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Release of plasminogen activator and stimulation of platelets by stable analogues of platelet activating factor (PAF)
thrmol~cr\ (1987) 1. 129 131 t 1987 Longman Group 11&Z Ltd
Release of Plasminogen Activator and Stimulation of Platelets by Stable Analogues of Platelet Activating Factor (PAF)
H.-P. KlGcking, A. Hoffmann, G. Ostermann, H. P. Kertscher SUMMAR Y. PAF and 9 stable analogues, racemic l-O-bexadecyl-2-(n-alkyl-propanediole)-3-pbosph~holin~ and 1-0-hexadecyl-2-0-alkylglycero-3-phosphocholines, were tested for plasminogen activator (PA) releasing activity in the isolated pig ear and aggregating effects on pig platelets. Structural analogues are considerably less effective than PAF in both test-systems. The most potent analogues are able to release PA already at a concentration of 10m8 mol/l. At this concentration no visual platelet aggregation was observed in pig platelet-rich plasma. The results suggest to look for an increase in blood librinolytic activity in animals. KEYWORDS. Platelet activating effect. Platelet-stimulating effect.
factor (PAF). Structural analogues
The platelet-activating factor (PAF) which is a potent mediator of allergic and inflammatory reactions has diverse biological effects on a variety of cells other than platelets.’ Thus, recently it was found that PAF is also a potent inducer of plasminogen activator (PA) release.2,3 Since PAF is rapidly inactivated in vivo by an enzymatical removal of the acetyl group4 structural analogues containing a nonhydrolysable residue at the 2-position may be of particular interest for increasing blood fibrinolytic activity. Therefore, racemic 1-Ohexadecyl-2-(n-alkyl-propanediole)-3-phosphocholines (I-V) and I-O-hexadecyl-2-O-alkyl-glycero-3-phosphocholines (VI-IX) were tested for PA releasing activity in the perfused isolated pig ear and for aggregating effects on pig platelets.
AND
activator-releasing
against human urokinase (UK) and IgG antibodies against human vascular plasminogen activator (V-PA) were kindly supplied by Prof. Binder (Vienna, Austria). PAF and the structural analogues were dissolved in albumin-PBS (2.5 mg human serum albumin per ml phosphate-buffered saline, pH 7.4) to a final concentration of 2.2 mM, stored at - 2O”C, and further diluted with albumin-PBS immediately before use. Activator release was studied in the isolated perfused pig ear at constant perfusion volume (for test design see *). The plasminogen activator content of the perfusate was estimated by measuring the fibrinolytic activity during application on plasminogenappearing containing fibrin-agar plates and converting it into Ploug-units by means of a urokinase calibration curve.8.9 Since the calibration curves of t-PA and urokinase are not parallel only percentage changes in the release of PA are given. For significance calculation the mean increase in activator release was related to the mean values of spontaneous release in the controls (14.5% f 12; n = 9). Statistical reliability was performed by student’s t-test. p > 0.05 was considered as not significant. For characterisation of the plasminogen activator activity a post PAF-analogues perfusate was incubated with IgG against UK and V-PA at 37°C for 30 min. The remaining activator activity was tested on plasminogen-rich fibrin plates. The preparation of platelet-rich plasma (PRP) from fresh pig blood anticoagulated with 10.6mmol/l citrate and the platelet aggregation were performed as described previously. lo The maximum change in
INTRODUCTION
MATERIALS
of PAF. Plasminogen
METHODS
The structural analogues of PAF (Table 1) were synthesised by Kertscher et al 5*6Semisynthetic PAF7 was a generous gift from Prof. H. K. Mangold (Miinster, FRG). Bovine fibrinogen (Behringwerke AG, Marburg/Lahn, FRG) and Bovine thrombin (VEB Arzneimittelwerk Dresden, GDR). IgG antibodies
H.-P. Klocking, A. Hoffmann, Institute of Pharmacology and Toxicology, G. Ostermann, Institute of Pathological Biochemistry, Medical Academy Erfurt, H. P. Kertscher, Section of Biosciences. Karl Marx University Leipzig, CDR. 129
130
Plasminogen
Activator
Release and Stimulation
Table 1 Plasminogen
Activator
of Platelets by Stable Analogues
Releasing
and Platelet Stimulating
of Platelet Activating
Factor
Effects of Stable PAF Analogues
CH,-0-Ci6H3a &H--R’
CHa
AH,-0-P-O-CH,-CH,-LR~ oH
R,
R*
PAF I II III IV V VI VII VIII IX control
OCOCHa
CH, CH, CH, CH, CH, CH, CH, CH, CH,CH,Br
(without
Q&H, QCzHs Q&H, substance)
CH3 Increase in activator release (%)1,2.3 (n)
Substance
CH, GH, CjH, GHs C,H,, OCH,
I
‘0s
105*65++ 31+ 5 10*12 12*25+++ 22*20 20*20 22+15 54*25+++ 62*44+ 32k30 14,5* 12
CH,
1. Increase in PA release was calculated as per cent of the spontaneous PAF-analogues. 2. All compounds were tested at the concentration of 1O-7 mol/l. 3. Significance to control: + p
transmission observed within 3 min after the addition of the substances tested served as a measure of aggregation. The maximum aggregation (100%) used as reference was assessed by the addition of PAF at a final concentration of 10 nmol/l.
RESULTS
PAF at a final concentration of lo-’ mol/l causes an increase in PA release of about 100’~ on perfusion of isolated pig ears. As shown in Table 1 structural analogues of PAF are considerably less effective under
1005
0.05
0.5 ul IgG
5
added
Fig. 1 Quenching of plasminogen activator activity in perfusates from isolated perfused pig ear, stimulated by lo-’ mol/l l-O-hexadecyl-2-(n-propyl-propanediole)-3-phosphocholine (0) by anti-human V-PA IgG (o), and by anti-human urokinase IgG. Mean values of the percentage of residual activity, as determined in three separate experiments are shown.
Platelet aggregation
(5) (3) (4) (5) (3) (3) (3) (10) (6) (10) (9)
ECsa (molil)
(n)
8.Ok3.7. lo-’ >2.0 .10-4 12.0 1O-4 >2.0 1o-4 z2.0 .10-4 > 2.0 1o-4 > 2.0 1o-4 2.9k1.2. 10-s 7.4_t1.5.10~h 8.1+1.6.10-s
(15) (4) (3) (4) (3) (3) (4) (4) (3) (3)
release observed
before the application
of
identical experimental conditions. Using a concentration of lo-’ mol/l only the compounds III, VII, and VIII release significant amounts of PA. The most potent analogues III, VII, and VIII are also capable of releasing PA at a concentration of lo-* mol/l. Quenching with anti-human V-PA (= tissue-type PA) shows that the activator released by analogues of PAF is tissue-type activator. An example is shown in PAF _-
4
2. lo-‘molll 5 * lo-’ mall L
lb 1
min
10m6 mol/I
10m5 mall 1
Fig. 2 Concentration-dependent aggregation of pig platelets by PAF. Final concentrations of PAF added to stirred PRP as indicated by arrow are given at the endings of the traces. One out of five experiments that gave similar results is shown.
Journal
Figure 1. As seen in Figure 2, PAF at a final concentration of lO-7-lO-s mol/l produces a concentrationdependent aggregation in pig PRP. However, considerable individual variations are observed in the response to PAF and also the responsiveness of pig PRP distinctly decreases with time after preparation. In comparison with PAF the structural analogues exhibit considerably lower platelet-aggregating effects. Thus, the most potent derivative VIII is shown to be about tenfold less efficient than PAF (Table 1). Moreover, at a final concentration of lo-’ mol/l which was used for studying the release of PA, none of the analogues tested caused visual platelet aggregation in pig PR P. DISCUSSION
AND
CONCLUSIONS
In the present study PAF and some structural analogues containing a nonhydrolysable residue at the 2-position were tested for both, platelet-aggregating potency and PA release from the perfused pig ear. Using titrated PRP from pigs PAF produces a concentration-dependent aggregation response which is somewhat lower than that obtained in human PRP under identical conditions.” Compared with PAF, the analogues tested are considerably less potent in both test systems. This result is in accordance with those obtained with human’ and rabbit” platelets and stresses the critical importance of the acetyl group of PAF for expression of high biological activity.’ *.l 3q14 The biological effects of the analogues reported here seem not to be due to membrane damage because a measurable increase in membrane permeability of Fogh and Lund (FL)-cells15 and erythrocytes’ has been seen only at concentrations higher than lo- 5 mol/l. Comparing the pattern of PA-releasing and platelet-aggregating activities no substantial differences are found. Similar results were also reported in a previous study that compared the platelet-stimulating and hypotensive activities of PAF and analogues.16 But in spite of similar structure-activity relationship the release of PA is observed already at concentrations of PAF and analogues which are not sufficient to produce a visual platelet aggregation under our experimental conditions. The 2-(n-propyl) derivative III and the 2-0-ethoxy derivatives VII and VIII exert the most pronounced PA-releasing effect among the substances tested in the isolated perfused pig ear.4.8,17,18,‘9.20 Because of their stability in plasma these compounds may be suitable to induce PA release in animals as was shown for PAF by Emeis and Kluft3 REFERENCES I. Benveniste J, Vargaftig B B 1983 Platelet-activating factor: an ether lipid with biological activity. In: Mangold H K, Paltauf F
orders to: Prof. Dr H.-P. Kliicking, Medical Academy Erfurt, Institute of Pharmacology and Toxicology, NordhPuser Stral3e 74, DDR-5010 Erfurt, GDR.
Olfprint
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. IX.
19. 20.
of Fibrinolysis
13 I
(eds), Ether Lipids: Biochemical and Biomedical Aspects, Academic Press, New York, 355-376 KlBcking H-P, Markwardt F, Hoffmann Anna 1985 Release of plasminogen activator by platelet-activating factor. Thromb Res 38:413416 Emeis J J. Kluft C 1985 PAF-Acether-induced release of tissuetype plasminogen activator from vessel walls. Blood 66:8&91 Blank M L, Cress E A, Whittle T, Snyder F 1981 In vivo metabolism of a new class of biologically active phospholipids: l-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating hypotensive phospholipid. Life Sci 29:769%775 Kertscher H-P. Ostermann G, Lang A, Weissflog W, Gawrich K 1985 Synthese und biologische Aktivitat einiger stabiler Strukturanaloga des Plattchenaktivierenden Faktors. Pharmazie 40:702Z704 Kertscher H P, Ostermann G 1986 Synthesis of I-O-hexadecyl2-0-methyl-rat-glycero-3-phosphocholine and analogues. Chemistry and physics of lipids (in press) Murdmatsu T, Totani N, Mangold H K 1981 A facile method for the preparation of l-O-alkyl-2.O-acetoyl-.sn-glycero-3phosphocholines (platelet activating factor). Chem Phys Lipids 29: 121-127 Kliicking H-P, Bock G, Drawert J, Hinsenbrock K-P, Kaiser B, Sedlarik U 1976 iiber die Freisetzung von Plasminogenaktivatoren. Folia Haematol (Leipz) 103:404422 Markwardt F, Kllicking H-P 1978 Einflun von Mediatoren auf die Freisetzung von Plasminogenaktivatoren. Acta Biologica MedGerman37:1603-1610 Ostermann G, Till U, Thielmann K 1983 Studies on the stimulation of human blood platelets by semisynthetic plateletactivating factor. Thromb Res 30: 127-136 Ostermann G, Kertscher H-P, Lang A, Till U 1986 Stimulation and desensitization of human and rabbit platelets by I-0-hexadecyl-2-0-methyl-r~~-glycero-3-phosphocholine and analogues. Thromb Res 43:675-680. Tence M, Coeffier E, Heymans F, Polonsky J, Godfoid J-J, Benveniste J 1981 Structural analogs of platelet-activating factor (PAF-acether). Biochimie 63:723%727 Hadvary P, Baumgartner H R 1983 Activation of human and rabbit platelets by synthetic analogs of platelet activating factor. Thromb Res 30: 143 I56 O’Flaherty J T, Salzer W L, Cousart S, McCall C E. Plantadose C, Surles J R. Hammelt M J, Wykle R L 1983 Platelet-activating factor and analogues: Comparative studies with human neutrophils and rabbit platelets. Res Commun Chem Pathol Pharmacol39:291~309 Klocking H-P. Hoffmann A, Ostermann G, Kertscher H-P, Kliicking R. Eichhorn U 1985 Studies on the biological activity of stable analogues of platelet activating factor. IVth International Meeting of Danubian League against Thrombosis and Hemorrhagic Diseases. September 25-28. Istanbul, Turkey Blank M L, Cress E A. Lee T-C, Malone B, Surles J R. Piantadosi C, Hadju J. Snyder F 1982 Structural features of platelet activating factor (I-alkyl-2-acetyl-sn-glycero-3phosphocholine) required for hypotensive and platelet serotonin response. Res Commun Chem Pathol Pharmacol 38: 3-20 Markwardt F, Kliicking H-P 1976 Studies on the release of plasminogen activator. Thrombosis Research 8:217-223 Kliicking H-P 1979 Release of plasminogen activator by acetylcholine from the isolated perfused pig ear. Thromb Res 16:261-264 Markwardt F. Kliicking H-P 1977 Heparin-induced release of plasminogen activator.-Haemostas 6:j7&374 Klijckine H-P. Kl(ickine R. Helbig B 1984 Einflul3 von Phenolk~rperpolymeris~ten auf die Freisetzung von Plasminogenaktivatoren. Farmakol Toksikol47:93-96 (Russian)
Release of Plasminogen Activator and Stimulation of Platelets by Stable Analogues of Platelet Activating Factor (PAF)
H.-P. KlGcking, A. Hoffmann, G. Ostermann, H. P. Kertscher SUMMAR Y. PAF and 9 stable analogues, racemic l-O-bexadecyl-2-(n-alkyl-propanediole)-3-pbosph~holin~ and 1-0-hexadecyl-2-0-alkylglycero-3-phosphocholines, were tested for plasminogen activator (PA) releasing activity in the isolated pig ear and aggregating effects on pig platelets. Structural analogues are considerably less effective than PAF in both test-systems. The most potent analogues are able to release PA already at a concentration of 10m8 mol/l. At this concentration no visual platelet aggregation was observed in pig platelet-rich plasma. The results suggest to look for an increase in blood librinolytic activity in animals. KEYWORDS. Platelet activating effect. Platelet-stimulating effect.
factor (PAF). Structural analogues
The platelet-activating factor (PAF) which is a potent mediator of allergic and inflammatory reactions has diverse biological effects on a variety of cells other than platelets.’ Thus, recently it was found that PAF is also a potent inducer of plasminogen activator (PA) release.2,3 Since PAF is rapidly inactivated in vivo by an enzymatical removal of the acetyl group4 structural analogues containing a nonhydrolysable residue at the 2-position may be of particular interest for increasing blood fibrinolytic activity. Therefore, racemic 1-Ohexadecyl-2-(n-alkyl-propanediole)-3-phosphocholines (I-V) and I-O-hexadecyl-2-O-alkyl-glycero-3-phosphocholines (VI-IX) were tested for PA releasing activity in the perfused isolated pig ear and for aggregating effects on pig platelets.
AND
activator-releasing
against human urokinase (UK) and IgG antibodies against human vascular plasminogen activator (V-PA) were kindly supplied by Prof. Binder (Vienna, Austria). PAF and the structural analogues were dissolved in albumin-PBS (2.5 mg human serum albumin per ml phosphate-buffered saline, pH 7.4) to a final concentration of 2.2 mM, stored at - 2O”C, and further diluted with albumin-PBS immediately before use. Activator release was studied in the isolated perfused pig ear at constant perfusion volume (for test design see *). The plasminogen activator content of the perfusate was estimated by measuring the fibrinolytic activity during application on plasminogenappearing containing fibrin-agar plates and converting it into Ploug-units by means of a urokinase calibration curve.8.9 Since the calibration curves of t-PA and urokinase are not parallel only percentage changes in the release of PA are given. For significance calculation the mean increase in activator release was related to the mean values of spontaneous release in the controls (14.5% f 12; n = 9). Statistical reliability was performed by student’s t-test. p > 0.05 was considered as not significant. For characterisation of the plasminogen activator activity a post PAF-analogues perfusate was incubated with IgG against UK and V-PA at 37°C for 30 min. The remaining activator activity was tested on plasminogen-rich fibrin plates. The preparation of platelet-rich plasma (PRP) from fresh pig blood anticoagulated with 10.6mmol/l citrate and the platelet aggregation were performed as described previously. lo The maximum change in
INTRODUCTION
MATERIALS
of PAF. Plasminogen
METHODS
The structural analogues of PAF (Table 1) were synthesised by Kertscher et al 5*6Semisynthetic PAF7 was a generous gift from Prof. H. K. Mangold (Miinster, FRG). Bovine fibrinogen (Behringwerke AG, Marburg/Lahn, FRG) and Bovine thrombin (VEB Arzneimittelwerk Dresden, GDR). IgG antibodies
H.-P. Klocking, A. Hoffmann, Institute of Pharmacology and Toxicology, G. Ostermann, Institute of Pathological Biochemistry, Medical Academy Erfurt, H. P. Kertscher, Section of Biosciences. Karl Marx University Leipzig, CDR. 129
130
Plasminogen
Activator
Release and Stimulation
Table 1 Plasminogen
Activator
of Platelets by Stable Analogues
Releasing
and Platelet Stimulating
of Platelet Activating
Factor
Effects of Stable PAF Analogues
CH,-0-Ci6H3a &H--R’
CHa
AH,-0-P-O-CH,-CH,-LR~ oH
R,
R*
PAF I II III IV V VI VII VIII IX control
OCOCHa
CH, CH, CH, CH, CH, CH, CH, CH, CH,CH,Br
(without
Q&H, QCzHs Q&H, substance)
CH3 Increase in activator release (%)1,2.3 (n)
Substance
CH, GH, CjH, GHs C,H,, OCH,
I
‘0s
105*65++ 31+ 5 10*12 12*25+++ 22*20 20*20 22+15 54*25+++ 62*44+ 32k30 14,5* 12
CH,
1. Increase in PA release was calculated as per cent of the spontaneous PAF-analogues. 2. All compounds were tested at the concentration of 1O-7 mol/l. 3. Significance to control: + p
transmission observed within 3 min after the addition of the substances tested served as a measure of aggregation. The maximum aggregation (100%) used as reference was assessed by the addition of PAF at a final concentration of 10 nmol/l.
RESULTS
PAF at a final concentration of lo-’ mol/l causes an increase in PA release of about 100’~ on perfusion of isolated pig ears. As shown in Table 1 structural analogues of PAF are considerably less effective under
1005
0.05
0.5 ul IgG
5
added
Fig. 1 Quenching of plasminogen activator activity in perfusates from isolated perfused pig ear, stimulated by lo-’ mol/l l-O-hexadecyl-2-(n-propyl-propanediole)-3-phosphocholine (0) by anti-human V-PA IgG (o), and by anti-human urokinase IgG. Mean values of the percentage of residual activity, as determined in three separate experiments are shown.
Platelet aggregation
(5) (3) (4) (5) (3) (3) (3) (10) (6) (10) (9)
ECsa (molil)
(n)
8.Ok3.7. lo-’ >2.0 .10-4 12.0 1O-4 >2.0 1o-4 z2.0 .10-4 > 2.0 1o-4 > 2.0 1o-4 2.9k1.2. 10-s 7.4_t1.5.10~h 8.1+1.6.10-s
(15) (4) (3) (4) (3) (3) (4) (4) (3) (3)
release observed
before the application
of
identical experimental conditions. Using a concentration of lo-’ mol/l only the compounds III, VII, and VIII release significant amounts of PA. The most potent analogues III, VII, and VIII are also capable of releasing PA at a concentration of lo-* mol/l. Quenching with anti-human V-PA (= tissue-type PA) shows that the activator released by analogues of PAF is tissue-type activator. An example is shown in PAF _-
4
2. lo-‘molll 5 * lo-’ mall L
lb 1
min
10m6 mol/I
10m5 mall 1
Fig. 2 Concentration-dependent aggregation of pig platelets by PAF. Final concentrations of PAF added to stirred PRP as indicated by arrow are given at the endings of the traces. One out of five experiments that gave similar results is shown.
Journal
Figure 1. As seen in Figure 2, PAF at a final concentration of lO-7-lO-s mol/l produces a concentrationdependent aggregation in pig PRP. However, considerable individual variations are observed in the response to PAF and also the responsiveness of pig PRP distinctly decreases with time after preparation. In comparison with PAF the structural analogues exhibit considerably lower platelet-aggregating effects. Thus, the most potent derivative VIII is shown to be about tenfold less efficient than PAF (Table 1). Moreover, at a final concentration of lo-’ mol/l which was used for studying the release of PA, none of the analogues tested caused visual platelet aggregation in pig PR P. DISCUSSION
AND
CONCLUSIONS
In the present study PAF and some structural analogues containing a nonhydrolysable residue at the 2-position were tested for both, platelet-aggregating potency and PA release from the perfused pig ear. Using titrated PRP from pigs PAF produces a concentration-dependent aggregation response which is somewhat lower than that obtained in human PRP under identical conditions.” Compared with PAF, the analogues tested are considerably less potent in both test systems. This result is in accordance with those obtained with human’ and rabbit” platelets and stresses the critical importance of the acetyl group of PAF for expression of high biological activity.’ *.l 3q14 The biological effects of the analogues reported here seem not to be due to membrane damage because a measurable increase in membrane permeability of Fogh and Lund (FL)-cells15 and erythrocytes’ has been seen only at concentrations higher than lo- 5 mol/l. Comparing the pattern of PA-releasing and platelet-aggregating activities no substantial differences are found. Similar results were also reported in a previous study that compared the platelet-stimulating and hypotensive activities of PAF and analogues.16 But in spite of similar structure-activity relationship the release of PA is observed already at concentrations of PAF and analogues which are not sufficient to produce a visual platelet aggregation under our experimental conditions. The 2-(n-propyl) derivative III and the 2-0-ethoxy derivatives VII and VIII exert the most pronounced PA-releasing effect among the substances tested in the isolated perfused pig ear.4.8,17,18,‘9.20 Because of their stability in plasma these compounds may be suitable to induce PA release in animals as was shown for PAF by Emeis and Kluft3 REFERENCES I. Benveniste J, Vargaftig B B 1983 Platelet-activating factor: an ether lipid with biological activity. In: Mangold H K, Paltauf F
orders to: Prof. Dr H.-P. Kliicking, Medical Academy Erfurt, Institute of Pharmacology and Toxicology, NordhPuser Stral3e 74, DDR-5010 Erfurt, GDR.
Olfprint
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17. IX.
19. 20.
of Fibrinolysis
13 I
(eds), Ether Lipids: Biochemical and Biomedical Aspects, Academic Press, New York, 355-376 KlBcking H-P, Markwardt F, Hoffmann Anna 1985 Release of plasminogen activator by platelet-activating factor. Thromb Res 38:413416 Emeis J J. Kluft C 1985 PAF-Acether-induced release of tissuetype plasminogen activator from vessel walls. Blood 66:8&91 Blank M L, Cress E A, Whittle T, Snyder F 1981 In vivo metabolism of a new class of biologically active phospholipids: l-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a platelet activating hypotensive phospholipid. Life Sci 29:769%775 Kertscher H-P. Ostermann G, Lang A, Weissflog W, Gawrich K 1985 Synthese und biologische Aktivitat einiger stabiler Strukturanaloga des Plattchenaktivierenden Faktors. Pharmazie 40:702Z704 Kertscher H P, Ostermann G 1986 Synthesis of I-O-hexadecyl2-0-methyl-rat-glycero-3-phosphocholine and analogues. Chemistry and physics of lipids (in press) Murdmatsu T, Totani N, Mangold H K 1981 A facile method for the preparation of l-O-alkyl-2.O-acetoyl-.sn-glycero-3phosphocholines (platelet activating factor). Chem Phys Lipids 29: 121-127 Kliicking H-P, Bock G, Drawert J, Hinsenbrock K-P, Kaiser B, Sedlarik U 1976 iiber die Freisetzung von Plasminogenaktivatoren. Folia Haematol (Leipz) 103:404422 Markwardt F, Kllicking H-P 1978 Einflun von Mediatoren auf die Freisetzung von Plasminogenaktivatoren. Acta Biologica MedGerman37:1603-1610 Ostermann G, Till U, Thielmann K 1983 Studies on the stimulation of human blood platelets by semisynthetic plateletactivating factor. Thromb Res 30: 127-136 Ostermann G, Kertscher H-P, Lang A, Till U 1986 Stimulation and desensitization of human and rabbit platelets by I-0-hexadecyl-2-0-methyl-r~~-glycero-3-phosphocholine and analogues. Thromb Res 43:675-680. Tence M, Coeffier E, Heymans F, Polonsky J, Godfoid J-J, Benveniste J 1981 Structural analogs of platelet-activating factor (PAF-acether). Biochimie 63:723%727 Hadvary P, Baumgartner H R 1983 Activation of human and rabbit platelets by synthetic analogs of platelet activating factor. Thromb Res 30: 143 I56 O’Flaherty J T, Salzer W L, Cousart S, McCall C E. Plantadose C, Surles J R. Hammelt M J, Wykle R L 1983 Platelet-activating factor and analogues: Comparative studies with human neutrophils and rabbit platelets. Res Commun Chem Pathol Pharmacol39:291~309 Klocking H-P. Hoffmann A, Ostermann G, Kertscher H-P, Kliicking R. Eichhorn U 1985 Studies on the biological activity of stable analogues of platelet activating factor. IVth International Meeting of Danubian League against Thrombosis and Hemorrhagic Diseases. September 25-28. Istanbul, Turkey Blank M L, Cress E A. Lee T-C, Malone B, Surles J R. Piantadosi C, Hadju J. Snyder F 1982 Structural features of platelet activating factor (I-alkyl-2-acetyl-sn-glycero-3phosphocholine) required for hypotensive and platelet serotonin response. Res Commun Chem Pathol Pharmacol 38: 3-20 Markwardt F, Kliicking H-P 1976 Studies on the release of plasminogen activator. Thrombosis Research 8:217-223 Kliicking H-P 1979 Release of plasminogen activator by acetylcholine from the isolated perfused pig ear. Thromb Res 16:261-264 Markwardt F. Kliicking H-P 1977 Heparin-induced release of plasminogen activator.-Haemostas 6:j7&374 Klijckine H-P. Kl(ickine R. Helbig B 1984 Einflul3 von Phenolk~rperpolymeris~ten auf die Freisetzung von Plasminogenaktivatoren. Farmakol Toksikol47:93-96 (Russian)