The antihypertensive drug, captopril, exerted an anticoagulant effect upon clotting time as followed by prothrombin time (PT) and activated partial thromboplastin time (APTT), with prolongation of clotting observed at 4.25 × 10−3 M and 4 × 10−3 M captopril for PT and APTT, respectively, using commercial level I plasma. Utilizing level III plasmas, PT and APTT values were both prolonged by 4.25 × 10−3 M captopril. Captopril (6 × 10−3 M) also directly prolonged the clotting of thrombin in a thrombin-factor II-deficient plasma assay, whereas 5 × 10−3 M captopril inhibited FIIDP in a thrombin-FIIDP assay. In thrombin-fibrinogen assays, pre-incubation of 2.5 × 10−3 M captopril with fibrinogen also prolonged clotting time, whereas 3 × 10−3 M captopril prolonged thrombin activity. These data suggest that thiol-disulfide exchange permits reduction of disulfide groups in thrombin and fibrinogen, altering their tertiary structure and physiological function. Lisinopril at a pharmacological 10−2 M also prolonged APTT, although it lacks a thiol group. Polylysine (1k-4k) affected a prolongation of APTT at 6.7 × 10−6 M, suggesting inhibition of clotting by a different mechanism.
From the Department of Chemistry, Bowling Green State University, Bowling Green, Ohio.
Received for publication October, 23, 2006; accepted August 24, 2007.
The support of the National Science Foundation through their Research Experiences for Undergraduates program helped to support KDG and the Office of Undergraduate Research at Bowling Green State University, which generously supported SJM.
The authors gratefully acknowledge the gift of captopril from the Squibb Institute for Medical Research (Princeton, NJ) and the gift of lisinopril from Merck Research Laboratories (Rahway, NJ).
The authors state that they have no financial interest in the products mentioned within this article.
Reprints: Arthur S. Brcher, PhD, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403 (e-mail: firstname.lastname@example.org).
Cardiovascular disease and hypertension have been linked together recently in an increasing number of ways as a consequence of studies on antihypertensive drugs. In several studies, it has been reported that angiotensin-converting enzyme (ACE) inhibitors such as captopril, enalapril, and lisinopril can lower both morbidity and mortality rates of patients with myocardial infarction (MI) and ischemic heart disease. Indeed, captopril is in current clinical use for post-MI therapy.1-11 Among its reported effects are the lowering of platelet aggregation,2,12 increasing the effect of endothelial-derived relaxing factor in decreasing platelet aggregation,12 and countering the thrombin-induced inhibition of fibrinolysis, which leads to pulmonary and renal insufficiency.1 Further known functions of captopril include potentiation of PGE2 production13 and attenuation of PAI-1 levels,14,15 attenuation of expression of tissue factor,16 and bradykinin (BK) degradation.14,17 The antithrombotic effects of captopril and enalapril were reported in a rat venous thrombosis model by Buczko et al,6 who noted further that neither drug affected the prothrombin time (PT) or the activated partial thromboplastin time (APTT). Pawlak et al subsequently noted that captopril brought on an increase in PT in blood that they collected at the site thrombus formation in rats.5
Thiol groups have been implicated in key biological interactions. Captopril with a thiol group has greater antithrombotic activity than does enalapril.5 N-Acetyl-cysteine also exhibited antithrombotic activity.5 However, the thiol compounds exerted no effect upon PT and APTT, as reported by those investigators.
Koterba et al18 examined the effect of thiols on PT as part of a study on their effectiveness on the influence of acetaldehyde on PT because it had been observed by Cederbaum and Rubin that cysteine exerted a protective effect on mitochondria by lowering inhibitory effects of acetaldehyde.19 In their research, Koterba et al reported that thiols such as cysteine, N-acetyl-cysteine, homocysteine, and penicillamine all prolonged the PT of level I plasma. Prolongation of PT was potentiated in an additive manner by cysteine and N-acetyl-cysteine in the presence of acetaldehyde and attenuated by homocysteine and penicillamine in the presence of acetaldehyde. Dithiothreitol also prolonged PT.
The present investigation was performed to clarify the discrepancies reported on the effects of thiols on clotting times and to gain insight into the dimensions of the prolongation of clotting by captopril. Accordingly, this report investigates the effect of captopril on the PT and APTT of level I plasma and level III plasma as well as the effect of captopril on thrombin in 2 assays: the thrombin-factor II-deficient plasma (FIIDP) assay and the thrombin-fibrinogen assay. In so doing, it is hypothesized that the thiol moiety of captopril may act as a coagulation inhibitor by reducing the disulfide bonds known to occur in coagulation factors II, VII, IX, X, XI, XII, and fibrinogen, thereby reducing their functionality. This would represent and as-yet-unreported influence of captopril in addition to its antithrombotic effects as represented by increased PGE2 production and decreased platelet adhesion/aggregation, decreased expression of TF, decreased bradykinin levels, and decreased PAI-1 levels. A further comparison of the effect of lisinopril, another ACE inhibitor, which does not contain a thiol, and poly-l-lysine upon APTT is also reported.
MATERIALS AND METHODS
Accuclot control #1 (level I) plasma (lot #108H6147 and lot #528106), accuclot control #III (level III) plasma (lot #070K6026), and factor II-deficient plasma (lot #088H613) were purchased from Sigma Diagnostics (St. Louis, MO). A second FIIDP (lot #P22713) supply was purchased from Amax Trinity Biotech (Wicklow, Ireland). Poly-L-lysine ·HBr (lot #015K5104) (M.W.:1000-4000 Da) was obtained from Sigma Aldrich Co. (St. Louis, MO). Dade Berhring (Marburg, Germany) supplied the Dade Thromboplastin C Plus (lot #TPC-161), Dade Actin activated cephaloplastin reagent (lot #527111 and lot #527313A), and the 0.025 M CaCl2 (lot #CC-441B and lot #506872). Thrombin, 515 U/mg (lot #46491) and fibrinogen, Fraction I, bovine plasma, 75% clottable (lot #1786C and lot #7358F) were received from ICN Biochemicals, Inc. (Costa Mesa, CA) and ICN, Biochemicals, Inc. (Aurora, OH), respectively. Aldrich Chemical Co. provided the acetaldehyde, 99%. The acetaldehyde was passed through a column of activated, basic, Brockman I aluminum oxide (lot #0791DY) from Aldrich Chemical Co. (Milwaukee, WI) to remove oxidation products. It was subsequently stored in aliquots at -20°C. The captopril (batch #02-705-47224) and the lisinopril utilized in these studies were generously donated by the Squibb Institute for Medical Resarch (Princeton, NJ) and Merck Research Laboratories (Rahway, NJ), respectively. Clotting assays were performed on a Fibrosystem Precision Coagulation Timer, Model 5 (Becton, Dickinson and Company, Cockeysville, MD).
PT Assays With Captopril
The PT assay was carried out as described earlier.20 The effect of captopril on the PT was performed whereby the control consisted of 90 μL of level I plasma to which 10 μL of imidazole saline buffer (ISB) was added. After standing in a fibrometer cup for 5 minutes at 37°C, 200 μL of thromboplastin C (TplC) at 37°C was added thereto to initiate clotting. Captopril (10 μL) at 8.5 × 10−2, 4.25 × 10−2, 8.5 × 10−3, and 4.25 × 10−3 M was substituted for the ISB in the control run to give final concentrations of 8.5 × 10−3, 4.25 × 10−3, 8.5 × 10−4, and 4.25 × 10−4 M captopril. In comparative studies, level III plasma was substituted for level I plasma.
APTT Assays With Captopril
The APTT assay was performed as described previously.21 In the control assay, 10 μL of ISB was added to 90 μL of level I plasma in a fibrometer cup. After warming for 5′ at 37°C in a stoppered cup, 100 μL of APTT reagent at 37°C was added and further incubated for 5′ at 37°C. Clotting was then initiated with 100 μL of 0.025 M CaCl2 at 37°C. Captopril (10 μL) at 10−1, 6 × 10−2, 4 × 10−2, 2 × 10−2, and 10−3 M to give 10−2, 6 × 10−3, 4 × 10−3, 2 × 10−3, and 10−4 M captopril in plasma was utilized to replace ISB in the experimental studies. In comparative studies, level III plasma was substituted for the level I plasma.
Thrombin-FIIDP Assay With Captopril
Mixtures of 100 μL FIIDP and 100 μL CaCl2 were brought to 37°C for 3 minutes in a fibrometer cup. Clotting was initiated by the addition of 90 μL of thrombin [0.093 U in 0.1% BSA/Tris buffer, 0.1M, pH 7.3 (TB)] plus 10 μL of TB, which had previously incubated at RT for 2 minutes and then 37°C for 5 minutes. In studies on the effect of captopril on thrombin in this assay, thrombin was preincubated at RT for 2 minutes in the presence of 10−1, 6 × 10−2, and 3 × 10−2 M captopril to yield 10−2, 6 × 10−3, and 3 × 10−3 M concentrations before raising it to 37°C for 5 minutes. Clotting was initiated with 100 μL aliquots of the thrombin solution. In contrasting studies, the effect of captopril upon FIIDP was investigated. To 90-μL aliquots of FIIDP were added 10 μL of Tris buffer or 10 μL of captopril (10−1, 5 × 10−2, 2 × 10−2, and 10−3 M) to bring the final drug concentrations in plasma to 10−2, 5 × 10−3, 2 × 10−3, and 10−4 M. After standing at 37°C for 5 minutes, 100 μL of CaCl2 (at 37°C) was added. This solution was incubated for 3 minutes at 37°C. In a separate fibrometer cup, 100 μL of thrombin in BSA/TB (0.1 U) was warmed to 37°C for 2 minutes and then added to the FIIDP/captopril solution to initiate clotting.
Thrombin-Fibrinogen Assays With Captopril
To 90 μL of thrombin (0.093 U) were added 10 μL of TB or 10 μL of 10−1, 6 × 10−2, 3 × 10−2, 1.5 × 10−2, and 10−2 M captopril to yield final concentrations of 10−2, 6 × 10−3, 3 × 10−3, 1.5 × 10−3, and 10−3 M captopril. The solutions in fibrometer cups were stoppered and raised to 37°C for 5 minutes. Subsequently, 100 μL of TB and 100 μL of 1% fibrinogen were mixed and raised to 37°C for 2 minutes. Clotting was initiated by addition of the thrombin/TB or thrombin/captopril to the TB/fibrinogen mixture. In a contrasting study, the effect of captopril on fibrinogen was carried out by mixing 90 μL of fibrinogen with 10 μL of TB or 10 μL of 10−1, 2.5 × 10−2, and 10−2 M captopril to yield final concentrations in fibrinogen of 10−2, 2.5 × 10−3, and 10−3 M captopril. The solutions in fibrometer cups were raised to 37°C for 5 minutes. Thrombin, 100 μL (0.1 U), was mixed with 100 μL of TB in a separate fibrometer cup and raised to 37°C for 2 minutes. Clotting was initiated by addition of the fibrinogen/Tris or fibrinogen/captopril mixture to the thrombin/TB mixture.
APTT Assays With Lisinopril
To 90-μL aliquots of plasma were added 10 μL of ISB or 10 μL of lisinopril solution to final concentrations in plasma of 5 × 10−4, 1 × 10−3, and 1 × 10−2 M. After standing 10 minutes at RT, 100 μL of APTT reagent was added. The mixture was raised to 37°C for 5 minutes after which time 100 μL of CaCl2 at 37°C was added to initiate clotting.
APTT With Polylysine (1000-4000 Da)
To 90 μL of plasma in a fibrometer cup was added 10 μL of ISB or 10 μL of poylysine to final concentrations of 2 × 10−4, 4 × 10−5, 2 × 10−5, 1 × 10−5, 6.7 × 10−6, 5 × 10−6, and 1 × 10−6 M. The mixture was stored at 37°C for five minutes, after which time 100 μL APTT was added. After another five minutes, clotting was initiated upon addition of 100 μL CaCl2.
All data were tested for significance by Student t test. Data were deemed statistically significant if P ≤ 0.05. Other data approached statistical significance when 0.05 < P < 0.1.
The effect of captopril on the PT of level I plasma is seen in Figure 1 (n = 4). Captopril at 8.5 × 10−3 M and 4.25 × 10−3 M prolonged PT in a statistically significant manner. The sensitivity of the assay was identical when level III plasma was used in lieu of level I plasma (Figure 2) (n = 4). When an APTT assay was substituted for PT, captopril at 4 × 10−3 M prolonged clotting time in a statistical manner (Figure 3) (n = 6). In a similar manner, the APTT data with level III plasma indicated that 4.25 × 10−3 M captopril was the lowest level at which a statistical prolongation of clotting time was observed (Figure 4) (n = 4). A similar clotting assay was performed with a thrombin-FIIDP system. Under these conditions, 6 × 10−3 M captopril inhibited thrombin function statistically when the 2 components were preincubated at 37°C for 5 minutes before mixing with FIIDP (Figure 5) (n = 4). The analogous, almost comparable captopril concentration of 5 × 10−3 M approached statistical significance with a P-value of 0.0796, which prolonged clotting time upon preincubation with FIIDP before the addition of thrombin (Figure 6) (n = 4). A comparative pattern was seen when captopril at 3 × 10−3 M was added to thrombin or 2.5 × 10−3 M captopril was added to fibrinogen before mixing and determining clotting times (Figures 7 and 8) (n = 4). In each case, statistically significant elevations of clotting times were noted. At lower concentrations of captopril, no significant differences from the controls were discernable. In comparison to the thiol-containing antihypertensive captopril, lisinopril was tested for its capacity to interfere with clotting as followed by an APTT assay. At a concentration of 0.01 M, lisinopril prolonged the APTT reaction in a statistically significant manner (Figure 9) (n = 4). However, 0.001 M lisinopril exhibited no effect on the APTT assay. When poly-L-lysine (1000 to 4000 Da) was substituted for lisinopril, it was noted that 6.7 × 10−6 M polylysine affected a statistically significant prolongation of the APTT (Figure 10) (n = 4).
Captopril was initially developed as an ACE inhibitor for use as an antihypertensive drug. In more recent years, the link between hypertension and coagulopathies has grown. Captopril has been reported to serve an antithrombotic function, decrease morbidity and mortality rates in coronary patients, decrease platelet aggregation at a 10−4 M concentration (but not lower), decrease thromboxane B2 levels in patients with stage I-II essential hypertension without affecting platelet aggregation, and increase fibrinolysis, and it is now employed in postmyocardial infarction therapy as well as in therapy for ischemic heart disease.3,6,7,22,23 The implications of captopril and other ACE-Is in anticoagulant mechanisms have been expanding. On the pathway to thrombosis, captopril, idapril, and fosinopril as ACE-Is and losartan as AngII AT1 receptor antagonist affect a lowering of the expression of TF by monocytes,16,24-25 thereby exhibiting an antithrombotic effect for patients susceptible to recurrent MIs. AngII promotes TF activity.25 ACE-Is reduce the levels of AngII. Additionally, platelet aggregation is enhanced by PGE2 leading to thromboembolism27 and atherothrombosis.28 Activated platelets synthesize PGE229 and have receptors on their plasma membranes.30 PGE2 also promotes the involvement of erythrocytes in the formation of the blood clot.31 AngII, a vasoconstrictor, stimulates the synthesis of PGE2, which has been reported to have vasodilatory actions.13 It also is the precursor of ANG1-7, which promotes generation of NO and prostacyclin, both of which inhibit thrombus formation.32 Captopril also increases PGE2 production.13 In addition to its promotion of platelet aggregation, PGE2 serves as an inhibitor of aggregation when administered orally to women in labor induced by secondary ADP.33
Strategies for the reduction of thrombi also include reduction in thrombus size. Fibrinolytic activity is the key to reduction in thrombus size. Endothelial tPA stimulates the activation of plasminogen into plasmin, which degrades clots. tPA levels are elevated by the action of bradykinin, which is generated by Factor XIIa of the intrinsic coagulation cascade.34 The ACE-I, quinapril, stimulates release of bradykinin; therefore, ACE-Is play an important role in reduction of thrombus size. Furthermore, ACE-Is inhibit/reduce the degradation of bradykinin.35 Interestingly, BK1-5, which is a stable degradation product of bradykinin, produced as a consequence of ACE activity, inhibits the thrombin-induced platelet aggregation, as does its parent compound.36 tPA activity, in turn, is inhibited by PAI-1,26,37 thereby decreasing fibrinolysis. PAI-1 levels are increased by AngII, which also stimulates an elevation in tPA.37 PAI-1 levels are lowered by ACE-Is such as lisinopril, captopril, and fosinopril, thereby maintaining fibrinolytic activity.14 PAI-1 levels are elevated after coronary artery bypass grafting, thereby increasing the risk of acute graft thrombosis.14 Accordingly, several natural components of the body, such as AngII and PGE2 may be involved in both stimulation and reduction of thrombosis. However, ACE-Is reduce both thrombus formation and stimulate thrombus dissolution.
The potential for sulfhydryl-disulfide interchange has been known and studied for decades. In this light, it was hypothesized that the sulfhydryl-containing ACE inhibitor, captopril, might prolong clotting time by a mechanism involving reduction of the disulfide bonds in the coagulation factors. Interestingly, Buczko et al have reported that it has no effect on PT and APTT.6 It was because of the lack of effect of captopril on PT and APTT that this current investigation was initiated. In the current investigators' hands, it was observed that captopril prolonged PT and APTT at plasma concentrations of 4.25 × 10−3 M and 4 × 10−3 M, respectively, with level I plasma and 4.25 × 10−3 M for both with level III plasma. Commercial level I plasma approximates normal plasma, whereas commercial level III plasma approximates plasmas with decreased coagulation factors. Both level I and level III plasmas exhibited comparable sensitivity to captopril. Pooled plasmas represent means of plasmas in lieu of differentiations on the basis of sex and age. In thrombin-fibrinogen assays, however, 3 × 10−3 M captopril exerted a statistical inhibition of coagulation on thrombin, as did 2.5 × 10−3 M captopril on fibrinogen. Umali and Simanek have recently reported on a chemical reaction of sulfhydryl-disulfide interchange38; the likely explanation for the phenomenon of prolongation of PT and APTT would appear to be the reduction of disulfide bonds in the coagulation factors such as thrombin (prothrombin), Factor Xa, and others. This suggestion has been made earlier that reduction of the disulfide bonds in the coagulation factors alters their tertiary structure, thereby impairing their ability to catalyze degradative reactions.18 The level of captopril used in this study was comparable to that of Maes et al, who employed 10−4 M captopril in studying the effect of the drug on the release of endothelin-1 by endothelial cells.39 Indeed, captopril inhibits the release of the vasoconstrictor, endothelin-1 from the endothelial cells.39
In these investigations, thrombin was taken up in BSA/TB solutions to increase the stability of the enzyme, particularly in the thrombin-fibrinogen assays. The BSA was added to simulate albumin in plasma. Assays of thrombin activity with a fibrinogen substrate indicated that the BSA/TB mixture exhibited higher activity than did the sole TB solvent. Similar results were obtained when replacing TB with ISB, the solvent used in many clinical assays (data not submitted).
The effect of captopril on thrombin activity was investigated separately with thrombin-fibrinogen assays and thrombin-FIIDP assays in order to more closely examine potential sulfhydryl-disulfide exchange effects. As stated above, 2.5 × 10−3 M captopril affected fibrinogen in the thrombin-fibrinogen reaction, whereas 3 × 10−3 M captopril showed a statistically significant effect on thrombin. Fibrinogen also has disulfide bonds that are apparently susceptible to reduction by thiols. Comparable results were obtained when fibrinogen was replaced with FIIDP. These results further support the suggestion that thiol-disulfide interchange is occurring when captopril is preincubated with proteins containing disulfide bonds.
Enalapril and lisinopril, neither of which contains thiol groups, each lower morbidity and mortality rates in patients with ischemic heart disease.3 Enalapril exhibits antithrombotic effects in young rats.6 In contrast to captopril, lisinopril displays no profibrinolytic activity.40 For comparative effects, lisinopril was also examined for its impact on APTT. While it prolonged clotting times at a pharmacological 10−2 M level, it had no effect at 10−3 or lower levels. Lisinopril also has no effect on morbidity and mortality rates in ischemic patients and patients with myocardial infarctions.3
Thrombin exhibits a wide spectrum of physiological activity. It has been reported to inhibit ACE activity, promote clot formation, promote platelet aggregation, activate coagulation cascade factors, and inhibit fibrinolysis1,12,23; therefore, inhibition of thrombin function by captopril can have profound consequences in nature. Assuredly, further studies will illuminate additional impacts of captopril as well as thrombin on hemostasis.
Protamine has been used for a number of years to neutralize the effects of overdosing patients with heparin.41 Brecher et al have studied the effect of protamine and acetaldehyde-modified protamine on PT activity.42 Polylysine has also been reported to extend clotting time.43 In a comparative study on the effect of polylysine (1k to 4k) on APTT it was determined that levels as low as 6.7 × 10−6 M prolonged the APTT. Polylysine is a basic synthetic polypeptide in contrast to the naturally occurring protamine isolated from salmon sperm. Polylysine can promote the activation of prothrombin by Factor Xa.44 It also exhibits anticoagulant activity by inhibiting the monocyte-derived tissue factor-dependent activation of Factor VII.43 It remains to be determined whether polylysine would serve as a (competitive) inhibitor of the reaction between thrombin and ATIII. Future studies should shed more light on the physiological/pharmacological differences between polylysine and the small lysine-containing lisinopril.
In conclusion, captopril prolongs the PT and APTT in standard clinical tests. Additionally, it inhibits thrombin activity in clotting assays with fibrinogen and FIIDP. It is likely that the inhibition of thrombin is a consequence of sulfhydryl-disulfide exchange whereby the tertiary structure of thrombin is affected. This role of captopril provides an additional means of serving as an anticoagulant in addition to those described above by other investigators. Lisinopril, an antihypertensive drug that does not contain a thiol moiety, is far weaker an anticoagulant than captopril. However, polylysine (1k to 4k) is a powerful anticoagulant as measured with the APTT.
We thank Mr. Robert Harr for graciously supplying PT and APTT reagents.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
captopril; anticoagulants; blood coagulation; thrombin; hypertension; lisinopril; polylysine