- Original basic research
- Open Access
Development of a perfusion chamber assay to study in real time the kinetics of thrombosis and the antithrombotic characteristics of antiplatelet drugs
© Stephens et al.; licensee BioMed Central Ltd. 2012
- Received: 28 February 2012
- Accepted: 18 July 2012
- Published: 1 August 2012
Arterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.
Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time.
Untreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen.
These results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.
- Real time
- Antiplatelet drugs
It is recognized that formation of arterial thrombi (thrombus growth) depends on platelet activation, resulting in the activation of the receptor function of GP IIb-IIIa for fibrinogen and von Willebrand factor to mediate platelet aggregation. Under shear stress conditions such as those found in stenosed arteries, platelet activation triggered by either von Willebrand factor interactions with GP Ibα and GP IIb-IIIa[1–4], GP VI binding to collagen, or ADP and TxA2 binding to their respective receptors (P2Y12, TP) have been shown to mediate thrombus growth. Unlike for the effectors of thrombus growth, our knowledge of the effectors of dethrombosis is limited, in part due to a lack of appropriate technology. It is known that fibrin stabilizes platelet-rich arterial thrombi (accounting for the ability of fibrinolytics to enhance vessel patency) and that inhibition of GP IIb-IIIa also has some efficacy in reversing arterial thrombi. Models utilizing intravital microscopy have opened a new era as they permitted real-time analysis of the kinetics of thrombosis and helped discover multiple secondary mediators of thrombus growth and stability (reviewed in). In light of the significant discoveries associated with the use of live microscopy, some laboratories have decided to couple the features of intravital microscopy with perfusion chamber technology to better understand the kinetics of human thrombosis under flow. As a result, it was demonstrated that continuous ADP-P2Y12 signaling is critical for maintaining the active conformation level of GP IIb-IIIa to promote thrombus stability[10, 11]. This observation, however, required specialized laboratories with expensive best-in-class technology.
Since drugs used to treat arterial thrombosis are believed to affect key mediators of thrombus growth and/or stability we decided to develop a simple perfusion chamber assay that allows for mechanistic studies but also continuous monitoring of the pharmacodynamic activity of antiplatelet agents on the kinetics of thrombosis in real time.
The thrombosis profiler (Additional file1: Figure S1A) consisted of a custom built epifluorescence microscope, a syringe pump and a holding stage (Additional file1: Figure S1B) in which sits a glass capillary (Vitrocom, 0.2 mm x 2 mm section) coated with human type III fibrillar collagen. In a first set of experiments, we analyzed the homogeneity of the collagen coating. To do so, thrombotic deposits were rinsed for 40 seconds by perfusion of a rinsing buffer, followed by perfusion of a phosphate buffer solution (used for collagen dialysis) supplemented with glutaraldehyde (final concentration of 2.5%). Fixation was continued for another 2 hours. Post-fixation with KMnO4, dehydration in ethanol was followed by epon embedding, removal of the glass surface and a second embedding in epon. Evaluation of the thrombotic deposits was performed on semi-thin cross-section cut perpendicular to the direction of the blood flow at the proximal part of the capillary. En Face examination indicated homogenous distribution in size and location of platelet-rich thrombi (Additional file1: Figure S1C). We next analyzed rhodamine 6 G uptake by platelets in whole blood. Immediately after collection, blood was labeled with rhodamine-6 G (1.25 μg/mL) and maintained at 37°C for the entire course of the experiment. In 10-minute intervals, 5 μL of blood was sampled and diluted into 1.4 mL of sheath fluid buffer (BD, San Jose, CA). Two minutes after the dilution, each sample was analyzed by BD FACSCalibur. The forward scatter (FSC), side scatter (SSC), and fluorescence channel (FL1) were set at logarithmic scale. A total of 10,000 events in the platelet region were collected. The geometric mean fluorescence of platelets was measured with FL1 gain set at 999 (to allow detection of rhodamine 6 G). A total of 5 different samples were analyzed. Data indicated that constant fluorescence of the platelets is achieved after 20 minutes and lasted for at least 70 minutes (Additional file1: Figure S1D).
Software development for quantification of platelet deposition
Segmentation, partitioning of an image into non-overlapping regions, was accomplished based on a method proposed by Otsu. This algorithm locates a point in the histogram to minimize the intra-class variance of the foreground and the background. Once the threshold is determined, pixels with values lower than the threshold (e.g., circulating blood) are classified as background and pixels with values greater than the threshold (e.g., platelets and thrombi) are marked as foreground. The success of this thresholding method centers upon whether the proper threshold exists and whether it can be inferred from the image histogram. If, for example, the surface reflectance of the objects to be segmented is not distinct from the background or if the scene is not evenly illuminated then the resulting image histogram would not produce a bimodal or multi-modal graph to allow the computation of best possible threshold. For this reason we adopted a multi-stage segmentation process. Thus after applying the threshold to generate a binary image, morphological operation “closing” (dilation followed by erosion-used to fill in holes and small gaps) followed by morphological operation “opening” (erosion followed by a dilation-used to eliminate all pixels in regions that are too small to contain the structuring element) is applied to join together the thrombi objects and clear the image of small artifacts. Next, a median filter was applied to further reduce the salt-and-pepper noise while simultaneously preserving the edges. The fluorescence intensity of the flowing whole blood that was perfused during the first 20 seconds was used for threshold determination, expressed as background object and not taken into consideration in the calculation of the mean fluorescence intensity. Lastly, a watershed algorithm was applied to identify individual thrombi in the image. Once the image was segmented, the sum of intensity values of pixels inside each foreground object was divided by the corresponding object area (μm2) for every frame (sec). Other parameters recorded were the time to reach the peak (sec) and the slope before and after the peak (Additional file1: Figures S1E, S1F). Similar technology and software analysis can be used for evaluation of the pro-coagulant activity of platelets under flow (data not shown).
Within donor variability of Peak Fluorescence (Pixels/Total Area (μm 2 )) and Slope (sBP-Arbitrary Units) of thrombotic profile assessed by RTTP
Peak thrombosis (t = 300 sec)
Within donor variability was established by perfusing 3 whole blood samples collected on different days from 6 individuals at 1000/sec. Maximum peak and slope were calculated for each sample, outlined in Table1. Coefficient of variation for each individual was calculated as SD/mean, ranging from 0.06 to 0.17 in maximum peak and from 0.02-0.19 in slope before peak.
Characterization of the kinetics of thrombosis
Thrombosis triggered by type III collagen followed the classical pattern described by the group of K.S. Sakariassen : i) an increase in thrombus size that parallels that of the wall shear rates (Figure1A;) an axial dependency as thrombi gradually decreased in size towards the distal part (data not shown). The continuous monitoring revealed a linear increase in thrombus growth for lower wall shear rates (125 to 750/sec), a profile that progressively becomes exponential for higher levels of shear. Similar findings were reported by the group of Dr. McIntire on a collagen type I-vWf-coated surface.
Antithrombotic activities of antiplatelet agents
Eptifibatide, inhibited thrombus growth independently of the shear rates (data not shown), and inhibited thrombus growth in a dose-dependent manner at 1000/sec, reaching a maximal effect at 2.4 μM (Figure2A).
Characteristics of the thrombotic profiles generated upon in vitro addition of saline, eptifibatide and 2-MeSAMP (2MeS.)
2-MeS. (10 μM)
2-MeS. (100 μM)
2-MeS. (10 μM) + asa
2-MeS. (100 μM) + asa
Eptifibatide (2.4 μM)
Maximum Peak (pixels/μm2)
596 ± 65 693
309 ± 54 * 318
160 ± 50 ** 109
455 ± 61 423
218 ± 530 **,† 189
127 ± 33 ***, †† 103
63.5 ± 12 *** 53
Time to the peak (sec)
240 ± 0.1 240
216 ± 16 240
197 ± 22 * 231
239 + 0.5 239
214 ± 12 228
204.6 ± 16 *,†† 233
234.4 ± 10 240
Slope before the peak
3.2 ± 0.3 3.1
1.96 ± 0.3 * 1.87
1 ± 0.24 ** 0.94
2.47 + 0.37 2.14
1.33 ± 0.4 **,† 1.13
0.77 ± 0.2 ***,†† 0.43
0.34 ± 0.07 *** 0.3
Slope after the peak
2/7# −0.3 ± 0.1
4/7# −0.5 ± 0.24
4/7# −1.4 ± 0.66
3/7# −0.07 ± .02
4/7# −0.13 ± 0.05
The importance of capturing the kinetics of thrombosis was further highlighted in Figure3A-D. Data showed that the antithrombotic activity (both qualitative and quantitative) of the different compounds and their combination varied according to the duration of the blood perfusion and the process (growth or stability) they affect. All capillaries perfused with untreated samples of blood occluded before 500 sec. While aspirin did not show activity at 150 sec, its curve reached a plateau beyond 200 sec. The combination of aspirin with PRT060128 showed stronger antithrombotic activity, reaching an inflexion point after 150 sec. The size of the error bars (sem smaller for the combination of drugs than for aspirin alone) indicated a constant formation and embolization of smaller thrombi (Figure3C, D). Eptifibatide (0.5 μM, the concentration achieved during the IMPACT-II trial) only affected thrombus growth and could not prevent occlusion in 2 out of the 5 capillaries over the 500 sec period (although the Figure3A suggested strong activity), indicating ability to delay but not to prevent vascular occlusion. This is in sharp contrast with the interpretation of the Figure3A that suggested that eptifibatide (0.5 μM) displayed ~ 50% inhibition while aspirin was ineffective. Greater concentration of eptifibatide (2.4 μM) abolished the thrombotic process throughout the entire duration of the experiment (Figure3D).
Constant signaling via P2Y12 is critical for thrombus stability
Antithrombotic profiles of clopidogrel, aspirin and their combinations
Characteristics of the thrombotic profile of healthy subjects
Clopidogrel + ASA
Max. Peak (pixels/μm2)
M ± sem median
800 ± 51 797
516 ± 48** 526
263 ± 53***,†, ‡ 243
484 ± 67* 460
Time to the peak (sec)
M ± sem Median
239 ± 1 240
203 ± 12*, † 208
234 ± 6 240
Slope before peak
M ± sem Median
4.3 ± 0.3 4.3
2.7 ± 0.2** 2.7
1.6 ± 0.2***,†, ‡ 1.6
2.7 ± 0.4 2.65
Slope after peak
M ± sem median
1 out of 8# −0.18
5 out of 8# −1.2 ± 0.8
2 out of 8# −0.5 ± 0.4
Thrombus volume (μm3/μm2)
M ± sem
37 ± 5.2
25.8 ± 3.7
5.4 ± 0.5
22.1 ± 4.8
Perfusion chambers were developed more than 30 years ago to study platelet thrombosis in samples of non-anticoagulated or anticoagulated blood exposed to physiological thrombogenic surfaces under defined conditions of shear (for review, see). Major contributions to the field of thrombosis have been reviewed by Sakariassen and coworkers[19, 20]. These include: - the understanding of the mechanisms of inherited clinical disorders associated with platelet pathology (e.g., von Willebrand disease, Glanzmann thrombasthenia, Bernard Soulier Syndrome, etc.…), - the relative role of coagulation factors in thrombosis (Tissue factor, Fibrinogen, FVII, FVIII, FIX, FXI and FXII), - the critical role of platelet membrane glycoproteins (GP IIb-IIIa, GPIb-IX-V and GPVI) in mediating platelet adhesion and thrombus growth under arterial shear rates, - the agonist activities of ADP and TxA2. Perfusion chamber technology has also proven instructive in monitoring the antithrombotic activity achieved by antithrombotic therapies and their combination, e.g. clopidogrel plus aspirin. Data showed that the clinical benefits achieved by the combination of these two drugs were superior to each drug used as monotherapy[6, 17, 21]. Recently published manuscripts by Hosokawa et al. measuring perfusion chamber flow pressure changes to evaluate the thrombotic profiles of whole blood support the methodology described here[22, 23]. Despite the different quantification methods, the measured effects on thrombus formation and stability were similar- specifically the activities of aspirin alone, P2Y12 inhibitor alone, the combination of aspirin and P2Y12 inhibitor and the GP IIb-IIIa inhibitor.
As highlighted in this manuscript, three considerations suggest that continuous monitoring of thrombosis at arterial rates of shear would be of value to understand the molecular mechanism regulating arterial thrombosis and to determine the antithrombotic activity achieved in individual patients: First, the assay provides immediate, real time evaluation of the kinetics of thrombosis, it is easy to use, reproducible, detects and differentiates the activity of mono and combination therapies, and can be used as a bedside device. Second, it has become clear from clinical conditions such as unstable angina and from experiments in animals such as those using the Folts model that platelet factors exist that mediate or inhibit thrombus stability. Our data obtained in blood from healthy volunteers confirmed the fact the clinical efficacy of the state-of-the art antiplatelet therapy (clopidogrel + aspirin) utilized in the management of post-atherothrombotic events certainly stems from a synergism in destabilization activities of the two drugs[11, 12, 24]. They also suggested that high levels of inhibition of the P2Y12 receptor may be sufficient to provide substantial benefits in agreement with others[17, 25]. The effect of eptifibatide related on the other hand, to the inhibition of thrombus growth. This is particularly important as we found that 0.5 μM eptifibatide (the plasma concentration achieved during the IMPACT-II trial that failed at demonstrating substantial clinical benefits) only delayed the growth of the thrombus, but could not prevent occlusion of the collagen-coated capillary upon prolonged perfusion (Figure3). Third, emerging data suggest that the antithrombotic response to aspirin and clopidogrel is variable, possibly including non-responders[27, 28]. Three out of 20 healthy individuals did not benefit from either aspirin or clopidogrel treatments, suggesting non-responsiveness. Interestingly however, all individuals showed robust inhibition while on combination regimen, indicating that the mechanisms targeted (COX-1 or P2Y12) while inhibited, could not account for antithrombotic activity without concomitant inhibition of the other signaling pathway.
While this methodology for the evaluation of anti-platelet therapies has obvious benefits, there are also limitations that warrant discussion: i) use of a single purified protein surface rather than damaged endothelial cells- the choice of collagen can affect the thrombotic process therefore careful standardization of the collagen preparation (or protein of choice) is required ii) use of anticoagulant- a method for evaluation in absence of anticoagulant at low and high shear would facilitate the study of existing therapies on both fibrin formation and platelet deposition simultaneously. Based on the optimized conditions used in this study (high shear, and Factor Xa anticoagulant), our results relate to platelet rich thrombi only.
Coupling the real time feature of intravital technology with perfusion chamber has been proposed by the group of McIntire almost 20 years ago. This concept was based on the simple idea that real time monitoring of perfusion chambers would circumvent the limitations of light transmittance aggregometry and other point of care devices which either: i) do not offer continuous monitoring of the kinetics of arterial thrombosis, ii) interrogate only one platelet signaling pathway at a time and therefore cannot provide reliable monitoring of combination therapies. The system described herein offers a relatively simple and reproducible technology that can be utilized to: i) characterize the mechanism of action and activity of antiplatelet or anticoagulant agents ii) possibly tailor patient therapy, iii) identify novel antiplatelet targets.
Real time perfusion chamber assay
Blood was collected from the antecubital vein of subjects who gave written informed consent to the protocol (Approval was obtained from Western Institutional Review Board or the California Pacific Medical Centre Human Subjects Committee) via butterfly 19 G on syringes containing 5 μM (final concentration) of the factor Xa inhibitor C921-78, a concentration sufficient to block any FXa activity.
Platelets were labeled in whole blood by adding an aliquot of Rhodamine 6 G (final concentration, 1.25 μg/ml). To visualize platelet deposition through the microscope, the dye was excited with light from a high-power light emitting diode with a spectral maximum at 530 nm and a spectral half width of 35 nm (Luxeon V, Lumileds Lighting, San Jose, CA). Excitation and emission lights were filtered with a set of fluorescence filters (31002, Chroma Technologies, Rockingham, VT). A microscope objective imaged an area of 360 x 270 μm on the internal wall of the capillary onto a Sony XCD X-710 digital camera (resulting magnification ca. 13 x). Images were recorded at a frequency of 1 Hz. Blood flow was established by a syringe pump withdrawing blood through the capillary (Harvard Apparatus, Holliston, MA). A personal computer with custom software was used to control the camera and the syringe pump, and to display and record images and experimental conditions.
Unless otherwise specified, whole blood was perfused through the capillary at 1000 or 1500/sec for a period of 5 minutes according to the following equation: γ = 1.03x6Q/ab2, where γ = shear rate; Q = volumetric flow rate (ml/60); b = height (cm); a width (cm).
Antithrombotic activities of antiplatelet agents
Eptifibatide (added in vitro at 0.125 to 2.4 μM) and 2-MeSAMP (a direct P2Y12 antagonist added in vitro at 10 and 100 μM, a concentration that fully antagonizes P2Y12) effects were tested in absence or presence of aspirin (325 mg QD for 3 days) at 1000/sec. Qualitative and quantitative changes in thrombotic profiles of 5 individuals treated with vehicle control, aspirin, aspirin + PRT060128 (2.5 μM,) or eptifibatide (0.5 and 2.4 μM) were also assessed over a 500 second perfusion period.
In a second set of experiments, we asked whether addition of inhibitors of the receptors for fibrinogen (GP IIb-IIIa) or ADP (P2Y12) to preformed thrombi could induce dethrombosis. Untreated blood samples were perfused at 1500/sec through the chamber for 210 seconds, followed by blood treated with saline, 2.4 μM eptifibatide, or 2-MeSAMP (10, 100 μM) for 100 seconds. Similar design was applied to the blood of normal volunteers who had been taking aspirin (325 mg/d) for 3 consecutive days.
Monitoring the antithrombotic activities of clopidogrel, aspirin and clopidogrel + aspirin in humans
Antithrombotic effects were evaluated in a sequential study at the California Pacific Medical Centre (CPMC, San Francisco, CA). Twenty individuals were evaluated before antithrombotic therapy, 2 weeks after a daily regimen of clopidogrel (75 mg/d), 1 week after a dual clopidogrel (75 mg/d) + aspirin (325 mg/d) therapy, and finally 2 weeks off clopidogrel therapy while continuing on aspirin regimen (325 mg/d).
Statistics were performed with Graph Pad Prism v4.03 and numerical data are presented as mean ± S.E.M. The significance between two data sets was tested using paired t-test for the CPMC study, and unpaired t-test for all other in vitro studies. p < 0.05 was considered to be statistically significant.
GS and PA designed and executed experiments. GS and PA wrote the manuscript. MH, CW, MJ conducted experiments. H-CL, GS, KM, and CM designed, executed hardware and software of the assay. PBC and DRP contributed to scientific design of the studies. All authors read and approved the final manuscript.
The authors would like to thank Nathan Frazee for his contribution to the mechanical design of the kinetic aggregometer.
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