Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
CLINICAL TOXICOLOGY 8(1), pp. 43-52 (1975)
Alkaloids of Tobacco and Blood Coagulation: Effect of Nicotine on Thrombin and Fibrinogen*
JASBIR M. SINGHt AND MELBRA DIANE SINGH Department of Pharmacology Xavier University College of Pharmacy New Orleans, Louisiana
INTRODUCTION The oral administration of nicotine equivalent to one pack of cigarettes per day and an atherogenic diet for a period of 12 to 24 weeks decreased the blood clotting time of rabbits, and this effect w a s greater with a nicotine and atherogenic diet than with nicotine or atherogenic diet alone [ 1, 21. Singh has reported also that nicotine and hypercholesterolemic diet decreased the blood-clotting time of r a t s [ 31. Nicotine decreased the blood-clotting time of human blood in vitro, and this effect was blocked partially by benodaine, an adrenergic blocking agent [ 4, 51. The intravenous administration of 0.01 o r 0.02 mg/kg of nicotine to rabbits significantly decreased the blood-clotting time as measured by the Lee-White capillary tube method, and this effect was also blocked by prior administration of benodaine [ 61. Benodaine also blocked the effect of epinephrine on
*A preliminary report appeared in The Pharmacologist, 12, 245, (1970). SPresent address: Alcoholism Services Unit, Department of Psychiatry, Charity Hospital of Louisiana, New Orleans, Louisiana. Reprint request: Jasbir M. Singh, Ph.D., Alcoholism Out Patient Treatment Clinic, 3901 Houma Blvd., Metairie, La. 70002.
43 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
SINGH AND SINGH
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
44
blood-clotting time. From these experiments it w a s suggested that nicotine possibly affected the blood-clotting time, either directly o r indirectly, through the release of epinephrine. Nicotine and epinephrine affected the blood-clotting and prothrombin time of human plasma [7]. In order for the blood to clot, prothrombin is converted to thrombin, thereby reacting with fibrinogen to form a fibrin clot [ 81. The purpose of the present investigation was to report the effect of nicotine on the clot-forming property of the enzyme, thrombin, on the substrate, fibrinogen (thrombin time). METHOD Plasma Tubes containing 5 to 10 ml of unexpired human plasma was stored in the freezer. The frozen plasma was thawed at 37.5"C before each experiment, and the experiment w a s completed within 40 min. Thrombin Thrombin (Thrombin, Upjohn, 1000 units) w a s dissolved in 10 ml of glycerine. The desired dilutions of the stock solution were made with saline, and the solution was kept at 4°C throughout the experiment. Fibrinogen
A 1%solution of fibrinogen (fibro-AHF, Merck, Sharp & Dohme) w a s prepared in vehicle provided by the pharmaceutical house (sterile, pyrogen-free distilled water). The tubes containing 1 ml of 1% solution of fibrinogen were stored in the freezer. Fibrinogen solution w a s thawed at 37.5"C before each experiment. Nicotine Tartarate The 1%stock solution of nicotine tartarate w a s made in saline.
NICOTINE, THROMBIN, AND FIBRJNOGEN
45
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
Procedure
Our experimental approach is based on the fact that fibrinogen in plasma reacts with thrombin to form a clot [ 81. In the following experiments, plasma o r purified fibrinogen substrate were used to observe the effect of thrombin, Frozen plasma was thawed and then incubated with various drug concentrations for 5 min at 37.5"C. Plasma o r fibrinogen (0.2 ml) was placed in the boat of the Machrolab Dual Clot Time (MDCT) while the thrombin solution (0.1 ml) was placed on the rotor. Dual determinations were made with the MDCT and both clotting times were determined automatically. This method is referred to as "one stage thrombin time" (OSTT), which is an indicator of the clot-forming property of thrombin on the fibrinogen. All the experiments were performed within 40 min. All the materials except thrombin were pre-incubated for 5 min at 37.5"C. The following series of experiments were performed. 1. Plasma as a substrate-. To 0.2 ml of plasma plus saline Icontroll o r plasma d u s various concentrations of nicotine bitartarate, 0.1 m l thrombin solution was added and the OSTT was determined with the automatic Machrolab Dual Clot Timer (MDCT). 2. To 0.2 ml of plasma, 0.1 ml of thrombin solution (0.5 to 1 units) plus saline (control) o r thrombin plus various concentrations of nicotine was added, and OSTT was determined using the MDCT. 3. Fibrinogen as a substrate. To 0.2 ml of fibrinogen containing saline (control) o r nicotine, 0.1 ml of thrombin was added, and OSTT was determined using the MDCT. 4. To 0.2 ml of fibrinogen containing nicotine, 0.2 ml of thrombincontaining saline (control)or thrombin-containing nicotine was added, and OSTT was determined with the MDCT. 5. Using incubated plasma o r fibrinogen as a substrate. In this series of experiments, plasma o r fibrinogen solution mixed with 0.1 ml of saline (control) o r 0.1 ml of nicotine was incubated for 30 min, To 0.2 ml of substrate, 0.1 ml of thrombin (1 unit) was added, and OSTT was determined using one unit of thrombin at various time intervals with the MDCT. RESULTS The effect of various concentrations of nicotine on the clot-forming activity of thrombin on the substrate, containing nicotine-plasma
SINGH AND SINGH
46
than
+551
+%t
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
+45
4. 51
400
\
\ \
NICOTINE
W
CONC
I N 0.1 cc of CM
FIG. 1. Effect of nicotine on the clot-forming activity of thrombin on the nicotine containing substrate p l a s m a The clotting time values are expressed in seconds. The mean percentage change (MPC)is calculated from the control values. The experiments were performed in duplicate, and if more than one second difference was observed between the two values, the experiments were repeated. (-) o r (+) signs indicate decreased or increased clotting time. In these experiments nicotine was added to the plasma. (thrombin time), is shown in Fig. 1. The clot-forming activity of thrombin is retarded when various concentrations of nicotine are added to the substrate plasma. OSTT was more than 355 sec when the nicotine concentrations were 333.3 and 166.62 p g per 0.1 cc of clotting mixture. By decreasing the concentration of nicotine in the clotting mixture, the clot-forming activity of the enzyme is accelerated. Clot-forming activity of nicotine-containing thrombin on plasma is shown in Fig. 2. Nicotine changed the clot-forming activity of
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
NICOTINE, THROMBIN, AND FIBRINOGEN
5
E
- 0
47
l66.6 833 41.6 2OB 1Q4 5.2 conc of nicotine in 0.1 cc of CM
FIG. 2. Reaction to the clot-forming activity of nicotinecontaining thrombin on the plasma substrate. The clotting values are expressed in seconds. The mean percentage (MPC) change is calculated from control values. The experiments are performed in duplicate and in our experiments a 1-sec difference was acceptable. A (+) sign indicates increased clotting time. In these experiments, nicotine was added to thrombin, and 0.1 ml (1 unit) of nicotine-containing thrombin was added to 0.2 ml of p l a s m a thrombin. No clot was detected when the concentration of thrombin containing various concentrations of nicotine was 0.2 units in the clotting mixture. However, a clot was detected when the concentration of thrombin was sufficiently high to react with fibrinogen, Reaction of thrombin on the nicotine-containing fibrinogen is shown in Fig. 3. In these experiments different concentrations of nicotine were added to fibrinogen, Nicotine retarded the clot-forming activity of thrombin when the fibrinogen contained nicotine. The effect on fibrinogen was dependent on the concentrations of thrombin and nicotine, and this effect was greater when 2 units of thrombin was added to the clotting mixture. Clot-forming activity of nicotine-containing thrombin on nicotinecontaining fibrinogen is shown in Fig. 4. Nicotine retarded the clotforming activity of thrombin and fibrinogen, and this effect was dose dependent. The effect of thrombin (1 unit) on plasma incubated for 30 min with various concentrations of nicotine is reported in Table 1. Nicotine retarded the clot-forming activity of thrombin at 10 and 30 min of incubation.
SINGH AND SINGH
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
48
FIG. 3. Reaction of thrombin on the fibrinogen substrate that contained nicotine. The clotting values are expressed in seconds. The mean percentage value is calculated from the control (saline) values. The experiments are performed in duplicate, and if more than a 1-sec difference between the two values was recorded with a MDCT, the experiment was repeated. In these experiments, nicotine was added to fibrinogen substrate (Fibro-AHF, Merck, Sharpe & Dohme). The effect of thrombin (1 unit) on the incubated nicotinecontaining fibrinogen i s shown in Table 2. The results of this series of experiments also showed the retarding effect of thrombin on the incubated fibrinogen and this effect was also dose dependent. DISCUSSION
Our previous work has shown that nicotine exerts a biphasic effect, retarded o r accelerated, on human blood coagulation and plasma prothrombin time [ 3-51. In order for the blood to clot,
49
NICOTINE, THROMBIN, AND FIBRINOGEN
f
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
.-c
MPC
greater than
500%
\
C 0
Q
E
166.6
11x3
91.6
20.H
10.6
conc of nicotine in CM, 0.1 ml
FIG. 4. Reaction of the clot-promoting activity of nicotinecontaining thrombin o r the nicotine-containing fibrinogen substrate. The clotting time values are expressed in seconds. The mean percentage change is calculated from control values. The experiments are performed in duplicate, and if more than 1-sec difference was observed between the two values, the experiment was repeated. (+) signs indicate increased clotting time. In these experiments 0.1 U of the thrombin-nicotine mixture (1 unit) was added to 0.2 ml of fibrinogen-nicotine mixture. prothrombin is converted to thrombin to react with the fibrinogen to form a fibrin clot [ 81. The purpose of the present investigation was to find out the effect of nicotine on the clot-forming activity of thrombin on fibrinogen. The thrombin-fibrinogen reaction, which the thrombin time measures, is a two-stage reaction. The first is enzymatic and consists of splitting fibrinopeptide A and (later) B from fibrinogen to form the fibrin monomer. The second step consists of the monomer polymerization forming a visible gel [ 81. Our model, which measures thrombin time o r clot-forming activity of thrombin on plasma or fibrinogen, indicates that nicotine exerts its effect on thrombin and fibrinogen. The thombin time is either retarded or accelerated by nicotine, depending upon the concentrations used. Therefore, it is proposed that nicotine retards or accelerates the blood coagulation time and by this process, in
21.3 24,l
60.40
90.60 +18.8
+3.9 26.0
27.0 -20.3
+22.2
+4.5
-
%change
30.9
30.1
24.8
23.7
30
+23.3
+27.0
+4.6
-
% change
The clotting time values a r e expressed in seconds. The percentage change was calculated from control values. The experiments were performed in duplicate in a dual MDCT and i f more than 1-sec difference was observed between the two values, the experiment was repeated. (+) o r (-) signs indicate retarded or accelerated blood-clotting effects.
a
19.1
30.20 23.1
22.1
-
20.5
Control
-6.8
10
%change
0
Conc. of nicotine in clotting mixture, pg/O. 1 ml
Incubation time, min
TABLE 1. Effect of Thrombin (1 unit) on the Incubated Nicotine-Containing Plasmaa
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
@ li
0
cn
18.9 32.7 37.1 56.1
Control
30.22
60.62 90.62 +96.0 +150.3
+73.0
-
%Change
39.8 107.7
27.0 37.0
10
+67.3
+47.0
+37.0
-
%change
Incubation time, min
48.6 76.3
38.8 47.9
30
+25.2 ~73.4
+23.4
-
% change
The clotting time values are expressed in seconds. The percentage change was calculated from control values. The experiments were performed in duplicate in a dual MDCT and if more than 1-sec difference w a s observed between the two values, the experiment was repeated. (+) sign indicates retarding effect. In these experiments nicotine-containing fibrinogen was incubated for 30 min.
a
0
Conc. of nicotine in clotting mixture, ml
TABLE 2. Effect of Thrombin (1 unit) on the Incubated Nicotine-Containing Fibrinogena
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
z
i3
0
Ez
8tl
->8
z
SINGH AND SINGH
52
addition to the effect on the prothrombin time, the clot-forming property of thrombin on fibrinogen is increased o r decreased.
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
SUMMARY An alkaloid of tobacco, nicotine, affected the clot-formation property of the enzyme, thrombin, on the substrate, plasma o r fibrinogen (thrombin time). Higher concentrations of nicotine retarded the clotformation property of thrombin. By decreasing the nicotine concentration in the clotting mixture, the clotting time of thrombin w a ~ accelerated. Nicotine also modified the clot formation property of plasma o r fibrinogen to thrombin, and this effect was dose dependent. From this experimental evidence, it is suggested that nicotine does alter the clot-forming properties of thrombin on fibrinogen. ACKNOWLEDGMENT Supported by an Edward G. Schlieder Foundation Grant. REFERENCES
[ 11 J. M. Singh, Effect of nicotine on blood clotting of albino rats fed atherogenic diet, Arch. Int. Pharmacodyn., 154,221 (1965). [2] J. M. Singh and Y. T. Oester, Possible effect of nicotine on in vitro human blood coagulation time, Arch. Int. Pharmacodyn.,
148 237 (1964). [ 31 T M . Singh and Y. T. Oester, Nicotine antagonism with heparin: Possible mode of action on human blood coagulation, Arch. Int. Pharmacodyn., 149, 354 (1964). [ 4 ] J. M. Singh and Y. T. Oester, Effect of nicotine on prothrombin time and its possible mode of action, Arch. Int. Pharmacodyn.,
154. 435 (1964). [ 51 T G . Wenzel, 'J. M. Singh, and J. A. Turner, Chronic effects of
[ 61 [ 71 [8]
[ 91
orally administered nicotine in cholesterol fed rabbits, Ann. N.Y. Acad. Sci., 3, 302 (1960). D. G. Wenzel and J. M. Singh, Effect of nicotine and epinephrine on in vivo blood coagulation time, J. Pharm. Sci., 5-,l 875 (1963). D. G. Wenzel, J. A. Turner, S. W. Jordan, and J. M. Singh, Cardiovascular interactions of nicotine, ergonovine, and hyper cholesterolemia in the rabbits, Circ. Res., 9, 694 (1961). D. G. Wenzel and J. M. Singh, Effect of nicotine and epinephrine on in vivo blood coagulation time, J. Pharm. Sci., 5J 875 (1963). W. H. Seegers, Prothrombin, Harvard University Press, Cambridge, Mass., 1962.
CLINICAL TOXICOLOGY 8(1), pp. 43-52 (1975)
Alkaloids of Tobacco and Blood Coagulation: Effect of Nicotine on Thrombin and Fibrinogen*
JASBIR M. SINGHt AND MELBRA DIANE SINGH Department of Pharmacology Xavier University College of Pharmacy New Orleans, Louisiana
INTRODUCTION The oral administration of nicotine equivalent to one pack of cigarettes per day and an atherogenic diet for a period of 12 to 24 weeks decreased the blood clotting time of rabbits, and this effect w a s greater with a nicotine and atherogenic diet than with nicotine or atherogenic diet alone [ 1, 21. Singh has reported also that nicotine and hypercholesterolemic diet decreased the blood-clotting time of r a t s [ 31. Nicotine decreased the blood-clotting time of human blood in vitro, and this effect was blocked partially by benodaine, an adrenergic blocking agent [ 4, 51. The intravenous administration of 0.01 o r 0.02 mg/kg of nicotine to rabbits significantly decreased the blood-clotting time as measured by the Lee-White capillary tube method, and this effect was also blocked by prior administration of benodaine [ 61. Benodaine also blocked the effect of epinephrine on
*A preliminary report appeared in The Pharmacologist, 12, 245, (1970). SPresent address: Alcoholism Services Unit, Department of Psychiatry, Charity Hospital of Louisiana, New Orleans, Louisiana. Reprint request: Jasbir M. Singh, Ph.D., Alcoholism Out Patient Treatment Clinic, 3901 Houma Blvd., Metairie, La. 70002.
43 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
SINGH AND SINGH
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
44
blood-clotting time. From these experiments it w a s suggested that nicotine possibly affected the blood-clotting time, either directly o r indirectly, through the release of epinephrine. Nicotine and epinephrine affected the blood-clotting and prothrombin time of human plasma [7]. In order for the blood to clot, prothrombin is converted to thrombin, thereby reacting with fibrinogen to form a fibrin clot [ 81. The purpose of the present investigation was to report the effect of nicotine on the clot-forming property of the enzyme, thrombin, on the substrate, fibrinogen (thrombin time). METHOD Plasma Tubes containing 5 to 10 ml of unexpired human plasma was stored in the freezer. The frozen plasma was thawed at 37.5"C before each experiment, and the experiment w a s completed within 40 min. Thrombin Thrombin (Thrombin, Upjohn, 1000 units) w a s dissolved in 10 ml of glycerine. The desired dilutions of the stock solution were made with saline, and the solution was kept at 4°C throughout the experiment. Fibrinogen
A 1%solution of fibrinogen (fibro-AHF, Merck, Sharp & Dohme) w a s prepared in vehicle provided by the pharmaceutical house (sterile, pyrogen-free distilled water). The tubes containing 1 ml of 1% solution of fibrinogen were stored in the freezer. Fibrinogen solution w a s thawed at 37.5"C before each experiment. Nicotine Tartarate The 1%stock solution of nicotine tartarate w a s made in saline.
NICOTINE, THROMBIN, AND FIBRJNOGEN
45
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
Procedure
Our experimental approach is based on the fact that fibrinogen in plasma reacts with thrombin to form a clot [ 81. In the following experiments, plasma o r purified fibrinogen substrate were used to observe the effect of thrombin, Frozen plasma was thawed and then incubated with various drug concentrations for 5 min at 37.5"C. Plasma o r fibrinogen (0.2 ml) was placed in the boat of the Machrolab Dual Clot Time (MDCT) while the thrombin solution (0.1 ml) was placed on the rotor. Dual determinations were made with the MDCT and both clotting times were determined automatically. This method is referred to as "one stage thrombin time" (OSTT), which is an indicator of the clot-forming property of thrombin on the fibrinogen. All the experiments were performed within 40 min. All the materials except thrombin were pre-incubated for 5 min at 37.5"C. The following series of experiments were performed. 1. Plasma as a substrate-. To 0.2 ml of plasma plus saline Icontroll o r plasma d u s various concentrations of nicotine bitartarate, 0.1 m l thrombin solution was added and the OSTT was determined with the automatic Machrolab Dual Clot Timer (MDCT). 2. To 0.2 ml of plasma, 0.1 ml of thrombin solution (0.5 to 1 units) plus saline (control) o r thrombin plus various concentrations of nicotine was added, and OSTT was determined using the MDCT. 3. Fibrinogen as a substrate. To 0.2 ml of fibrinogen containing saline (control) o r nicotine, 0.1 ml of thrombin was added, and OSTT was determined using the MDCT. 4. To 0.2 ml of fibrinogen containing nicotine, 0.2 ml of thrombincontaining saline (control)or thrombin-containing nicotine was added, and OSTT was determined with the MDCT. 5. Using incubated plasma o r fibrinogen as a substrate. In this series of experiments, plasma o r fibrinogen solution mixed with 0.1 ml of saline (control) o r 0.1 ml of nicotine was incubated for 30 min, To 0.2 ml of substrate, 0.1 ml of thrombin (1 unit) was added, and OSTT was determined using one unit of thrombin at various time intervals with the MDCT. RESULTS The effect of various concentrations of nicotine on the clot-forming activity of thrombin on the substrate, containing nicotine-plasma
SINGH AND SINGH
46
than
+551
+%t
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
+45
4. 51
400
\
\ \
NICOTINE
W
CONC
I N 0.1 cc of CM
FIG. 1. Effect of nicotine on the clot-forming activity of thrombin on the nicotine containing substrate p l a s m a The clotting time values are expressed in seconds. The mean percentage change (MPC)is calculated from the control values. The experiments were performed in duplicate, and if more than one second difference was observed between the two values, the experiments were repeated. (-) o r (+) signs indicate decreased or increased clotting time. In these experiments nicotine was added to the plasma. (thrombin time), is shown in Fig. 1. The clot-forming activity of thrombin is retarded when various concentrations of nicotine are added to the substrate plasma. OSTT was more than 355 sec when the nicotine concentrations were 333.3 and 166.62 p g per 0.1 cc of clotting mixture. By decreasing the concentration of nicotine in the clotting mixture, the clot-forming activity of the enzyme is accelerated. Clot-forming activity of nicotine-containing thrombin on plasma is shown in Fig. 2. Nicotine changed the clot-forming activity of
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
NICOTINE, THROMBIN, AND FIBRINOGEN
5
E
- 0
47
l66.6 833 41.6 2OB 1Q4 5.2 conc of nicotine in 0.1 cc of CM
FIG. 2. Reaction to the clot-forming activity of nicotinecontaining thrombin on the plasma substrate. The clotting values are expressed in seconds. The mean percentage (MPC) change is calculated from control values. The experiments are performed in duplicate and in our experiments a 1-sec difference was acceptable. A (+) sign indicates increased clotting time. In these experiments, nicotine was added to thrombin, and 0.1 ml (1 unit) of nicotine-containing thrombin was added to 0.2 ml of p l a s m a thrombin. No clot was detected when the concentration of thrombin containing various concentrations of nicotine was 0.2 units in the clotting mixture. However, a clot was detected when the concentration of thrombin was sufficiently high to react with fibrinogen, Reaction of thrombin on the nicotine-containing fibrinogen is shown in Fig. 3. In these experiments different concentrations of nicotine were added to fibrinogen, Nicotine retarded the clot-forming activity of thrombin when the fibrinogen contained nicotine. The effect on fibrinogen was dependent on the concentrations of thrombin and nicotine, and this effect was greater when 2 units of thrombin was added to the clotting mixture. Clot-forming activity of nicotine-containing thrombin on nicotinecontaining fibrinogen is shown in Fig. 4. Nicotine retarded the clotforming activity of thrombin and fibrinogen, and this effect was dose dependent. The effect of thrombin (1 unit) on plasma incubated for 30 min with various concentrations of nicotine is reported in Table 1. Nicotine retarded the clot-forming activity of thrombin at 10 and 30 min of incubation.
SINGH AND SINGH
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
48
FIG. 3. Reaction of thrombin on the fibrinogen substrate that contained nicotine. The clotting values are expressed in seconds. The mean percentage value is calculated from the control (saline) values. The experiments are performed in duplicate, and if more than a 1-sec difference between the two values was recorded with a MDCT, the experiment was repeated. In these experiments, nicotine was added to fibrinogen substrate (Fibro-AHF, Merck, Sharpe & Dohme). The effect of thrombin (1 unit) on the incubated nicotinecontaining fibrinogen i s shown in Table 2. The results of this series of experiments also showed the retarding effect of thrombin on the incubated fibrinogen and this effect was also dose dependent. DISCUSSION
Our previous work has shown that nicotine exerts a biphasic effect, retarded o r accelerated, on human blood coagulation and plasma prothrombin time [ 3-51. In order for the blood to clot,
49
NICOTINE, THROMBIN, AND FIBRINOGEN
f
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
.-c
MPC
greater than
500%
\
C 0
Q
E
166.6
11x3
91.6
20.H
10.6
conc of nicotine in CM, 0.1 ml
FIG. 4. Reaction of the clot-promoting activity of nicotinecontaining thrombin o r the nicotine-containing fibrinogen substrate. The clotting time values are expressed in seconds. The mean percentage change is calculated from control values. The experiments are performed in duplicate, and if more than 1-sec difference was observed between the two values, the experiment was repeated. (+) signs indicate increased clotting time. In these experiments 0.1 U of the thrombin-nicotine mixture (1 unit) was added to 0.2 ml of fibrinogen-nicotine mixture. prothrombin is converted to thrombin to react with the fibrinogen to form a fibrin clot [ 81. The purpose of the present investigation was to find out the effect of nicotine on the clot-forming activity of thrombin on fibrinogen. The thrombin-fibrinogen reaction, which the thrombin time measures, is a two-stage reaction. The first is enzymatic and consists of splitting fibrinopeptide A and (later) B from fibrinogen to form the fibrin monomer. The second step consists of the monomer polymerization forming a visible gel [ 81. Our model, which measures thrombin time o r clot-forming activity of thrombin on plasma or fibrinogen, indicates that nicotine exerts its effect on thrombin and fibrinogen. The thombin time is either retarded or accelerated by nicotine, depending upon the concentrations used. Therefore, it is proposed that nicotine retards or accelerates the blood coagulation time and by this process, in
21.3 24,l
60.40
90.60 +18.8
+3.9 26.0
27.0 -20.3
+22.2
+4.5
-
%change
30.9
30.1
24.8
23.7
30
+23.3
+27.0
+4.6
-
% change
The clotting time values a r e expressed in seconds. The percentage change was calculated from control values. The experiments were performed in duplicate in a dual MDCT and i f more than 1-sec difference was observed between the two values, the experiment was repeated. (+) o r (-) signs indicate retarded or accelerated blood-clotting effects.
a
19.1
30.20 23.1
22.1
-
20.5
Control
-6.8
10
%change
0
Conc. of nicotine in clotting mixture, pg/O. 1 ml
Incubation time, min
TABLE 1. Effect of Thrombin (1 unit) on the Incubated Nicotine-Containing Plasmaa
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
@ li
0
cn
18.9 32.7 37.1 56.1
Control
30.22
60.62 90.62 +96.0 +150.3
+73.0
-
%Change
39.8 107.7
27.0 37.0
10
+67.3
+47.0
+37.0
-
%change
Incubation time, min
48.6 76.3
38.8 47.9
30
+25.2 ~73.4
+23.4
-
% change
The clotting time values are expressed in seconds. The percentage change was calculated from control values. The experiments were performed in duplicate in a dual MDCT and if more than 1-sec difference w a s observed between the two values, the experiment was repeated. (+) sign indicates retarding effect. In these experiments nicotine-containing fibrinogen was incubated for 30 min.
a
0
Conc. of nicotine in clotting mixture, ml
TABLE 2. Effect of Thrombin (1 unit) on the Incubated Nicotine-Containing Fibrinogena
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
z
i3
0
Ez
8tl
->8
z
SINGH AND SINGH
52
addition to the effect on the prothrombin time, the clot-forming property of thrombin on fibrinogen is increased o r decreased.
Clinical Toxicology Downloaded from informahealthcare.com by Kainan University on 04/05/15 For personal use only.
SUMMARY An alkaloid of tobacco, nicotine, affected the clot-formation property of the enzyme, thrombin, on the substrate, plasma o r fibrinogen (thrombin time). Higher concentrations of nicotine retarded the clotformation property of thrombin. By decreasing the nicotine concentration in the clotting mixture, the clotting time of thrombin w a ~ accelerated. Nicotine also modified the clot formation property of plasma o r fibrinogen to thrombin, and this effect was dose dependent. From this experimental evidence, it is suggested that nicotine does alter the clot-forming properties of thrombin on fibrinogen. ACKNOWLEDGMENT Supported by an Edward G. Schlieder Foundation Grant. REFERENCES
[ 11 J. M. Singh, Effect of nicotine on blood clotting of albino rats fed atherogenic diet, Arch. Int. Pharmacodyn., 154,221 (1965). [2] J. M. Singh and Y. T. Oester, Possible effect of nicotine on in vitro human blood coagulation time, Arch. Int. Pharmacodyn.,
148 237 (1964). [ 31 T M . Singh and Y. T. Oester, Nicotine antagonism with heparin: Possible mode of action on human blood coagulation, Arch. Int. Pharmacodyn., 149, 354 (1964). [ 4 ] J. M. Singh and Y. T. Oester, Effect of nicotine on prothrombin time and its possible mode of action, Arch. Int. Pharmacodyn.,
154. 435 (1964). [ 51 T G . Wenzel, 'J. M. Singh, and J. A. Turner, Chronic effects of
[ 61 [ 71 [8]
[ 91
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