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Cardiovascular Research 2002 53(4):984-992; doi:10.1016/S0008-6363(01)00514-4
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Copyright © 2002, European Society of Cardiology

Thrombostatin, a bradykinin metabolite, reduces platelet activation in a model of arterial wall injury

Alejandro R Prietoa, Hongbao Maa, Ruiping Huanga, Gauhar Khana, Kenneth A Schwartza, Elie E Hage-Korbana, Alvin H Schmaierb,c, John M Davisa, Ahmed A.K Hasanb,c and George S Abelaa,*

aDepartment of Internal Medicine, Divisions of Cardiology and Hematology and Oncology, Michigan State University, East Lansing, MI, USA
bDepartment of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
cThromgen, Inc., Ann Arbor, MI, USA

* Corresponding author. Present address: Department of Medicine/Cardiology, Michigan State University, B-208 Clinical Center, East Lansing, MI 48824, USA. Tel.: +1-517-353-4832; fax: +1-517-432-4039 abela{at}pilot.msu.edu

Received 16 February 2001; accepted 18 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Thrombin activates platelets and contributes to the occlusion of arteries following thrombolytic therapy or angioplasty. Thrombostatin (RPPGF), the angiotensin converting enzyme degradation product of bradykinin, inhibits {alpha}-thrombin induced platelet activation. We hypothesized that thrombostatin prevents platelet aggregation and adhesion after balloon angioplasty (BA). Methods: Platelet-rich plasma (PRP) was obtained from 22 Beagle dogs before sacrifice and 10% of the PRP was labeled with 111In. Carotid arteries were then removed from each dog and mounted in a dual perfusion chamber and intimal injury was performed with BA. 111In-PRP with or without thrombostatin or aspirin alone was perfused through the arteries for 60 min. During perfusion, platelet volume was measured using a Coulter counter and a laser-light scattering technique. Platelet adhesion to arteries was measured by radioactivity count. Results: Arterial injury alone compared to non-injury increased platelet volume in the circuit by 1.4 times (x) (P<0.05) using a Coulter counter or 1.8x (P<0.05) using laser-light scattering and increased platelet adhesion by 2.3x (P<0.01). When compared to BA injury alone, the addition of thrombostatin reduced platelet volume by 1.8x (P<0.03) as measured by Coulter counter or 1.9x (P<0.01) by laser-light scattering and platelet adhesion by 4.2x (P<0.05). Compared to BA injury alone, aspirin reduced platelet volume by 1.2x (P<0.01) as assessed by Coulter counter or 1.5x (P<0.03) using laser-light scattering and platelet adhesion by 1.8x (P<0.02). Conclusion: Thrombostatin or aspirin independently decreases evidence of platelet activation in the canine carotid artery model of BA injury.

KEYWORDS Angioplasty; Anticoagulants; Arteries; Platelets; Thrombosis/embolism


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
A major factor in the development of thrombosis following angioplasty is thrombin-induced platelet activation. Aspirin and heparin are routinely administered during interventional procedures to prevent acute arterial occlusion. Heparin, however, is unable to prevent platelet activation from clot bound thrombin [1–3]. Although potent direct thrombin inhibitors, such as hirudin, inhibit clot bound thrombin in vitro, clinical studies have not demonstrated hirudin to be more effective than heparin in preventing clots during acute coronary syndromes [4–6]. The effectiveness of hirudin as well as the platelet glycoprotein IIb/IIIa inhibitors in the presence of heparin are limited by increased risk for bleeding [7,8]. Thus, the development of agents that selectively target thrombin activation of platelets may be one way to achieve inhibition of thrombin without excessive bleeding.

Thrombostatin (RPPGF) is the angiotensin converting enzyme metabolite of bradykinin and is at least a bifunctional inhibitor of {alpha}-thrombin-induced platelet activation [9,10]. It binds to both the active site of thrombin and at 175-fold tighter affinity to protease activator receptor 1 (PAR 1), preventing {alpha}-thrombin from cleaving the tethered ligand between arginine-41 and serine-42 [11–13]. In vitro, RPPGF inhibits 100% {gamma}-thrombin-induced human and canine platelet aggregation at 0.33±0.28 and 0.6±0.12 mM, respectively. After a single intravenous infusion, thrombostatin inhibits 50% or more of 20 nM {gamma}-thrombin-induced platelet activation ex vivo for 165 min and 40 nM {gamma}-thrombin-induced platelet activation for 60 min [13].

The effect of thrombostatin to prevent platelet activation following balloon angioplasty was investigated in an ex vivo canine carotid artery model by continuous measurement of platelet volume change and detecting platelet deposition on the arterial wall. Measurement of platelet volume change was determined by both Coulter counter and a novel technique of continuous detection of laser-light scattering of platelet aggregates circulating in the perfusate [14,15]. 111In-labeled platelets were used to determine the amount of platelet deposition on arterial walls as an indirect measure of platelet activation. These studies indicate that thrombostatin is as good as aspirin in this ex vivo model of platelet activation after balloon angioplasty.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals and blood collection
The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Twenty-two Beagle dogs were used in the study. Whole blood (450 ml) was collected in 50 ml of sodium citrate (150 mM) from each dog after general anesthesia with intravenous injection of sodium pentobarbital (65 mg/ml, 0.5 ml/kg). After the collection of whole blood, the dogs were sacrificed with an intravenous infusion of euthanasia solution. Platelet rich plasma (PRP) was obtained by centrifugation of whole blood at 2000 rpm for 20 min at room temperature.

2.2. Preparation of 111indium-labeled platelets
Platelet labeling with 111In-tropolone was performed as described by Dewanjee et al. [16]. The labeled platelets were examined for their ability to respond to ADP- and collagen-induced platelet aggregation in a Lumi Aggregometer (Chrono-log Corp., Havertown, PA, USA). The 111In-labeled platelets were 60% reactive following the labeling compared to their pre-labeling responsiveness.

2.3. Circulation system
The materials used for the circulation setup consisted of 3 mm I.D. silicon tubing connected to and from a plastic box with two parallel chambers separated by a plastic divider modified from a previously described setup [17]. A roller pump was used to propel the fluid through the circuit at 70 ml/min for a period of 60 min. The total volume of PRP circulated in each chamber was 60 ml. The exterior of the artery was bathed in an oxygenated physiologically buffered solution (PBS). The temperature of the solution and the circuit was maintained at 37°C.

2.4. Preparation of the carotid artery and balloon angioplasty
Both carotid arteries from each dog were carefully isolated and all arterial branches were ligated with 4-0 silk suture. A 5-cm segment from each carotid artery was mounted side-by-side in a dual perfusion chamber immersed in PBS. The intimal surface of both carotid artery segments was injured using a 3x30 mm balloon catheter inflated to 10 Atm at three different sites starting most distal and moving proximally to avoid overlapping injury. The duration of each balloon inflation lasted for 1 min. During balloon inflation, surgical umbilical tape was wrapped around the artery at the center of the balloon to create a resistance between the balloon and the arterial wall ensuring intimal vascular injury. Following injury the PRP with 111In-labeled platelets was perfused through the system. Three treatments were evaluated: (1) group I: three arteries were used to test the effect of balloon injury alone and these were compared to another three control non-injured arteries; (2) group II: ten balloon injured arteries were exposed to thrombostatin (0.5 mM) in the PRP perfusate and another ten balloon injured arteries alone served as control; (3) group III: five balloon injured arteries were exposed to aspirin (1.1 mM) [18] in the PRP perfusate and five other balloon injured arteries alone served as control.

2.5. Measurement of platelet activation and adhesion
2.5.1. Coulter counter
Platelet number and volume were measured by a Coulter counter (Models Zf and MHR, Coulter Electronics, Inc., Hialeah, FL, USA) using 50 µl of PRP sampled at 5–10 min intervals of perfusion. Changes in platelet volume indicated a change in platelet shape and/or platelet aggregates, an indirect sign of in vivo platelet activation. Calibration for both Coulter counter and laser-light scattering techniques was performed using known particle size standards (latex beads of 2, 3 and 5 µm in diameter; Coulter Corp., Miami, FL, USA).

2.5.2. Laser-light scattering
Laser-light scattering was utilized simultaneously to indirectly examine volume changes in the circulating platelets using a customized system. This newly described method allows continuous measurement of platelet volume changes [14]. Briefly, a He–Ne laser beam was split and passed through cuvettes in the tubes draining the carotid artery segments. The scattering light from particles in the cuvettes was spread on the diode array of a multichannel analyzer (Model ST-120, Princeton Instruments Inc., Princeton, NJ, USA). From the angle of incidence, the ratio of scattering light at 1° to 5° represented the particle size distribution. Continuous measurement of the particle size distribution provided an indirect estimation of circulating platelet activation throughout the experiment. A similar system to assess platelet activation has been used in patients with acute vascular syndromes [19]. After perfusion of the arterial segment for 60 min, the arteries were removed and cut into 2-cm long pieces for radioactivity count and processing for histological analysis.

2.5.3. P-selectin expression analysis by flow cytometry
To determine if platelets were activated following vascular injury canine platelet P-selectin expression was measured. P-selectin (CD62) is a trans-membrane protein specific to alpha granules of platelets that is expressed on the surface of only activated platelets. P-selectin expression on platelets was determined in PRP that was used to perfuse balloon injured and non-injured arteries. Detection of canine P-selectin was performed with a fluorescein-labeled mouse monoclonal antibody, MDP-1, that was provided by Dr Samuel Burstein, University of Oklahoma (Oklahoma City, OK, USA). The antibody was labeled with fluorescein using N-hydroxysuccinamide fluorescein (NHS-F, Pierce Chemical Co., Rockville, IL, USA). Briefly, NHS-F was dissolved in DMSO (1 mg/ml) and added in 20-fold molar excess to 1 mg MDP-1 and allowed to react on ice for 2 h. Unreacted NHS-F was removed by diluting with phosphate-buffered saline and ultrafiltration through a Centricon-10 ultrafiltration device (Amicon Inc, Beverly, MA, USA). Test platelets (100 µl) were labeled with 10 µg of fluorescein conjugated MDP-1 for 20 min at room temperature. The labeled platelets were then diluted with PBS containing 0.025% NaN3 and incubated at room temperature until analyzed. Flow cytometry was performed on a Vantage flow cytometer (Becton Dickinson and Co., San Jose, CA, USA). Excitation was performed at 488 nm and detection at 530 nm. Data were analyzed by Cell Quest analysis software (Becton Dickinson and Co., Immunocytometry Systems, San Jose, CA, USA). All samples were analyzed within 2–3 h of collection.

2.5.4. 111Indium-labeled platelet deposition
The platelet deposition was measured by radioactivity associated with each arterial segment as determined by counting in a {gamma}-counter. This radioactivity was presumably due to platelet adhesion/aggregation to the injured arterial wall.

2.6. Gross and microscopic examination
The external surface of the arterial segment was inspected for tears and perforation. To determine the status and thrombogenicity of treated sites, the perfusion-fixed arterial segments were examined using light and scanning electron microscopy (SEM). Samples for SEM were taken from each of the arterial segments and subjected to critical point drying in liquid carbon dioxide, mounted on stubs and gold-coated in a sputter coater. The intimal surface was examined with a SEM (JEOL JSM-6400 V scanning electron microscope, JEOL Ltd., Tokyo, Japan) and representative sections were photographed. The other arterial homografts were embedded in paraffin and mounted on glass slides. The sections were stained with hematoxylin and eosin, and Masson's trichrome and examined using a light microscope.

2.7. Statistical analysis
Intra group analysis between injured and non injured as well as between injured and injured with platelet inhibitor was performed using a paired Student's t-test. The data are reported as mean±S.D. with P<0.05 accepted as the level of significance. Also, regression analysis was performed between platelet deposition and platelet volume change.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Changes in platelet volume by laser-light scattering
Initial investigations determined the optimal time to measure platelet laser-light scattering, a technique to assess platelet volume changes, an indirect measure of platelet activation. Peak changes in platelet volume as detected by platelet laser-light scattering occurred at 15 min after initiating the circulation of PRP. This time for detection of maximal platelet volume change was the same for uninjured and injured arteries. Injured arteries, however, resulted in more platelet volume change (Fig. 1). Therefore, platelet volume measurement by Coulter counter and laser-light scattering were recorded at 15 min in subsequent comparisons below.


Figure 1
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  		Fig. 1 Graph of continuous measurement of circulating platelet activation following balloon injury in one arterial segment and one uninjured artery. Platelet activation peaked at 15 min followed by a gradual reduction in particle volume.

 
3.2. P-selectin expression
Further studies were performed (four balloon injured and four uninjured arteries) to determine if platelet volume studies performed by Coulter counter and laser-light scattering changes were indicative of platelet activation. Using the monoclonal antibody MDP-1, the percent expression of P-selectin in the PRP perfusing the arteries was higher at 15 min versus the immediate baseline after stabilization at 10 min (3.62±0.65 vs. 1.12±0.18, P<0.0001, Fig. 2). These data indicated that laser-light scattering increased simultaneously with platelet activation. However, there was no difference between injured and uninjured arteries.


Figure 2
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  		Fig. 2 Graph of P-selectin expression comparing baseline to peak activation following PRP perfusion. This demonstrates a significant activation of P-selectin expression at 15 min compared to the immediate stabilized baseline at 10 min. The peak effect of P-selectin activation is coincident with the peak of the platelet volume curve shown in Fig. 1.

 
3.3. Group I: balloon injury vs. no balloon injury
In this group six arteries were studied (three balloon injured and three uninjured arteries, Fig. 3). The mean platelet volume as measured by the Coulter counter was increased significantly with balloon injury compared to the uninjured arteries (69.2±20.6 vs. 48.9±16.6 µm3; ratio of injured/uninjured was 1.4:1, P<0.05). Using laser-light scattering, platelet volume represented by the ratio at 1°/5° increased significantly with injury compared to no injury (44.7±25.8 vs. 25.4±14.2 µm3; ratio of injured/uninjured was 1.8:1, P<0.05). Radioactive counts from 111In deposition on the artery was increased significantly with arterial injury compared to no injury (8414±1770 vs. 3637±331 cpm; ratio of injured/uninjured was 2.3:1, P<0.01).


Figure 3
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  		Fig. 3 Bar graph demonstrating the effect of arterial wall injury on platelet volume and arterial wall deposition using (Left) Coulter counter, (Center) laser-light scattering, and (Right) arterial wall radioactivity. All techniques demonstrated a significant increase in platelet activation following balloon injury of the arterial intima.

 
3.4. Group II: thrombostatin vs. injury alone
In this group, 20 injured arteries were studied (10 balloon injured plus thrombostatin and 10 balloon injured alone, Fig. 4). The mean platelet volume as determined by the Coulter counter was significantly reduced with thrombostatin treatment when compared to the injured arteries alone (20.9±6.7 vs. 36.9±15.1 µm3; ratio of thrombostatin/injury alone was 1:1.8, P<0.03). Using laser-light scattering platelet volume was significantly decreased with thrombostatin when compared to injured arteries (26.9±17.2 vs. 50.1±35.3 µm3; ratio of thrombostatin/injury alone was 1:1.9, P<0.01). Platelet deposition on the arterial wall as measured by radioactive 111In counts was significantly reduced with thrombostatin treated circuits compared to untreated, injured arteries alone (1223±584 vs. 5164±1027 cpm; ratio of thrombostatin/injury alone was 1:4.2, P<0.05).


Figure 4
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  		Fig. 4 Bar graph demonstrating effect of thrombostatin on platelet volume and arterial wall deposition using (Left) Coulter counter, (Center) laser-light scattering, and (Right) arterial wall radioactivity. All techniques demonstrated a significant decrease in platelet activation following balloon injury of the intima in the presence of thrombostatin.

 
3.5. Group III: aspirin vs. injury alone
In this group ten arteries were studied (five balloon injured plus aspirin and five balloon injured alone, Fig. 5). Mean platelet volume changes as measured by the Coulter counter was reduced significantly with aspirin treatment of the PRP compared to untreated injured arteries (41.3±12.4 vs. 50.5±7.3 µm3; ratio of aspirin/injury alone was 1:1.2, P<0.01). Also, using laser-light scattering, platelet volume change was decreased significantly with aspirin treatment when compared to injured arteries alone (49.6±16.1 vs. 73.3±26.2 µm3; ratio of aspirin/injury alone was 1:1.5, P<0.03). Radioactive counts from 111In deposition on the artery was significantly reduced with aspirin treatment compared to injury alone (3280±2519 vs. 5810±4715; ratio of aspirin/injury alone was 1:1.8, P<0.02).


Figure 5
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  		Fig. 5 Bar graph demonstrating effect of aspirin on platelet volume and arterial wall deposition using (Left) Coulter counter, (Center) laser-light scattering, and (Right) arterial wall radioactivity. All techniques demonstrated a significant decrease in platelet activation following balloon injury of the intima in the presence of aspirin.

 
3.6. Histology
3.6.1. Light microscopy
Light microscopy of balloon-treated arterial segments showed medial dissection and stretching of the arterial wall when compared to uninjured specimen. These injuries were prominent at sites where the umbilical tape was used to create resistance to the balloon inflation. Also, platelet aggregates were seen at sites of disrupted endothelium. These findings are consistent with previous reports [20].

3.6.2. Scanning electron microscopy
SEM examination of the balloon-injured arterial segments showed wedge-shaped intimal surface tears (Fig. 6). Extensive platelet and fibrin adhesion was observed on the arterial surface at the intimal injury sites. In the injured arteries alone, the wedge-shaped tears were filled with platelets and fibrin thrombi. In the circuit that had PRP treated with thrombostatin or aspirin, the arteries had less platelet adhesion on their arterial surface when compared to balloon injured arteries alone (Fig. 6). Also, fibrin thrombi seen on the arterial lumen had markedly reduced strand thickness when the circuit had aspirin or thrombostatin present (Fig. 7). Normal control arterial segments showed an intact endothelial surface with a typical cobblestone appearance without thrombi (data not shown).


Figure 6
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  		Fig. 6 Scanning electron micrographs of arterial lumen. (A) Micrograph of balloon injured artery demonstrating a break in the endothelial lining with platelet aggregates and fibrin thrombi filling the intimal tear. (B) Micrograph of balloon injury in a thrombostatin treated artery. The endothelium was interrupted and only few deposited platelets could be seen. (C) Micrograph of balloon injury in an aspirin treated artery. The endothelial lining was interrupted and only a few deposited platelets were noted.

 

Figure 7
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  		Fig. 7 Scanning electron micrographs of arterial lumen. (A) Fibrin thrombus in a balloon injured only artery. The fibrin mesh was dense with interspersed platelets. (B) Fibrin thrombus in a balloon injured artery treated with thrombostatin. The fibrin strands were thinner and had fewer platelets.

 
There was a high correlation between platelet deposition on the arterial wall and platelet volume change in circulation (r=0.77; P<0.01). This result suggested that the severity of the arterial wall injury is correlated with the degree of platelet activation and aggregate formation.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
These results demonstrate that thrombostatin was effective in decreasing circulating platelet activation and adhesion following balloon angioplasty. The amount of platelet deposition in the artery is presumed to be a measurement of both platelet adhesion and aggregation. The laser-light scattering technique provided additional information about platelet activity as a function of time following balloon angioplasty. It was observed that with or without a platelet inhibitor, platelet activation peaked at 15 min after which there was a gradual decline (Fig. 1). The use of the laser-light scattering technique and a change in platelet volume as a sign of platelet activation was confirmed by increased P-selectin expression on the circulating platelets. Overall, however, the percent of platelets with P-selectin expression was low and showed no difference between injured and non-injured arterial segments. These results are consistent with literature that indicates that P-selectin is a variable marker of injury after balloon angioplasty [21,22].

As expected, there was a greater amount of platelet adhesion and aggregation with balloon injury compared to no balloon injury (group I). This result confirms the validity of the model to evaluate balloon injury. The disruption in the vascular endothelial surface initiates a series of events leading to platelet activation and thrombus formation. The initial event that occurs at the site of injury is platelet adhesion followed by platelet activation with granule release and platelet aggregation. Meanwhile plasma coagulation proteins are activated on or around the platelet surface to initiate secondary hemostasis that leads to the conversion of prothrombin to thrombin [23].

The high correlation between radioactivity on the arterial wall, due to platelet deposition, and platelet shape and volume changes in the circulation suggest that the extent of arterial damage correlates with the amount of platelet activation in the circulation. It has been presumed that platelet volume changes are due to platelet shape change occurring in individual platelets and also the formation of circulating platelet aggregates. Thus, the presence of circulating platelet aggregates may be a good surrogate indicator of arterial damage. This notion has been demonstrated in acute coronary syndromes where there also appears to be increased platelet aggregation [24,25].

The clinical implication of this study is that thrombostastin, a relatively platelet-selective thrombin inhibitor, is efficacious in inhibiting platelet activation and deposition on the arterial wall after balloon injury. Thrombostatin at high concentrations weakly interacts with the active site of thrombin and at much lower concentrations interacts with PAR 1. In addition thrombostatin also has the potential to be thromboprotective by the fact that its amino terminal arginine is a substrate of nitric oxide synthase [26]. These mechanisms of action for thrombostatin are significantly different than that proposed by Morinelli et al. [27] who showed that RPPGF at nM concentrations protected rats from the lethal effect of lipopolysaccharide. The present findings parallel a study by Hasan et al. [13] indicating that thrombostatin is as potent as aspirin in inhibiting acute coronary thrombosis and combined agents are synergistic to prolong time to thrombosis.

To date there are no known platelet selective thrombin inhibitors. A platelet-selective thrombin inhibitor may provide advantages over a non-selective thrombin inhibitor like hirudin or hirulog. By inhibiting platelet activation only, agents like thrombostatin may prevent arterial thrombosis but may not have the degree of bleeding complications associated with direct thrombin inhibitors like hirudin that inhibit all actions of thrombin. This kind of agent combined with aspirin and a platelet ADP receptor inhibitor may provide sufficient inhibition of platelet function to allow for favorable outcomes in patients with acute coronary syndromes and those undergoing interventional procedures. In conclusion, this study has demonstrated that the arterial wall injury-induced platelet activation in circulation and deposition on the intimal surface can be decreased by the use of aspirin or thrombostatin. Thrombin activation of platelets ex vivo appears to contribute to platelet adhesion to the arterial wall under high flow conditions.

Time for primary review 39 days.


   Acknowledgements
 
The work was supported in-part by grants from the National Institutes of Health, SBIR Phase II (HL55907) as a subcontract from Thromgen, Inc., Ann Arbor, MI, USA, and A.W. Ford Memorial Institute Grant, Wassau, WI, USA. The flow cytometry was performed at the Michigan State University Flow Cytometry Facility.


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

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