Eglin C and Heparin Inhibition of Platelet Activation Induced by Cathepsin G or Human Neutrophils P. RENESTO, P. FERRER-LOPEZ, AND M. CHIGNARD Unitk de Pharmacologit Cellrrlaire UnitP associee IPIINSERM U28.5 In.siirut Pastertr. Paris , Fra n c.e
When purified human polymorphonuclear neutrophils (PMNs) and washed platelets are incubated together and challenged with N-formyl-Met-Leu-Phe (fMLP), platelet activation as measured by aggregation and serotonin release ensues. It has previously been demonstrated that such cell-to-cell cooperation is due to cathepsin G (cat G), a serine proteinase stored in the azurophilic granules of PMNs.’” Experiments with purified cat G have determined that the range of concentrations for platelet activation is between 2 and 8 ~ g / m l . ~ Eglin C, a proteinase inhibitor extracted from leeches, has the capability to interact with cat G.4 We have indeed shown that 2 pgiml(250 n M ) of recombinant human eglin C prevented the biochemical activity of 180 nM purified cat G , as measured spectrophotometrically by hydrolysis of N-succinyl-Ala-Ala-Pro-Pheparanitroanilide, a specific synthetic substrate. The very same eglin C concentration was able to inhibit platelet activation induced by 200 n M cat G, that is, a concentration inducing a submaximal platelet response. In fact, strictly comparable concentration-inhibition curves for biochemical and biologic cat G activities were obtained. Inhibition was specific (200 pg/ml eglin C failed to inhibit platelet activation induced by thrombin) and surmountable (1.2pg/ml eglin C inhibited, by 100% and 50%, platelet activation triggered by 150 and 200 nM cat G, respectively). Heparin is a glycosaminoglycan that, like eglin C, is able to inhibit cat G enzymatic activity.5 However, the maximal inhibition that we observed was only 60% (heparin concentration up to 100 U/ml). As for eglin C, we investigated the capability of heparin to suppress cat G-induced platelet activation. A concentration-dependent response curve within the range of 10 to 70 mU/ml was obtained. Contrary to what was observed with the biochemical activity of cat G , the biologic activity was totally suppressed by high heparin concentrations. In fact, this inhibition resulted most probably from an electrostatic interaction between the anionic heparin and the cationic cat G, inasmuch as the addition of 180 rnU/ml of protamine sulfate restored platelet activation. The inhibitory effects of eglin C and heparin were then tested on the PMNplatelet cooperation model stimulated by 5 x lo-.’ M fMLP. As expected, each substance displayed a concentration-dependent inhibition curve, with 25 pg/ml eglin C or 200 mUfml heparin totally inhibiting platelet stimulation induced by 1 and 2 ) . activated PMNs (TABLES These experiments show that eglin C and heparin can prevent platelet activation induced by fMLP-activated PMNs and confirm the participation of cat G in the interaction between PMNs and platelets. I n addition, as this cell-to-cell com321
8.1 1.1
0.6 3.2 0.7
2 -t
2
?
2
8.0 8.3
2 2
ND ND ND ND
37.0 63.7 86.3 90.3 95.1 97.0 98.5
Cat G Enzymatic Activitv 2.6 ? 3.7 37.4 2 15.4 62.3 2 13.3 82.9 14.4 91.6 2 14.1 93.7 2 3.7
ND ND ND ND ND
ND ND ND ND ND
*
Cat G-lnduced Serotonin Release
9.2 f 8.7 30.6 f 9.5 54.6 2 13.1 74.0 f 11.0 90.3 2 12.2 100.0 2 0.0
Cat G-Induced Platelet Aaregation
0.5 2 0.9 8.8 f 2.2 27.6 2 5.1
1.9 2 2.4 6.3 f 7.3 27.3 2 3.0 40.5 ? 8.9 48.5 2 12.1 96.3 f 5.4 100.0 .t 0.0
33.8 ? 9.5 56.4 2 10.1 83.4 t 23.3 94.8 ? 4.6
ND
ND ND ND
ND ND ND
ND
fMLP-Induced Serotonin Release
fMLP-Induced Platelet Aggregation
Each value is the mean
* SD of three to six distinct experiments. ND = not determined.
The enzymatic activity of 180 nM cat G was measured by hydrolysis of 1 mM N-succinyl-Ala-Ala-Pro-Phe-paranitroanilide (column 2). Cat G induced platelet aggregation (column 3) and serotonin release (column 4) were measured (2 x 108 platelets/ml) following challenge with 200 nM of the proteinase. fMLP-induced aggregation (column 5 ) and serotonin release (column 6) were measured on a mixture of PMNs ( 5 x 106 cells/ml) and platelets (2 x 108 cells/ml) preincubated with cytochalasin B ( 5 &mi) and challenged by 5 x lo-' M fMLP.
Eglin C was added at different concentrations (column 1) to different experimental systems, and its inhibitory effect, expressed as a percentage of the control, was calculated.
0.2 0.4 0.8 I .2 I .6 2.0 2.4 2.8 3.6 12.0 24.0
Eglin C (udml)
TABLE I. Inhibition by Eglin C of Biochemical and Biologic Cathepsin G Effects"
8
3
P m
v1
2
5
?
.e 0 w x
3s
*2 2 *6
Cat G Enzymatic Activity
ND 17.5 -t 1.2 ND 38.1 2 1.5 31.2 2 1.5 56.1 t 1.9 ND ND 60.6 2 4.3 61.7 -t 2.0 57.0 2 9.0
Heparin (U/ml)
0.02 0.03 0.04 0.05 0.07 0.1 0.2 0.3 1 .O 10 100
18.6 48.2 ? 73.3 2 94.8 ? 98.2 ? 100.0 ? ND ND ND ND ND 8.0 14.3 5.3 3.6 0.0
* 12.0
Cat G-Induced Platelet Aggregation 29.2 ? 8.9 42.4 ? 9.2 56.0 ? 14.0 79.4 ? 19.3 100.0 2 0.0 100.0 2 0.0 ND ND ND ND ND
Cat G-Induced Serotonin Release 1.5 5.6 9.6 9.3 13.3 12.7 12.3 0. ND ND ND 11.6 ? 14.5 ? 18.4 ? 29.1 ? 59.5 ? 80.6 ? 87.8 t 100.0 ?
fMLP-Induced Platelet Aggregation
Inhibition by Heparin of Biochemical and Biologic Cathepsin G Effects"
Same legend as in TABLEI . Each value is the mean 2 S D of five to seven distinct experiments.
TABLE 2.
11.4 ? 9.0 22.2 t 6.6 43.9 ? 26.7 36.1 ? 30.1 69.2 ? 16.3 63.2 ? 12.3 83.5 ? 16.5 91.2 ? 8.8 ND ND ND
fMLP-Induced Serotonin Release
5 2
324
ANNALS NEW YORK ACADEMY OF SCIENCES
munication may be relevant to some pulmonary diseases such as adult respiratory distress syndrome, antiproteinases could have a therapeutic role in such pathology . REFERENCES I. 2. 3. 4.
5.
CHIGNARD, M . , M. A. SELAK& J . B. SMITH.1986. Proc. Natl. Acad. Sci. USA 8 3 8609. SELAK,M. A., M. CHICNARD & J . B. SMITH.1988. Biochem. J . 251: 293. FERRER-LOPEZ, P., P. RENESTO, M . SCHATTNER, S. BASSOT,P. LAURENT& M . CHICNARD. 1990. Am. J . Physiol. 258: CI 100. SEEMULLER, U . , M. EULITZ, H. FRITZ& A. STROBL. 1980. Hoppe-Seyler's Z Physiol. Chem. 261: 1841. MAROSSY, K. 1981. Biochem. Biophys. Acta 659: 351.
When purified human polymorphonuclear neutrophils (PMNs) and washed platelets are incubated together and challenged with N-formyl-Met-Leu-Phe (fMLP), platelet activation as measured by aggregation and serotonin release ensues. It has previously been demonstrated that such cell-to-cell cooperation is due to cathepsin G (cat G), a serine proteinase stored in the azurophilic granules of PMNs.’” Experiments with purified cat G have determined that the range of concentrations for platelet activation is between 2 and 8 ~ g / m l . ~ Eglin C, a proteinase inhibitor extracted from leeches, has the capability to interact with cat G.4 We have indeed shown that 2 pgiml(250 n M ) of recombinant human eglin C prevented the biochemical activity of 180 nM purified cat G , as measured spectrophotometrically by hydrolysis of N-succinyl-Ala-Ala-Pro-Pheparanitroanilide, a specific synthetic substrate. The very same eglin C concentration was able to inhibit platelet activation induced by 200 n M cat G, that is, a concentration inducing a submaximal platelet response. In fact, strictly comparable concentration-inhibition curves for biochemical and biologic cat G activities were obtained. Inhibition was specific (200 pg/ml eglin C failed to inhibit platelet activation induced by thrombin) and surmountable (1.2pg/ml eglin C inhibited, by 100% and 50%, platelet activation triggered by 150 and 200 nM cat G, respectively). Heparin is a glycosaminoglycan that, like eglin C, is able to inhibit cat G enzymatic activity.5 However, the maximal inhibition that we observed was only 60% (heparin concentration up to 100 U/ml). As for eglin C, we investigated the capability of heparin to suppress cat G-induced platelet activation. A concentration-dependent response curve within the range of 10 to 70 mU/ml was obtained. Contrary to what was observed with the biochemical activity of cat G , the biologic activity was totally suppressed by high heparin concentrations. In fact, this inhibition resulted most probably from an electrostatic interaction between the anionic heparin and the cationic cat G, inasmuch as the addition of 180 rnU/ml of protamine sulfate restored platelet activation. The inhibitory effects of eglin C and heparin were then tested on the PMNplatelet cooperation model stimulated by 5 x lo-.’ M fMLP. As expected, each substance displayed a concentration-dependent inhibition curve, with 25 pg/ml eglin C or 200 mUfml heparin totally inhibiting platelet stimulation induced by 1 and 2 ) . activated PMNs (TABLES These experiments show that eglin C and heparin can prevent platelet activation induced by fMLP-activated PMNs and confirm the participation of cat G in the interaction between PMNs and platelets. I n addition, as this cell-to-cell com321
8.1 1.1
0.6 3.2 0.7
2 -t
2
?
2
8.0 8.3
2 2
ND ND ND ND
37.0 63.7 86.3 90.3 95.1 97.0 98.5
Cat G Enzymatic Activitv 2.6 ? 3.7 37.4 2 15.4 62.3 2 13.3 82.9 14.4 91.6 2 14.1 93.7 2 3.7
ND ND ND ND ND
ND ND ND ND ND
*
Cat G-lnduced Serotonin Release
9.2 f 8.7 30.6 f 9.5 54.6 2 13.1 74.0 f 11.0 90.3 2 12.2 100.0 2 0.0
Cat G-Induced Platelet Aaregation
0.5 2 0.9 8.8 f 2.2 27.6 2 5.1
1.9 2 2.4 6.3 f 7.3 27.3 2 3.0 40.5 ? 8.9 48.5 2 12.1 96.3 f 5.4 100.0 .t 0.0
33.8 ? 9.5 56.4 2 10.1 83.4 t 23.3 94.8 ? 4.6
ND
ND ND ND
ND ND ND
ND
fMLP-Induced Serotonin Release
fMLP-Induced Platelet Aggregation
Each value is the mean
* SD of three to six distinct experiments. ND = not determined.
The enzymatic activity of 180 nM cat G was measured by hydrolysis of 1 mM N-succinyl-Ala-Ala-Pro-Phe-paranitroanilide (column 2). Cat G induced platelet aggregation (column 3) and serotonin release (column 4) were measured (2 x 108 platelets/ml) following challenge with 200 nM of the proteinase. fMLP-induced aggregation (column 5 ) and serotonin release (column 6) were measured on a mixture of PMNs ( 5 x 106 cells/ml) and platelets (2 x 108 cells/ml) preincubated with cytochalasin B ( 5 &mi) and challenged by 5 x lo-' M fMLP.
Eglin C was added at different concentrations (column 1) to different experimental systems, and its inhibitory effect, expressed as a percentage of the control, was calculated.
0.2 0.4 0.8 I .2 I .6 2.0 2.4 2.8 3.6 12.0 24.0
Eglin C (udml)
TABLE I. Inhibition by Eglin C of Biochemical and Biologic Cathepsin G Effects"
8
3
P m
v1
2
5
?
.e 0 w x
3s
*2 2 *6
Cat G Enzymatic Activity
ND 17.5 -t 1.2 ND 38.1 2 1.5 31.2 2 1.5 56.1 t 1.9 ND ND 60.6 2 4.3 61.7 -t 2.0 57.0 2 9.0
Heparin (U/ml)
0.02 0.03 0.04 0.05 0.07 0.1 0.2 0.3 1 .O 10 100
18.6 48.2 ? 73.3 2 94.8 ? 98.2 ? 100.0 ? ND ND ND ND ND 8.0 14.3 5.3 3.6 0.0
* 12.0
Cat G-Induced Platelet Aggregation 29.2 ? 8.9 42.4 ? 9.2 56.0 ? 14.0 79.4 ? 19.3 100.0 2 0.0 100.0 2 0.0 ND ND ND ND ND
Cat G-Induced Serotonin Release 1.5 5.6 9.6 9.3 13.3 12.7 12.3 0. ND ND ND 11.6 ? 14.5 ? 18.4 ? 29.1 ? 59.5 ? 80.6 ? 87.8 t 100.0 ?
fMLP-Induced Platelet Aggregation
Inhibition by Heparin of Biochemical and Biologic Cathepsin G Effects"
Same legend as in TABLEI . Each value is the mean 2 S D of five to seven distinct experiments.
TABLE 2.
11.4 ? 9.0 22.2 t 6.6 43.9 ? 26.7 36.1 ? 30.1 69.2 ? 16.3 63.2 ? 12.3 83.5 ? 16.5 91.2 ? 8.8 ND ND ND
fMLP-Induced Serotonin Release
5 2
324
ANNALS NEW YORK ACADEMY OF SCIENCES
munication may be relevant to some pulmonary diseases such as adult respiratory distress syndrome, antiproteinases could have a therapeutic role in such pathology . REFERENCES I. 2. 3. 4.
5.
CHIGNARD, M . , M. A. SELAK& J . B. SMITH.1986. Proc. Natl. Acad. Sci. USA 8 3 8609. SELAK,M. A., M. CHICNARD & J . B. SMITH.1988. Biochem. J . 251: 293. FERRER-LOPEZ, P., P. RENESTO, M . SCHATTNER, S. BASSOT,P. LAURENT& M . CHICNARD. 1990. Am. J . Physiol. 258: CI 100. SEEMULLER, U . , M. EULITZ, H. FRITZ& A. STROBL. 1980. Hoppe-Seyler's Z Physiol. Chem. 261: 1841. MAROSSY, K. 1981. Biochem. Biophys. Acta 659: 351.
