abscissa) to the DEF of normal plasma ( 0 ) , Hageman trait plasma (x) or Fletcher trait plasma (O). Concentrations of added [3-factor Xlla are expressed in percentage relative to the normal plasma level of factor XII. Panel B shows the effect of additions of large amounts of [3-factor Xlla to normal (O) and Fletcher trait (O)-
DISCUSSION In accordance with previous report, 3'9'~2 the plasminogen activator activity of the DEF of plasma is shown to comprise a factor XII-dependent activator activity which is unrelated to u-PA and t-PA. The factor XIIdependent activator activity can be assayed in the DEF by using quenching antibodies against t-PA and u-PA, and amounts to about 40 BAU/ml of a total activity in a normal DEF of 100 BAU/ml. ACTIVATOR A C T I V I T Y (BAU/ml) 120
m•••O
NORMAL P L A S M A
~
100
80
60,
40
FLETCHER TRAIT EMAN TRAIT
20
0 0
50
100
150
K A L L I K R E I N (%)
Fig. 2 Plasminogen activator activity changes induced by additions of plasma kallikrein to the DEF of normal (O), Hageman trait plasma (x) or Fletcher trait plasma (C)). Concentrations of added kallikrein are expressed in percentage relative to the normal plasma level of prekallikrein.
The activation of the factor XII-dependent activator activity can be studied using congenitally deficient plasmas. Both Hageman and Fletcher traits show absence of the factor XII-dependent plasminogen activator activity. The restoration of activity in the these plasma can be elecited by addition of factor XIIa (Fig. 1) and kallikrein (Fig. 2), respectively. In both cases, the activator activity that appears is larger than the intrinsic plasminogen activating activity of the added factors. This phenomenon has formed the basis for postulation of the presence of an entity (proactivator) that needs activation 2'3 and is different from factor XII and prekallikrein. Evidence for a molecular entity that might represent this component has been reported. ~2.~3In the present report, we further defined the route of activation of this putative plasminogen proactivator. To circumvent the complication of the mutual dependence of factor XII and prekallikrein for their activation, we used purified activated components to reconstitute the deficient plasmas. The reconstitution could be done in the DEE but not in plasma (results not shown) because of the presence of protease inhibitors for the active enzymes in the plasma. In the DEE the reconstitution was followed by immediate addition of flufenamate, which further eliminates inhibitors (notably, C 1inactivator) in euglobulin fractions which are already poor in inhibitors. 14 Our results show that the factor XII-dependent activator activity can be restored in both Hageman and Fletcher trait plasma by addition of active kallikrein. This indicates firstly that active factor XII is not required, and secondly that kallikrein directly or indirectly activates the putative proactivator. It is further shown that the activation of factor XII-dependent activity cannot very effectively be achieved by p-factor XIIa when prekallikrein is absent (Fig. 1).
Fibrinolysis
ACTIVATOR ACTIVITY (BAU/rnl)
ACTIVATOR ACTIVITY (BAU/ml)
ACTIVATOR ACTIVITY (BAUIml)
200"
200'
200"
100
100
100
0
0
o
;
175
0
o
o.s
PLASMIN (CU/ml)
1.o
is
o
u-PA (PL/ml)
15
3o
I-PA (IUIrnl)
Fig. 3 Changes in fibrinolytic activity induced by additions of plasmin, u-PA or t-PA to the DEF of normal ( 0 ) or Hageman trait (C)) plasma. The effects of the additions are all converted to apparent activator activity expressed in BAU/ml using the dose-response curve for the DEF (see ref. 4). CU=Casein units; Pl=Plough units; IU=lnternational units.
Comparing the activation of the factor Xll-dependent fibrinolytic pathway, subject of the present study, and the activation of pro-u-PA, similarities and differences can be noted. Similarities concern the activation of both routes/components by kallikrein and weak activation by activated factor XII. 15 A clear difference is the ability of plasmin to activate pro-u-PA, while plasmin is ineffective in activating the factor XII-dependent process directly (in the absence of factor XII). Thus, although both mentioned pathways can be activated by kallikrein and might be called factor XII-related processes, pro-u-PA activation can proceed independently via plasmin, in contrast to the factor XII-dependent activation we studied, which is fully dependent upon factor XII/prekallikrein.
The addition of plasmin, t-PA or u-PA to Hageman trait plasma showed only recovery of added activity, similar to that of normal plasma. This indicates that neither plasmin, t-PA nor u-PA can replace kallikrein. Further, they cannot directly activate prekallikrein. It is therefore concluded that the factor XII-dependent plasminogen activator activity is generated predominantly by the route 'factor Xlla - kallikrein - fibrinolysis', as indicate in Figure 4. Also indicated in Figure 4 is that factor XII activation can elicit fibrinolysis activation by more than one route. In addition to the process studied in the present manuscript, generation of bradykinin in vivo can result in release of t-PA, 2 and kallikrein can also activate pro-u-PA. 15
FACTOR X,, FAOTi X"a KALLIKRE!N ~
BRADYKININ
PROACTIVATOR~
PREKALLIKREIN
ACTIVATOR
FIBRINOLYSIS
Fig. 4 Factor Xll-dependent fibrinolytic mechanisms.
PRO-u-PA ~
u-PA
176
Factor XII-dependent Fibrinolysis
T h u s o n l y the p a t h w a y s t u d i e d in the p r e s e n t i n v e s t i g a tion is really f a c t o r X I I - ' d e p e n d e n t ' .
REFERENCES I. Bachmann F. Fibrinolysis. In: Verstraete M, Vermylen J, Lijnen H R, Amour J, eds. Thrombosis and haemostasis. Leuven: International Society on Thrombosis and Haemostasis and Leuven University Press, 1987: 227-265. 2. Kluft C, Dooijewaard G, Emeis J J. Role of the contact system in fibrinolysis. Semin Thromb Haemostas 1987; 13: 50-68. 3. Kluft C, Trumpi-Kalshoven M M, Jie A F H, Veldhuyzen-Stolk E C. Factor XII-dependent fibrinolysis: A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII- and kallikrein-dependent plasminogen proactivator. Thromb Haemostas 1979; 41 : 756-773. 4. Kluft C. Studies on the fibrinolytic system in human plasma: Quantitative determination of plasminogen activators and proactivators. Thromb Haemostas 1979; 41: 365-383. 5. Brakman P. Fibrinolysis, a standardized fibrin plate method and a fibrinolytic assay of plasminogen [Thesis] Amsterdam: Scheltema & Holkema, 1967. 6. Kluft C, Van Wezel A L, Van der Velden C A M, Emeis J J, Verheijen J H, Wijngaards G. Large-scale production of extrinsic (tissue-type) plasminogen activator from human melanoma cells. In: Mizrahi A, Van Wezel A L, eds. Advances in biotechnological processes, Vol 2. New York: Alan R Liss, 1983: 97.
Received: I I January 1993 Accepted after revision: 7 December 1993 Offprint orders to" Dr C. Kluft, Gaubius Laboratory IVVO-TNO, PO Box 430, 2300 AK Leiden, The Netherlands.
7. Kluft C, Brakman P, Veldhuyzen-Stolk E C. Screening of fibrinolytic activity in plasma euglobulin fractions on the fibrin plate. In: Davidson J F, Samama M M, Desnoyers P C, eds. Progress in chemical fibrinolysis and thrombolysis, Vol 2. New York: Raven Press, 1976: 57. 8. Hattersley P G, Hayse D. Fletcher factor deficiency: A report of three unrelated cases. BrJ Haematol 1970; 18:411-416. 9. Kluft C, Wijngaards G, Jie A F H. Intrinsic plasma fibrinolysis: Involvement of urokinase-related activity in the factor XIIindependent plasminogen proactivator pathway. J Lab Clin Med 1984; 103: 408418. 10. Rijken D C, Wijngaards G, Welbergen J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb Res 1980; 18:815-830. I 1. Haverkate F, Brakman P. Fibrin plate assay. In: Davidson J F, Samama M M, Desnoyers P C, eds. Progress in chemical fibrinolysis and thrombolysis, Vol 1. New York: Raven Press, 1975: 151. 12. Binnema D J, Dooijewaard G, Van lersel J J L. Identification of two types of protein immunochemically related to urinary urokinase occurring in human plasma. Biochem Biophys Res Commun 1987; 142: 162-168. 13. Binnema D J, Dooijewaard G, Van Iersel J J L, Turion P N C, Kluft C. The contact-system dependent plasminogen activator from human plasma: Identification and characterization. Thromb Haemos 1990; 63: 390-397. 14. Kluft C. Elimination of inhibition in euglobulin fibrinolysis by use of flufenamate: Involvement of C 1-inactivator. Haemostasis 1977; 6:351-369. 15. lchinose A, Fujikawa K, Suyama T. The activation of prourokinase by plasma kallikrein and its inactivation by thrombin. J Biol Chem 1986; 261: 3486-3489.