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Aspirin effect on the release of plasminogen activators inhibitors by human platelets
THROMBOSIS RESEARCH 52; 119-125,1988 0049-3848/88$3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.
ASPIRIN EFFECT ON THE RRT.RASE OF PLASMINOGKN INEIIBITORS BY EIUMAN PLATELETS
Adriana
ACTIVATORS
I. Woods and Maria A. Lazzari.
Department of Hemostasis and Thrombosis, Instituto de Investigaciones Hematologicas "Mariano R. Castex", Academia National de Medicina, Buenos Aires, Argentina (1425). (Received 25.11.1987; accepted in revised form 10.8.1988 by Editor 6. Casillas)
Intrinsic plasminogen activators in (PA) were tested euglobulins (eug) of platelet poor plasma (PPP) with and without washed platelets or not with (WP), treated urokinase (UK), streptokinase (SK), collagen (Col) and aspirin (ASA) using fibrin plates method. A significant decrease of the fibrinolytic activity related to the presence and number of platelets was observed. We confirm the presence of platelet anti-UK and anti-SK activities. The former appears to be higher than the other activity. Low and high concentrations of Co1 stimulated the release of plasminogen activator-inhibitors (PA-I) from platelets, and ASA could not modify this release. Besides ASA might inhibit some PA release. The high concentration of Co1 was capable to release anti-UK and anti-SK activities from platelets and perhaps intrinsic PA-I. The low other concentration of Co1 was only capable release to intrinsic PA-I, suggesting that anti-UK and anti-SK needed a stronger stimuli to be released than intrinsic PA-I. We must consider the possibility that the PA-I and/or activators different could be released by metabolic pathways other than cyclooxygenase pathway.
LKTRODUCTION Evidences from in vitro studies suggest that platelets may affect the fibrinolytic system thought to be one of the important -----------------------
Key-words: plasminogen fibrinolysis, aspirin,
activator inhibitors, fibrin plates assay. 119
blood
platelets,
120
PLATELET PA-I AND ASA
Vol. 52, No. 2
hemostatic regulatory mechanisms preventing inappropiate extension of the hemostatic plug which may lead to thrombotic vessel occlusion Several workers have repeatedly (1). demonstrated the presence of fibrinolytic inhibitors associated to platelets (2) against t-PA, UK-induced plasminogen activation (3, 4, 5, 6). Others identified it as endothelial cell type plasminogen activator inhibitor (PA-I) (7, 8, 9). The inhibitory levels of activity was released from platelets by physiological thrombin and in parallel with the release of PF4 and blocked by pretreating the platelet with PGEl and theophylline (8). Others described the release of PA-I by collagen, ADP, adrenalin and arachidonic acid (10). That release occurred quite rapid and prior or simultaneously with platelet aggregation. their The presence of PA-I from platelets and possible modification in pathological conditions have been appreciated by interesting other groups of research (3, 8, 10) and raised questions with respect to antiplatelet drug therapy in arterial and venous thrombosis; if the platelet PA-I presence are inhibition might be physiologically considerable, their important. The aim of this work was evaluate the effect of aspirin upon the collagen-induced platelet PA-I release. MATERIALS
AND METHODS
Materials: 0.1 M buffered citrate (0.1 M citric acid and 0.1 M sodium citrate) pH 4.5; flufenamic acid was a generous gift from (Fibrindex, Ortho Parke Davis, Argentina; 2 U/ml human thrombin bovine fibrinogen (Sigma); Diagnostic System); 300 mg% 1 IU/ml; final concentration streptokinase (SK) (Kabi Vitrum) 1 IU/ml; urokinase (UK) (Serono, Argentina) final concentration dextran sulfate (Sigma) MW 500,000; collagen (Cal) (Horm) final concentrations 2 and 20 Hg/ml; aspirin (ASA) (Egalgic, Armstrong, Argentina) final concentration 80 ug/ml; 2% EDTA; 100 mg% dextran sulfate solution; sodium flufenamate solution (0.5 M flufenamic acid in equivalent COnCerJtratiOrJ of NaOH diluted to 14 mM with EDTA buffer) final concentration 2 mM; EDTA buffer ( 0.05 M 0.25% (w/v) sodium diethylbarbiturate, 2.7 mM EDTA, 0.1 M NaCl, gelatin) pH 7.8; 1 g/l sodium borate; 1% acetic acid. Blood collection procedure: Nine volumes of blood from healthy fasting volunteers between 8 and 9 am, who had not received any antiplatelet drug 10 days before the study, were drawn into plastic tubes containing 1 volume of 0.1 M buffered citrate. Plasmas and washed platelets: Platelet rich plasma (PRP) were obtained by centrifugation of blood at 150 g for 10 min, and platelet poor plasma (PPP) by centrifugation at 2000 g for 15 min. Washed platelets (WP): platelets were obtained from PRP and washed twice in 2% EDTA and resuspended in the euglobulins from autologous PPP at the following concentations: 3x10ZJ > 1 0 1.:z!ei,.,(11,
2:,..,I,!:‘I’2 wp/ 1
Dextran sulfate euglobulins fractions ( tz) They were precipitated according to Kluft (15). In some zzperiments UK and SK were used to activate eug of PPP alone and containing WP
Vol. 52, No. 2
PLATELET PA-I AND ASA
121
platelets followed by the addition of Col. In other experiments, activation was inhibited by the addition of ASA to the eug of PPP cases, samples containing WP prior to Co1 stimulation; in these were incubated with the inhibitor for 10 min. at 37°C prior to of co1 and activation. Stimulation was made by the addition stirred for 5 min at 37°C. Untreated eug were used as control. fibrinogen Preparation of fibrin plates: Nine ml of bovine in Petri plates. solution was clotted by 1 ml of thrombin Aliquots of 30 ~1 of the eug samples were placed on the fibrin plate. The plates were incubated at 37°C for 18-24 hours. by measuring the two Fibrinolytic activity was determined diameters of the circular lysis areas of the fibrin. RESULTS *F‘b
'no ti P containing WP. WP The lysis areas produced by eug of PPP alone and containing whether or not treated with UK, SK and Co1 (2 and 20 M/ml) are of the shown in Table 1. It can observed a progressive decrease lysis area related with the number of platelets in the untreated and UK, SK and Co1 (2 and 20 ug/ml) treated eug of PPP + WP. When eug were treated with UK it was observed an increase of fibrinolytic activity in the eug of PPP with regard to untreated eug of PPP (p
Lysis areas (mm"') of eug of PPP containing WP (n:ZO). mean + SD Eug from PPP
untreated
UK
SK
Cal
2 ug/ml 20 M/ml -----------___--------------_-----_-_-_~~~~~~~~~~~~~-~-~-------alone 232278 284+77 303_+99 231256 234297
+3x101"WP/ml 217271 235270 252266 160+52 +lO,.XWP,ml 189t75 172255 203268 139+44 +2x101XWP/ml 157269 154k48 174286 126t21 +2xlO""WP/ml+ASA 158+66 156250 171+78 120+30 ____________________-_-_-_-_-_------------_-_-_~_~~~~~~~~~~
160273 141273 129281 106237 _----
PPP + WP with regard to untreated eug of PPP + WP (p:ns). The higher the number of platelets, the lower the lysis area; (eug of PPP + 3x101"WP/1 p:ns, eug of PPP + lO""WP/l p
PLATELET PA-I AND ASA
122
Vol. 52, No. 2
WP with regard to eug of PPP alone (eug of PPP + 3~10"~WP/l p
,
lbrinolvtic activity with K and SK Co1 (i and 20 bg/ml) pretreated with UK or (Table 2). ASA effect
in Co1 activated eug of PPP + WP pretreated activation of eug of PPP + 2xlOlVP/l SK were made and lysis area were measured. was evaluated upon these samples. TABLE 2
Lysis area (mm"") of Co1 (2 and 20 ug/ml) activated eug of PPP + 2xlO"'WP/l pretreated with UK and SK. (n:20) eug of PPP +2xlO""WP/l
untreated
UK
SK
SD X X SD X _-_-------------_----~-----~-~-~---~------~---~--~------------~alone 157 69 154 48 174 +2ug/ml Co1 126 21 157 50 174 +ASA and 2ug/mlCol 118 28 133 49 142 +20flg/ml Co1 130 81 135 25 144 +ASA and 20ug/ml Co1 106 31 116 65 129 --_-------------_-------------_----_----_-_--------------------_
SD 86 66 36 49 60
Co1 (20 fig/ml) produced a slight decrease of the lysis area in UK and SK pretrated eug of PPP + 2~lO"~'wP/l (p:ns). Co1 (2ubWml) did not modify the lysis area of UK and SK pretreated eug of PPP + 2xlO*VP/l. None of all these different combined treatments were modified by,ASA addition to the eug prior to treatments.
To evaluate the effect of drugs upon PA-I release from platelets, we used washed platelets resuspended in eug of PPP. A significant decrease of the lysis area related to the presence and number of platelets were observed. Our results using UK and SK confirmed a platelet anti-UK and anti-SK activities; the former appears to be higher than the other activity (Table 1). Low and high concentrations of Co1 (2 and 20 fig/ml respectively) stimulated the release of PA-I from platelets. Pretreatment of eug of PPP + WP with ASA did not inhibit the release of PA-I produced by both concentrations of Col. Besides, the lower lysis area observed in eug of PPP + WP + ASA with regard to eug without ASA suggested that it is possible that platelets contain some potentiating activity of plasminogen activation, as described by
Vol. 52, No. 2
PLATELET PA-I AND ASA
123
Deguchi (12), probably released by high and low concentrations of Col, and that ASA could inhibit its release. Tacking into account of Co1 was the results from Table 2, the high concentration capable to release anti-UK and anti-SK activities from platelets with them (intrinsic inhibitors other unrelated and perhaps PA-I). The low concentration of Co1 was only capable to release intrinsic PA-I; it could not release anti-UK and anti-SK activities. Several authors (13, 14, 15) observed that the release of a-granules content by high concentrations of thrombin and Co1 could not be inhibited by ASA, but ASA could inhibit the release induced by low concentrations of these agents (16). In a previous report (17) we found that ASA could not inhibit platel;? antiplasmins release with low and high concentrations of Col. the present work, ASA could not inhibit the PA-I release by low and high concentrations of Col. There are observations that platelets can undergo the release reaction in the presence of EDTA or EGTA in which aggregation does not occur (18, 19). Platelet adherent to collagen undergo the release reaction without aggregation and this release reaction is not dependent upon the arachidonic acid pathway (20). In conclusion, from our reports, the high concentration of Co1 was capable to release anti-UK and anti-SK activities and other PA-I from platelets, and the low concentration of Co1 could only release intrinsic PA-I, suggesting that anti-UK and anti-SK needed a stronger stimuli to be released than intrinsic PA-I. We must consider the possibility that PA-I and/or activators could be released by different metabolic other pathways than cyclooxygenase pathway. This point and the possible role of different antiplatelet drugs will be subject of further investigations.
This work was supported by a grant from the Consejo National de Investigaciones Cientificas y Tecnicas (Conicet), Republica Argentina.
1. JOIST, J.H. Platelets %,955-962, 1977.
and
fibrinolysis.
momb.
Hasmost
2. ECKERT, H., FRIELANDER,I. and HARDISTY, R.M. The role of platelets in fibrinolysis. Studies on the plasminogen activator and antiplasmin activity of platelets. Br. J. -01. 18, 575-584, 1970. 3. MURRAY, J., CRAWFORD, G.P.M., OGSTON, D. and DOUGLAS, A.S. Studies on an inhibitor of plasminogen activators in human platelets. Br. J. Haemtol. 26, 661-668, 1974. 4. BOOTH, N.A., plasma ANDERSON, J.A. and BENNET, B. The inhibitors of plasminogen activators, studied by a zymographic technique. Thromb. Res. 38, 261-268, 1985.
124
PLATELET PA-I AND ASA
Vol. 52, No. 2
5.
JUHAN-VAGUE, I., MOERMAN, B., DE COCK, F., AILLAUD, M.F. and COLLEN, D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathologic conditions. Thromb. Res. 33, 523-530, 1984
6.
KWAAN, H.C. and SUWANWELLA, N. Inhibitors of platelets in polycythemia and thrombocytosis. 21, 313-322, 1971.
fibrinolysis in Br. J. Haematol.
J.W.N. 7. SPRENGERS, E.D., AKKERMAN, and JANSEN, B.G. Blood platelet plasminogen activator inhibitor: two different pools of endothelial cell type plasminogen activator inhibitor in human blood. Thromb. Haemost. 55, 325-329, 1986. D.J. Detection 8. ERICKSON, L.A., GINSBERG, M.H. and LOSKUTOFF, and partial characterization of an inhibitor of plasminogen activator in human platelets. J. Clin. Invest. 74, 1465-1472, 1984. 9. ERICKSON, L.A., HEKMAN, C.M. and LOSKUTOFF, D.J. The primary endothelial cells, plasminogen-activator inhibitors in platelets, serum and plasma are immunologically related. Proc. Natl. Acad. Sci. USA 82,8710-8714, 1985. on lO.KRUITHOF, E.K.O., TRAN-THANG, C. and BACHMANN, F. Studies activator inhibitor by human the release of a plasminogen platelets. Thromb. Haemost. 55, 201-205, 1986. ll.KLUFT, C. Studies on the fibrinolytic system in human plasma: and quantitative determination of plasminogen activators proactivators. Thromb. Haemost. 41, 365-382, 1979. 12.DEGUCH1, K., MURASHIMA, S., SHIRAKAWA, S., SORIA, J DUNN, F. and TOBELEM, G. The potentiating activation by tissue piatelet on plasminogen activator. Tromb. Res. 40, 853-861, 1985.
C., SORIA, effect of plasminogen
13.KAPLAN, K.L., BROEKMAN, M.J., CHERNOFT, A., LESZNIK, G.R. and DRILLINGS, M. Platelet cy-granule proteins: studies on release and subcellular localization. Blood 53, 604-618, 1979. 14,HOOGENDIJK, E.M.G., CATE, J., LUDLAM, C.A. and BRUIN, T. No effect of aspirin on /3-tromboglobulin plasma levels in healthy volunteers. Thromb. Res. 19, 257-262, 1980. 15.SANTOS, M.T., VALLES, J., AZNAR, J. and VILLA, P. Platelet fl-TG release in vitro induced by mechanical and chemical stimulus: correlation with the aggregation curve parameters. Stand. J. Haematol. 29, 368-372, 1982. iG.SIESS, W., CUATRECASAS, P. and LAPETINA, E.G. A role of cyclooxygenase products in the formation of phosphatidic acid in stimulated human platelets. Differential mechanisms of action of thrombin and collagen. J. Biol. Chem. 258, 4683-4686, 1986. 17.WOODS, A.I. and
LAZZARI,
M. A.
Aspirin
effect
on
platelet
Vol. 52, No. 2
antiplasmin
PLATELET PA-I AND ASA
release. Thromb.,
269-277,
125
1987.
18.FEINMAN, R.D., and DETWILER, T.C. Absence of requirement for extracellular calcium for secretion from platelets.Thromb. Res. 7, 677-679; 1975. lS.HOLMSEN, H. Platelet secretion. Current concepts and methodological aspects. In: Day, H.J., Holmsen, H., Zucker, M.B. (eds). Platelet function testing. DHEW publication N" (NIH) 78-1087, US Goverment Printing Office, Washington DC, p 112, 1978. 20.KINLOUGH-RATHBONE, R.L., CAZENAVE, J.P., PACKHAM, M.A. and MUSTARD, J.F. Effect of inhibitors of the arachidonic pathway on the release of granules contents from rabbit platelet adherent to collagen. Lab. Invest. 42, 28-31, 1980.
ASPIRIN EFFECT ON THE RRT.RASE OF PLASMINOGKN INEIIBITORS BY EIUMAN PLATELETS
Adriana
ACTIVATORS
I. Woods and Maria A. Lazzari.
Department of Hemostasis and Thrombosis, Instituto de Investigaciones Hematologicas "Mariano R. Castex", Academia National de Medicina, Buenos Aires, Argentina (1425). (Received 25.11.1987; accepted in revised form 10.8.1988 by Editor 6. Casillas)
Intrinsic plasminogen activators in (PA) were tested euglobulins (eug) of platelet poor plasma (PPP) with and without washed platelets or not with (WP), treated urokinase (UK), streptokinase (SK), collagen (Col) and aspirin (ASA) using fibrin plates method. A significant decrease of the fibrinolytic activity related to the presence and number of platelets was observed. We confirm the presence of platelet anti-UK and anti-SK activities. The former appears to be higher than the other activity. Low and high concentrations of Co1 stimulated the release of plasminogen activator-inhibitors (PA-I) from platelets, and ASA could not modify this release. Besides ASA might inhibit some PA release. The high concentration of Co1 was capable to release anti-UK and anti-SK activities from platelets and perhaps intrinsic PA-I. The low other concentration of Co1 was only capable release to intrinsic PA-I, suggesting that anti-UK and anti-SK needed a stronger stimuli to be released than intrinsic PA-I. We must consider the possibility that the PA-I and/or activators different could be released by metabolic pathways other than cyclooxygenase pathway.
LKTRODUCTION Evidences from in vitro studies suggest that platelets may affect the fibrinolytic system thought to be one of the important -----------------------
Key-words: plasminogen fibrinolysis, aspirin,
activator inhibitors, fibrin plates assay. 119
blood
platelets,
120
PLATELET PA-I AND ASA
Vol. 52, No. 2
hemostatic regulatory mechanisms preventing inappropiate extension of the hemostatic plug which may lead to thrombotic vessel occlusion Several workers have repeatedly (1). demonstrated the presence of fibrinolytic inhibitors associated to platelets (2) against t-PA, UK-induced plasminogen activation (3, 4, 5, 6). Others identified it as endothelial cell type plasminogen activator inhibitor (PA-I) (7, 8, 9). The inhibitory levels of activity was released from platelets by physiological thrombin and in parallel with the release of PF4 and blocked by pretreating the platelet with PGEl and theophylline (8). Others described the release of PA-I by collagen, ADP, adrenalin and arachidonic acid (10). That release occurred quite rapid and prior or simultaneously with platelet aggregation. their The presence of PA-I from platelets and possible modification in pathological conditions have been appreciated by interesting other groups of research (3, 8, 10) and raised questions with respect to antiplatelet drug therapy in arterial and venous thrombosis; if the platelet PA-I presence are inhibition might be physiologically considerable, their important. The aim of this work was evaluate the effect of aspirin upon the collagen-induced platelet PA-I release. MATERIALS
AND METHODS
Materials: 0.1 M buffered citrate (0.1 M citric acid and 0.1 M sodium citrate) pH 4.5; flufenamic acid was a generous gift from (Fibrindex, Ortho Parke Davis, Argentina; 2 U/ml human thrombin bovine fibrinogen (Sigma); Diagnostic System); 300 mg% 1 IU/ml; final concentration streptokinase (SK) (Kabi Vitrum) 1 IU/ml; urokinase (UK) (Serono, Argentina) final concentration dextran sulfate (Sigma) MW 500,000; collagen (Cal) (Horm) final concentrations 2 and 20 Hg/ml; aspirin (ASA) (Egalgic, Armstrong, Argentina) final concentration 80 ug/ml; 2% EDTA; 100 mg% dextran sulfate solution; sodium flufenamate solution (0.5 M flufenamic acid in equivalent COnCerJtratiOrJ of NaOH diluted to 14 mM with EDTA buffer) final concentration 2 mM; EDTA buffer ( 0.05 M 0.25% (w/v) sodium diethylbarbiturate, 2.7 mM EDTA, 0.1 M NaCl, gelatin) pH 7.8; 1 g/l sodium borate; 1% acetic acid. Blood collection procedure: Nine volumes of blood from healthy fasting volunteers between 8 and 9 am, who had not received any antiplatelet drug 10 days before the study, were drawn into plastic tubes containing 1 volume of 0.1 M buffered citrate. Plasmas and washed platelets: Platelet rich plasma (PRP) were obtained by centrifugation of blood at 150 g for 10 min, and platelet poor plasma (PPP) by centrifugation at 2000 g for 15 min. Washed platelets (WP): platelets were obtained from PRP and washed twice in 2% EDTA and resuspended in the euglobulins from autologous PPP at the following concentations: 3x10ZJ > 1 0 1.:z!ei,.,(11,
2:,..,I,!:‘I’2 wp/ 1
Dextran sulfate euglobulins fractions ( tz) They were precipitated according to Kluft (15). In some zzperiments UK and SK were used to activate eug of PPP alone and containing WP
Vol. 52, No. 2
PLATELET PA-I AND ASA
121
platelets followed by the addition of Col. In other experiments, activation was inhibited by the addition of ASA to the eug of PPP cases, samples containing WP prior to Co1 stimulation; in these were incubated with the inhibitor for 10 min. at 37°C prior to of co1 and activation. Stimulation was made by the addition stirred for 5 min at 37°C. Untreated eug were used as control. fibrinogen Preparation of fibrin plates: Nine ml of bovine in Petri plates. solution was clotted by 1 ml of thrombin Aliquots of 30 ~1 of the eug samples were placed on the fibrin plate. The plates were incubated at 37°C for 18-24 hours. by measuring the two Fibrinolytic activity was determined diameters of the circular lysis areas of the fibrin. RESULTS *F‘b
'no ti P containing WP. WP The lysis areas produced by eug of PPP alone and containing whether or not treated with UK, SK and Co1 (2 and 20 M/ml) are of the shown in Table 1. It can observed a progressive decrease lysis area related with the number of platelets in the untreated and UK, SK and Co1 (2 and 20 ug/ml) treated eug of PPP + WP. When eug were treated with UK it was observed an increase of fibrinolytic activity in the eug of PPP with regard to untreated eug of PPP (p
Lysis areas (mm"') of eug of PPP containing WP (n:ZO). mean + SD Eug from PPP
untreated
UK
SK
Cal
2 ug/ml 20 M/ml -----------___--------------_-----_-_-_~~~~~~~~~~~~~-~-~-------alone 232278 284+77 303_+99 231256 234297
+3x101"WP/ml 217271 235270 252266 160+52 +lO,.XWP,ml 189t75 172255 203268 139+44 +2x101XWP/ml 157269 154k48 174286 126t21 +2xlO""WP/ml+ASA 158+66 156250 171+78 120+30 ____________________-_-_-_-_-_------------_-_-_~_~~~~~~~~~~
160273 141273 129281 106237 _----
PPP + WP with regard to untreated eug of PPP + WP (p:ns). The higher the number of platelets, the lower the lysis area; (eug of PPP + 3x101"WP/1 p:ns, eug of PPP + lO""WP/l p
PLATELET PA-I AND ASA
122
Vol. 52, No. 2
WP with regard to eug of PPP alone (eug of PPP + 3~10"~WP/l p
,
lbrinolvtic activity with K and SK Co1 (i and 20 bg/ml) pretreated with UK or (Table 2). ASA effect
in Co1 activated eug of PPP + WP pretreated activation of eug of PPP + 2xlOlVP/l SK were made and lysis area were measured. was evaluated upon these samples. TABLE 2
Lysis area (mm"") of Co1 (2 and 20 ug/ml) activated eug of PPP + 2xlO"'WP/l pretreated with UK and SK. (n:20) eug of PPP +2xlO""WP/l
untreated
UK
SK
SD X X SD X _-_-------------_----~-----~-~-~---~------~---~--~------------~alone 157 69 154 48 174 +2ug/ml Co1 126 21 157 50 174 +ASA and 2ug/mlCol 118 28 133 49 142 +20flg/ml Co1 130 81 135 25 144 +ASA and 20ug/ml Co1 106 31 116 65 129 --_-------------_-------------_----_----_-_--------------------_
SD 86 66 36 49 60
Co1 (20 fig/ml) produced a slight decrease of the lysis area in UK and SK pretrated eug of PPP + 2~lO"~'wP/l (p:ns). Co1 (2ubWml) did not modify the lysis area of UK and SK pretreated eug of PPP + 2xlO*VP/l. None of all these different combined treatments were modified by,ASA addition to the eug prior to treatments.
To evaluate the effect of drugs upon PA-I release from platelets, we used washed platelets resuspended in eug of PPP. A significant decrease of the lysis area related to the presence and number of platelets were observed. Our results using UK and SK confirmed a platelet anti-UK and anti-SK activities; the former appears to be higher than the other activity (Table 1). Low and high concentrations of Co1 (2 and 20 fig/ml respectively) stimulated the release of PA-I from platelets. Pretreatment of eug of PPP + WP with ASA did not inhibit the release of PA-I produced by both concentrations of Col. Besides, the lower lysis area observed in eug of PPP + WP + ASA with regard to eug without ASA suggested that it is possible that platelets contain some potentiating activity of plasminogen activation, as described by
Vol. 52, No. 2
PLATELET PA-I AND ASA
123
Deguchi (12), probably released by high and low concentrations of Col, and that ASA could inhibit its release. Tacking into account of Co1 was the results from Table 2, the high concentration capable to release anti-UK and anti-SK activities from platelets with them (intrinsic inhibitors other unrelated and perhaps PA-I). The low concentration of Co1 was only capable to release intrinsic PA-I; it could not release anti-UK and anti-SK activities. Several authors (13, 14, 15) observed that the release of a-granules content by high concentrations of thrombin and Co1 could not be inhibited by ASA, but ASA could inhibit the release induced by low concentrations of these agents (16). In a previous report (17) we found that ASA could not inhibit platel;? antiplasmins release with low and high concentrations of Col. the present work, ASA could not inhibit the PA-I release by low and high concentrations of Col. There are observations that platelets can undergo the release reaction in the presence of EDTA or EGTA in which aggregation does not occur (18, 19). Platelet adherent to collagen undergo the release reaction without aggregation and this release reaction is not dependent upon the arachidonic acid pathway (20). In conclusion, from our reports, the high concentration of Co1 was capable to release anti-UK and anti-SK activities and other PA-I from platelets, and the low concentration of Co1 could only release intrinsic PA-I, suggesting that anti-UK and anti-SK needed a stronger stimuli to be released than intrinsic PA-I. We must consider the possibility that PA-I and/or activators could be released by different metabolic other pathways than cyclooxygenase pathway. This point and the possible role of different antiplatelet drugs will be subject of further investigations.
This work was supported by a grant from the Consejo National de Investigaciones Cientificas y Tecnicas (Conicet), Republica Argentina.
1. JOIST, J.H. Platelets %,955-962, 1977.
and
fibrinolysis.
momb.
Hasmost
2. ECKERT, H., FRIELANDER,I. and HARDISTY, R.M. The role of platelets in fibrinolysis. Studies on the plasminogen activator and antiplasmin activity of platelets. Br. J. -01. 18, 575-584, 1970. 3. MURRAY, J., CRAWFORD, G.P.M., OGSTON, D. and DOUGLAS, A.S. Studies on an inhibitor of plasminogen activators in human platelets. Br. J. Haemtol. 26, 661-668, 1974. 4. BOOTH, N.A., plasma ANDERSON, J.A. and BENNET, B. The inhibitors of plasminogen activators, studied by a zymographic technique. Thromb. Res. 38, 261-268, 1985.
124
PLATELET PA-I AND ASA
Vol. 52, No. 2
5.
JUHAN-VAGUE, I., MOERMAN, B., DE COCK, F., AILLAUD, M.F. and COLLEN, D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathologic conditions. Thromb. Res. 33, 523-530, 1984
6.
KWAAN, H.C. and SUWANWELLA, N. Inhibitors of platelets in polycythemia and thrombocytosis. 21, 313-322, 1971.
fibrinolysis in Br. J. Haematol.
J.W.N. 7. SPRENGERS, E.D., AKKERMAN, and JANSEN, B.G. Blood platelet plasminogen activator inhibitor: two different pools of endothelial cell type plasminogen activator inhibitor in human blood. Thromb. Haemost. 55, 325-329, 1986. D.J. Detection 8. ERICKSON, L.A., GINSBERG, M.H. and LOSKUTOFF, and partial characterization of an inhibitor of plasminogen activator in human platelets. J. Clin. Invest. 74, 1465-1472, 1984. 9. ERICKSON, L.A., HEKMAN, C.M. and LOSKUTOFF, D.J. The primary endothelial cells, plasminogen-activator inhibitors in platelets, serum and plasma are immunologically related. Proc. Natl. Acad. Sci. USA 82,8710-8714, 1985. on lO.KRUITHOF, E.K.O., TRAN-THANG, C. and BACHMANN, F. Studies activator inhibitor by human the release of a plasminogen platelets. Thromb. Haemost. 55, 201-205, 1986. ll.KLUFT, C. Studies on the fibrinolytic system in human plasma: and quantitative determination of plasminogen activators proactivators. Thromb. Haemost. 41, 365-382, 1979. 12.DEGUCH1, K., MURASHIMA, S., SHIRAKAWA, S., SORIA, J DUNN, F. and TOBELEM, G. The potentiating activation by tissue piatelet on plasminogen activator. Tromb. Res. 40, 853-861, 1985.
C., SORIA, effect of plasminogen
13.KAPLAN, K.L., BROEKMAN, M.J., CHERNOFT, A., LESZNIK, G.R. and DRILLINGS, M. Platelet cy-granule proteins: studies on release and subcellular localization. Blood 53, 604-618, 1979. 14,HOOGENDIJK, E.M.G., CATE, J., LUDLAM, C.A. and BRUIN, T. No effect of aspirin on /3-tromboglobulin plasma levels in healthy volunteers. Thromb. Res. 19, 257-262, 1980. 15.SANTOS, M.T., VALLES, J., AZNAR, J. and VILLA, P. Platelet fl-TG release in vitro induced by mechanical and chemical stimulus: correlation with the aggregation curve parameters. Stand. J. Haematol. 29, 368-372, 1982. iG.SIESS, W., CUATRECASAS, P. and LAPETINA, E.G. A role of cyclooxygenase products in the formation of phosphatidic acid in stimulated human platelets. Differential mechanisms of action of thrombin and collagen. J. Biol. Chem. 258, 4683-4686, 1986. 17.WOODS, A.I. and
LAZZARI,
M. A.
Aspirin
effect
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