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Cardiovascular Research 2000 47(4):707-714; doi:10.1016/S0008-6363(00)00126-7
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Copyright © 2000, European Society of Cardiology

Fibrinolytic actions of intra-arterial angiotensin II and bradykinin in vivo in man

Catherine Labinjoha, David E. Newbya,b,*, Pamela Dawsonc, Neil R. Johnstona, Christopher A. Ludlamc, Nicholas A. Boonb and David J. Webba

aClinical Pharmacology Unit and Research Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK
bDepartment of Cardiology, Royal Infirmary, Lauriston Place, Edinburgh, EH3 9YW, Scotland, UK
cDepartment of Haematology, Royal Infirmary, Lauriston Place, Edinburgh, EH3 9YW, Scotland, UK

* Corresponding author. Tel. +44-131-536 2749; fax: +44-131-536 2744 d.e.newby{at}ed.ac.uk

Received 21 February 2000; accepted 26 April 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Angiotensin II and bradykinin are potent endogenous vasoactive peptides which may play a role in the regulation of endogenous fibrinolysis and, thereby, contribute to the beneficial actions of ACE inhibitors. The aims of the study were to determine the acute effect of angiotensin II and bradykinin on the local vascular release of tissue plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), and the endothelium-derived haemostatic factor, von Willebrand factor (vWf) from the forearm. Methods: Blood flow, and plasma haemostatic and fibrinolytic factors, were measured in both forearms of sixteen healthy men: eight subjects received intra-arterial angiotensin II (5, 50 and 500 pmol/min) which was coinfused with sodium nitroprusside (SNP; 0.3, 1.5 and 7.5 µg/min, respectively), and eight received intra-arterial bradykinin at 10–3000 pmol/min. Results: Despite substantial rises in plasma angiotensin II concentrations (P<0.001) which caused pressor effects (P<0.003) at the highest dose, angiotensin II infusion did not affect local plasma t-PA, PAI-1 or vWf concentrations. In contrast, bradykinin caused substantial dose-dependent increases in blood flow and t-PA release (>100 ng/100 ml of tissue/min) in the infused forearm (P<0.001 for both) without affecting plasma PAI-1 or vWf concentrations. Conclusions: Despite high local concentrations with breakthrough of significant systemic effects, angiotensin II did not affect acute endothelial cell t-PA, PAI-1 or vWf release in healthy men. In contrast, bradykinin is a potent vasodilator and selective stimulus for acute local t-PA release. This may, at least in part, explain the fibrinolytic actions of ACE inhibitors in heart failure and ischaemic heart disease.

KEYWORDS Thrombolysis; Endothelial Factors; Blood Flow; Angiotensin

This article is referred to in the Editorial by L. Dézsi (pages 642–644) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Tissue plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor type 1 (PAI-1), are potentially important endothelium derived mediators that are intimately linked to the risk of thrombosis. Acute t-PA release results from the rapid translocation of a dynamic intracellular storage pool present in the endothelium [1] of the precapillary arterioles and post capillary venules [2]. The time course of t-PA release is important since clot dissolution is much more effective if t-PA is incorporated during clot formation rather than following completion [3,4].

In patients with hypertension, an elevated plasma renin activity for a given urinary sodium excretion is independently associated with an increased risk of acute myocardial infarction [5]. Moreover, large scale clinical trials in patients with heart failure, ischaemic heart disease or a recent myocardial infarction suggest a reduction in reinfarction rates with angiotensin converting enzyme (ACE) inhibitor therapy. The mechanisms underlying the association of renin–angiotensin system activation with coronary thrombotic events are unknown but may relate, in part, to an effect on fibrinolytic parameters. Indeed, activation of the renin–angiotensin system by sodium depletion causes an increase in early morning plasma PAI-1 concentrations that can be reversed by ACE inhibition [6]. Furthermore, in patients with heart failure [7] or a recent myocardial infarction [8,9], ACE inhibitor therapy causes marked reductions in plasma PAI-1 concentrations.

The renin–angiotensin and kinin systems have a direct influence on the regulation of the fibrinolytic system. Cell culture studies demonstrate marked up-regulation and release of PAI-1 in response to angiotensin II administration [10,11] whilst ex vivo animal models have suggested that bradykinin is one of the most potent stimulants for the release of t-PA [12,13]. Studies in man have also indicated that systemic infusions of angiotensin II and bradykinin are associated with elevated levels of PAI-1 [14] and t-PA [15,16]. However, systemic drug administration of vasoactive agents can change blood pressure and regional blood flow, as well as having widespread effects on many tissues. Thus, altered systemic fibrinolytic parameters might be attributable, for instance, to changes in hepatic release and clearance of t-PA and PAI-1, and the concomitant release of other stimulatory, vasoactive and humoral mediators [15,17]. To avoid these potentially confounding systemic effects, the use of locally active intra-arterial infusion of drugs can be used to investigate the acute local release of t-PA and PAI-1 in the forearm of man [18–24].

We have previously been able to demonstrate that substance P, an endothelium-dependent vasodilator, causes a selective dose-dependent release of t-PA from the human forearm without causing significant release of PAI-1 or the endothelium-derived haemostatic factor, von Willebrand factor (vWf) [21,22]. In contrast, despite causing equivalent increases in blood flow, the potent endothelium-independent vasodilator, sodium nitroprusside, does not affect t-PA release from the forearm [19,21,24]. The aim of the present study was, therefore, to determine the local effect of intra-arterial angiotensin II and bradykinin administration on the local forearm release of t-PA, PAI-1 and vWf.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Subjects
Sixteen healthy male non-smokers, aged between 21 and 35 years, participated in the study which was undertaken with the approval of the local research ethics committee and in accordance with the Declaration of Helsinki. The written informed consent of each subject was obtained before entry into the study. None of the subjects received vasoactive or non-steroidal antiflammatory drugs in the week before each phase of the study, and all abstained from alcohol for 24 h, and from food and caffeine-containing drinks for at least 12 h before each study. All studies were carried out in a quiet, temperature controlled room maintained at 22–24°C.

2.2 Drugs
Pharmaceutical grade bradykinin (Clinalfa, Läufelfingen, Switzerland), angiotensin II (Celinalfa) and sodium nitroprusside (Nipride; Roche, Welwyn Garden City, UK) were administered following dissolution in saline (0.9%: Baxter Healthcare, Thetford, UK).

2.3 Intra-arterial administration
The brachial artery of the non-dominant arm was cannulated with a 27-standard wire gauge steel needle (Cooper's Needle Works, Birmingham, UK) under 1% lignocaine (Xylocaine: Astra Pharmaceutical, Kings Langley, UK) local anaesthesia and attached to a 16-gauge epidural catheter (Portex, Hythe, UK). Patency was maintained by infusion of saline via an IVAC P1000 syringe pump (IVAC, Basingstoke, UK). The total rate of intra-arterial infusions was maintained constant throughout all studies at 1 ml/min.

2.4 Measurements
2.4.1 Forearm blood flow and haemodynamics
Blood flow was measured in both forearms by venous occlusion plethysmography using mercury-in-silastic strain gauges applied to the widest part of the forearm [25]. During measurement periods, the hands were excluded from the circulation by rapid inflation of the wrist cuffs to a pressure of 220 mmHg using E20 rapid cuff inflators (D.E. Hokanson, Washington, USA). Upper arm cuffs were inflated intermittently to 40 mmHg for 10 s in every 15 s to achieve venous occlusion and obtain plethysmographic recordings. Analogue voltage output from an EC-4 strain gauge plethysmograph (D.E. Hokanson) was processed by a MacLab® analogue-to-digital converter and CHARTTM v3.3.8 software (AD Instruments, Castle Hill, Australia) and recorded onto a MacIntosh Classic II computer (Apple Computers, Cupertino, CA, USA). Calibration was achieved using the internal standard of the plethysmograph.

Blood pressure and heart rate were monitored in the non-infused arm at intervals throughout each study using a semi-automated non-invasive oscillometric sphygmomanometer (Takeda UA 751, Takeda Medical, Tokyo, Japan) [26].

2.5 Assays
Venous cannulae (17G) were inserted into large subcutaneous veins of the antecubital fossa in both arms. A 10–20 ml volume of of blood was withdrawn simultaneously from each arm and collected into acidified buffered citrate (Biopool® StabilyteTM, Umeå, Sweden; for t-PA assays), citrate (Monovette®, Sarstedt, Nümbrecht, Germany; for PAI-1 and vWf assays) and 0.45% o-phenanthroline–4.65% disodium ethylene diamine tetraacetic acid (for angiotensin II assays) tubes, and kept on ice before being centrifuged at 2000 g for 30 min at 4°C. Platelet free plasma was decanted and stored at –80°C before assay [27].

Plasma PAI-1 and t-PA antigen concentrations were determined using an ELISA; Coaliza® PAI-1 [28] and Coaliza® t-PA [29] (Chromogenix, Mölndal, Sweden), respectively. Plasma PAI-1 and t-PA activities were determined by a photometric method, Coatest® PAI-1 [30] and Coaset® t-PA [31] (Chromogenix). Intra-assay coefficients of variation were 5.5 and 7.0% for t-PA and PAI-1 antigen, and 2.4 and 4.0% for activity, respectively. Inter-assay coefficients of variability were 4.0, 7.3, 4.0 and 7.6%, respectively. The sensitivities of the assays were 0.5 ng/ml, 2.5 ng/ml, 0.10 I.U./ml and 5 AU/ml, respectively. Determination of vWf antigen [32] was undertaken using an ELISA (Dako, Glostrup, Denmark) with a sensitivity of 0.05 I.U./ml. The intra-assay and inter-assay coefficients of variability were 5.2 and 7.3%, respectively. Following extraction using Bond Elut® columns (Varian, Harbor City, CA, USA), plasma angiotensin II (Peninsula Labs. Europe, St. Helens, UK) concentrations were determined by radioimmunoassay as previously described [33]. The intra- and inter-assay coefficients of variability were 7.2 and 9.3%, respectively. Haematocrit was determined by capillary tube centrifugation of blood anticoagulated by ethylene diamine tetraacetic acid and was obtained from the infused forearm at baseline and at the end of the study protocol.

2.6 Study design
Subjects rested recumbent, and strain gauges and cuffs were applied. The brachial artery of the non-dominant arm was cannulated and forearm blood flow measured every 10 min or between 3 and 6 min for shorter (6 min) infusion periods during the bradykinin protocol. Before bradykinin or combined angiotensin II and sodium nitroprusside administration, saline was infused for 30 min to allow time for equilibration. The final blood flow measurement during saline infusion was taken as the basal forearm blood flow.

2.6.1 Angiotensin II protocol
There is a modest basal release of t-PA from the forearm and, in the presence of a reduction in blood flow, an apparent rise in t-PA concentrations may occur without a change in the rate of t-PA release [34]. To correct for any flow dependent effects, sodium nitroprusside, which does not cause t-PA release [19,21,24], was coinfused with angiotensin II to offset the angiotensin II mediated vasoconstriction and maintain forearm blood flow greater than or equal to that during saline infusion. Eight subjects received three incremental infusions of angiotensin II and sodium nitroprusside for 30 min at each dose in the following order: angiotensin II 5 pmol/min+sodium nitroprusside 0.3 µg/min; angiotensin II 50 pmol/min+sodium nitroprusside 1.5 µg/min; and angiotensin II 500 pmol/min+sodium nitroprusside 7.5 µg/min. The doses of sodium nitroprusside were determined from preliminary pilot studies (data on file). Venous blood samples were obtained from both forearms at baseline and every 10 min thereafter.

2.6.2 Bradykinin protocol
Eight subjects received an incremental infusion of bradykinin at 10, 30, 100, 300, 1000 and 3000 pmol/min followed by a 20-min saline infusion. Venous blood samples were obtained from both forearms at baseline, during bradykinin infusion at 30, 300 and 3000 pmol/min, and 10 and 20 min after cessation of the final bradykinin infusion of 3000 pmol/min. Bradykinin was infused for 6 min at each dose but was extended to 10 min at the 30, 300 and 3000 pmol/min doses because of venous sampling following blood flow measurements.

2.7 Data analysis and statistics
The study population size, based on power calculations derived from previous studies [21], gives 90% power of detecting a 21% difference in t-PA release at a significance level of 5%. Coefficients of repeatability [35] for plasma concentrations of t-PA antigen and activity during substance P infusion at 8 pmol/min are 1.6 ng/ml and 1.4 I.U./ml, respectively (data on file).

Plethysmographic data were extracted from the CHART data files. Forearm blood flows were calculated for individual venous occlusion cuff inflations by use of a template spreadsheet (EXCEL v5.0; Microsoft, Cambridge, USA). Recordings from the first 60 s after wrist cuff inflation were not used because of the variations in blood flow that this causes. Usually, the last five flow recordings in each 3-min measurement period were calculated and averaged for each arm. Estimated net release of t-PA activity and antigen was defined previously [21] as the product of the infused forearm plasma flow (based on the mean haematocrit, Hct, and the infused forearm blood flow, FBF) and the concentration difference between the infused ([t-PA]Inf) and non-infused arms ([t-PA]Non-inf)

Formula
Data were examined, where appropriate, by multifactorial analysis of variance (ANOVA) with repeated measures and two-tailed paired Student's t-test using EXCEL v5.0 (Microsoft). All results are expressed as mean±standard error of the mean. Statistical significance was taken at the 5% level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Subject characteristics are shown in Table 1.


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  		Table 1 Subject characteristics

 
3.1 Angiotensin II protocol
There were no significant changes in blood pressure, heart rate or blood flow in the non-infused arm throughout the study protocol except during the final 30 min infusion (Fig. 1).


Figure 1
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  		Fig. 1 Mean arterial pressure (solid squares), heart rate (open squares), and blood flow in the infused (solid circles) and non-infused (open circles) forearms during saline, bradykinin (pmol/min), and combined angiotensin II (Ang II, pmol/min) and sodium nitroprusside (SNP, µg/min) infusions. Paired t-test:*, P<0.001, {dagger}, P≤0.02, {ddagger}, P≤0.05 (vs. baseline); One-way ANOVA: P<0.001 for infused forearm blood flow (bradykinin and angiotensin II infusions). Two-way ANOVA:P<0.001 (infused vs. non-infused forearm blood flow, bradykinin and angiotensin II infusions); {dagger}, P=0.006, {ddagger}, P=0.02 (saline vs. angiotensin II 500 pmol/min); four-way ANOVA: {dagger}, P<0.003, {ddagger}, P=0.05 (four groups: saline, angiotensin II 5 pmol/min, angiotensin II 50 pmol/min and angiotensin II 500 pmol/min).

 
In comparison with the preceding 30-min infusion periods, administration of angiotensin II 500 pmol/min with sodium nitroprusside 7.5 µg/min was associated with an increase in mean arterial pressure (four-way ANOVA, P<0.003), and infused (four-way ANOVA, P<0.001) and non-infused (four-way ANOVA, P=0.05) forearm blood flow without a change in heart rate.

Venous plasma angiotensin II concentrations increased in the infused (ANOVA, P<0.001 for all doses) and non-infused (ANOVA, P<0.001 at 500 pmol/min only) forearms (Table 2). In comparison to the non-infused arm, plasma angiotensin II concentrations were substantially greater in the infused arm (Table 2; two-way ANOVA, P<0.001). However, despite substantial increases in local angiotensin II concentrations, there were no significant changes in plasma t-PA, PAI-1 or vWf antigen concentrations in either arm (Table 2).


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  		Table 2 Estimated net release and plasma concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) antigen, and venous plasma von Willebrand factor (vWf) and angiotensin II concentrations during angiotensin II and sodium nitroprusside infusion

 
3.1.1 Bradykinin protocol
Consistent with the actions of other kinins [21], transient and patchy flushing and skin oedema of the infused arm occurred with bradykinin infusion at doses ≥300 pmol/min. The oedema had an urticarial appearance, taking the form of a raised wheal with a yellow hue. The extent of oedema varied between subjects, beginning at the level of the elbow and extending distally with increasing dose. However, there was no associated pruritis or tenderness and the oedema resolved completely within 1–2 h of stopping the infusion. No local effects were seen with angiotensin II and sodium nitroprusside infusions.

There were no significant changes in blood pressure, heart rate or blood flow in the non-infused arm throughout the study protocol (Fig. 1). Bradykinin increased blood flow in the infused arm (ANOVA, P<0.001) in a dose dependent manner (Fig. 1) reaching a maximum increase of 22.8±2.7 ml/100 ml/min at 3000 pmol/min.

Bradykinin caused a dose-dependent increase in venous plasma t-PA activity and antigen concentrations of the infused (ANOVA, P<0.001) and non-infused (ANOVA, P=0.01) forearms (Fig. 2). In comparison to the non-infused arm, plasma t-PA antigen and activity concentrations were substantially greater in the infused arm (two-way ANOVA, P<0.001), consistent with a marked net local release of t-PA (ANOVA, P<0.001; Table 3).


Figure 2
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  		Fig. 2 Plasma tissue plasminogen activator (t-PA) antigen (solid lines; upper panel) and activity (dashed lines; lower panel) concentrations in the infused (solid circles) and non-infused (open circles) forearms during saline and bradykinin infusions. One-way ANOVA: infused arm; P<0.001 for t-PA antigen and activity. Non-infused arm; P=0.003 for t-PA antigen, P=0.01 for t-PA activity. Two-way ANOVA: infused vs. non-infused arm; P<0.001 for t-PA antigen and activity. Paired t-test: *, P<0.05, {dagger}, P<0.001 (vs baseline).

 

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  		Table 3 Estimated net release of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) antigen and activity during bradykinin infusion

 
Baseline plasma PAI-1 antigen, PAI-1 activity and vWf concentrations were 36±4 ng/ml, 12±2 and 0.64±0.05 I.U./ml in the infused arm, and 40±4 ng/ml, 13±1 and 0.67±0.09 I.U./ml in the non-infused arm, respectively. There were no significant changes in plasma PAI-1 antigen, PAI-1 activity or vWf concentrations in either arm during bradykinin infusion (data on file).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have shown that, despite very high local concentrations, ultimately causing significant systemic pressor effects, angiotensin II infusion does not directly affect acute local endothelial cell t-PA or PAI-1 release. In contrast, we found that the potent vasodilator, bradykinin, is a selective stimulus for acute local t-PA release. These findings suggest that bradykinin, but not angiotensin II, may contribute to the acute local regulation of endogenous fibrinolysis in man. This may, in part, contribute to altered endogenous fibrinolysis seen with the use of ACE inhibitor therapy [8,9].

In vitro studies have suggested that angiotensin II influences endothelial [11] and vascular smooth muscle cell [10] PAI-1 synthesis and release. However, in vitro cell techniques have limitations because the phenotype of cells in culture, and the ability to release PAI-1, may change, particularly with increasing passages. There has been conflicting in vivo evidence with regard to the role of angiotensin II in the regulation of endogenous fibrinolysis, with angiotensin II either increasing plasma PAI-1 concentrations without affecting t-PA [14] or increasing plasma t-PA concentrations without affecting PAI-1 [16]. However, systemic neurohumoral responses to the infusion of vasoactive agents may contribute to, or even mediate, the subsequent fibrinolytic response [17]. To avoid these confounding systemic effects, locally active intra-arterial infusion of drugs can be used to investigate the acute local release of t-PA in the forearm circulation of man [18–24]. Using high but locally vasoactive doses of angiotensin II (5 and 50 pmol/min), we were unable to detect alterations in plasma t-PA or PAI-1 concentrations in the forearm vascular bed. Moreover, the absence of a fibrinolytic response is unlikely to be a temporal effect since the blood vessels of the infused forearm were exposed to markedly elevated plasma angiotensin II concentrations for 90 min.

In order to counteract the development of forearm vasoconstriction and subsequent flow-dependent changes in plasma t-PA concentrations [34], sodium nitroprusside was co-infused with angiotensin II. Sodium nitroprusside is unlikely to have influenced the fibrinolytic response to angiotensin II infusion because a number of previous in vitro [36] and in vivo studies [19–21,24] have failed to demonstrate any effect of sodium nitroprusside administration on the acute release of t-PA or PAI-1. At the highest dose, angiotensin II did not affect plasma t-PA or PAI-1 concentrations despite increasing systemic blood pressure and increasing forearm blood flow in the non-infused arm. Increased blood flow of the infused forearm may, at least in part, reflect relative dominance of the vasodilatation associated with sodium nitroprusside co-infusion. However, vasodilatation of the non-infused forearm with systemic pressor doses of angiotensin II is consistent with previous work [37] and is related to the relatively greater vasoconstrictor response to angiotensin II infusion in other resistance vascular beds, such as the splanchnic circulation.

In addition to potential actions on plasma fibrinogen [38] and platelet function [39], the major benefits associated with ACE inhibitor therapy may, at least in part, be mediated through actions on endogenous fibrinolytic function. Following myocardial infarction, basal fibrinolytic parameters are improved by the use of ACE inhibitors [8,9], principally through a reduction in plasma PAI-1 concentrations. The absence of a direct effect of high local concentrations of angiotensin II in the forearm circulation suggests that this is not mediated by a direct action of angiotensin II on the endothelium. However, PAI-1 is synthesised and released by many tissues, including megakaryocytes, liver and vascular smooth muscle, and the present study does not exclude a systemic role of angiotensin II in the regulation of endogenous fibrinolysis.

Animal models suggest that bradykinin is an extremely potent stimulus for the release of t-PA from the vascular endothelium in vitro [1], ex vivo [13] and in vivo [40]. We have been able to demonstrate marked and substantial release of t-PA with intra-arterial infusions of bradykinin in humans. Assuming a forearm volume of ~1000 ml [25], 3000 pmol/min of bradykinin caused the release of over 1 µg/min of t-PA from the infused forearm. Plasma t-PA concentrations, but not blood flow, increased in the non-infused forearm at the highest dose of bradykinin, suggesting the release of t-PA from the infused forearm exceeded the capacity to clear it immediately from the systemic circulation.

Recently, Brown et al. [41] have described the forearm release of t-PA with intra-arterial bradykinin infusion using doses up to 400 pmol/min. Using similar techniques, we have confirmed their findings, shown a greater release of t-PA at higher doses of bradykinin, and extended these observations to demonstrate the absence of vWf release. In contrast to the animal studies [13], bradykinin does not appear to release vWf from the forearm circulation in man. This finding is consistent with similar observations obtained with substance P [21] and desmopressin infusions [20], and suggests the presence of separate and distinct endothelial storage pools and release mechanisms for t-PA and vWf.

Small areas of denudation and thrombus deposition are a common finding on the surface of atheromatous plaques [42,43] and are usually sub-clinical. However, in the presence of an imbalance in the coagulation or fibrinolytic systems, such microthrombi may propagate, ultimately leading to arterial occlusion. Bradykinin is not only an inflammatory mediator but is also released during the contact phase of coagulation when high-molecular-weight kininogen is cleaved by kallikrein to produce a disulphide-linked light and heavy chain [44,45]. This liberation of bradykinin may represent an important negative feedback loop in which bradykinin induced t-PA release inhibits thrombus formation within the vascular lumen when localised endothelial denudation occurs. Furthermore, given that bradykinin induced forearm vasodilatation is potentiated by ACE inhibition [46], such actions may be enhanced in the presence of ACE inhibition and may, in part, explain the anti-ischaemic action of this therapy [47]. However, although inferred, the potentiation of bradykinin induced t-PA release by ACE inhibition has yet to be established.

In conclusion, it would appear that bradykinin, but not angiotensin II, is a potent vasodilator and selective stimulus for acute local t-PA release. This may, in part, explain the beneficial fibrinolytic actions of ACE inhibition in heart failure and ischaemic heart disease.

Time for primary review 22 days.


   Acknowledgements
 
This work has been supported by a grant from the British Heart Foundation (PG/97197). CL is the recipient of a British Heart Foundation Junior Research Fellowship (FS/97007). DJW is supported by a Research Leave Fellowship from the Wellcome Trust (WT 0526330).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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