In Vivo Efficacy of SM-20302, a GP IIb/IIIa Receptor Antagonist, Correlates With Ex Vivo Platelet Inhibition in Heparinized Blood but Not in Citrated Blood
Abstract—We tested the hypothesis that the in vivo antithrombotic efficacy of SM-20302, a GP IIb/IIIa receptor antagonist, correlates with the ex vivo platelet inhibition in heparinized platelet rich plasma (hPRP) but not in citrated PRP (cPRP). The studies were performed in a canine model of carotid artery thrombosis in which thrombus formation was induced by electrolytic injury. Thrombosis of the right carotid artery was induced immediately after the administration of saline (n=12). Thirty minutes after persistent occlusive thrombosis was obtained, the vessel segment was ligated, and the time to occlusion and thrombus weight were noted. Subsequently, thrombosis of the left carotid artery was initiated in the presence of SM-20302 (100, 300, 600, or 1000 μg/kg IV; n=4 to 6). All the doses of SM-20302 inhibited (by ≥90%) the ex vivo platelet aggregation induced by ADP and arachidonic acid (AA) in cPRP. In hPRP, a dose-dependent inhibition of ex vivo platelet aggregation was observed. The maximal inhibition produced by 100 to 1000 μg/kg SM-20302 ranged from 18% to 80% for ADP and 44% to 88% for AA. Maximal prolongation of the template bleeding time induced by the 100-, 300-, 600-, and 1000-μg/kg doses were 2.5-, 9.5-, 10-, and >10-fold, respectively. All the injured carotid arteries (n=12) in the saline-treated group occluded. SM-20302 pretreatment produced a dose-dependent maintenance of the carotid artery patency, and the incidence of occlusion at 4 hours was 5/6, 3/6, 0/6, and 0/6 for the 100-, 300-, 600-, and 1000-μg/kg doses, respectively. The results indicate that SM-20302 prevents carotid artery thrombosis in response to electrolytic arterial wall injury and that its in vivo antithrombotic efficacy can be correlated accurately with the ex vivo platelet inhibition in PRP prepared from heparinized blood but not from citrated blood.
- Received October 7, 1997.
- Accepted January 6, 1998.
Circulating platelets exert their hemostatic effect through the interaction with their glycoprotein receptors and adhesive proteins. The platelet GP IIb/IIIa receptor, also known as the αIIbβ3 receptor, consists of α- and β-transmembrane subunits. The interaction of GP IIb/IIIa receptors with soluble fibrinogen is a well-recognized phenomenon in platelet aggregation and subsequent intravascular thrombus development. With the exception of chimeric 7E3 (abciximab), most of the newer GP IIb/IIIa receptor antagonists selectively inhibit the GP IIb/IIIa receptor without affecting receptors for vitronectin (αvβ3 and αvβ5) and fibronectin (α5β1).1 Preclinical2 3 4 and clinical5 6 experience with abciximab has demonstrated unequivocally that antagonism of the GP IIb/IIIa receptor is an attractive pharmacological target for preventing platelet-induced thrombosis. For this reason, efforts have been directed toward developing newer, low-molecular-weight drugs to serve as specific and potent GP IIb/IIIa receptor antagonists.
Preclinical evaluation of GP IIb/IIIa receptor antagonists relies importantly on ex vivo platelet inhibition assays and in vivo antithrombotic efficacy. For the ex vivo platelet aggregation assay, trisodium citrate has been a conventional anticoagulant of choice for the collection of whole blood to be used for the preparation of PRP. Unfortunately, the removal of ionized calcium from PRP by trisodium citrate may induce a morphological change in the platelets7 and/or dissociation of GP IIb/IIIa complex into free GP IIb and GP IIIa subunits.8 9 10 11 Under such circumstances, the ex vivo platelet aggregation studies may falsely reveal a greater antiplatelet potency of an antagonist. Intracellular calcium influences the platelet shape change, exposure of GP IIb/IIIa receptors, and fibrinogen binding.1 12 13 To maintain the GP IIb/IIIa receptor complex in an active conformation to bind later to soluble fibrinogen, the extracellular calcium concentration is crucial. In addition, binding of calcium ions to the 4 repetitive domains (resembling calmodulin or troponin C) on the GP IIb subunit of the integrin receptor plays a role in the stability of the receptor complex.14 15 Although trisodium citrate may not alter the intracellular calcium ion concentration, it can certainly do so extracellularly in the platelet suspension and thereby alter receptor function. Such low-calcium conditions may introduce an artifact in evaluation of GP IIb/IIIa receptor antagonists. We first observed this phenomenon with TP-9201, a small, peptidomimetic, cyclic GP IIb/IIIa receptor antagonist.16 In a canine model of carotid artery rethrombosis, we demonstrated that the in vivo efficacy of TP-9201 correlated with the ex vivo platelet inhibition in hPRP but not in cPRP.
SM-20302 is a synthetic GP IIb/IIIa receptor antagonist that has been shown to produce inhibition of the ex vivo platelet aggregation in response to various agonists in rhesus monkeys, guinea pigs, and mice.17 The primary aim of the present investigation was to find an effective dose of SM-20302 that could prevent occlusive arterial thrombus development in response to vessel wall injury. For this dose-finding study, we used the carotid artery of anesthetized dogs for thrombus formation and tested intravenous doses of 100, 300, 600, and 1000 μg/kg of SM-20302. Our experience with GP IIb/IIIa receptor antagonists indicates that reduction in the concentration of ionized calcium in the citrate-anticoagulated blood samples gives a false indication of the inhibitory potency of the drug and does not correlate reliably to its in vivo efficacy. Inhibition of platelet aggregation by SM-20302 in heparinized blood samples, however, may provide a more accurate prediction of the antithrombotic efficacy of the drug in vivo. To test this hypothesis, we performed platelet aggregation studies in citrated as well as heparinized blood samples and correlated the ex vivo aggregometry observations with the in vivo studies on intravascular thrombosis.
The study conforms to the position of the American Heart Association on research animal use adopted November 11, 1984, by the American Heart Association. The procedures followed in this study are in accordance with the guidelines of the University of Michigan (Ann Arbor) Committee on the Use and Care of Animals and with the Guide for the Care and Use of Laboratory Animals, US Department of Health, Education, and Welfare publication NIH 78-23.
SM-20302 was supplied by Sumitomo Pharmaceuticals, Inc. The chemical structure of SM-20302 is shown in Figure 1⇓. The drug was supplied as a white powder that was dissolved in 0.02N HCl-saline mixture and administered in 1- to 3-mL volumes. Trisodium citrate, ADP, AA, epinephrine, and standard reagents were purchased from Sigma Chemical Co. Heparin Sodium Injection, USP (1000 U/mL), was purchased from Elkins-Sinn, Inc.
Model of Vessel Occlusion
Healthy, male or female, purpose-bred dogs (9 to 15 kg) were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated, and ventilated with room air at a tidal volume of 30 mL/kg at a rate of 12 breaths per minute (Harvard Apparatus). The right and left common carotid arteries were exposed by blunt dissection with care taken not to injure the vessel. Catheters were inserted into the right and left femoral veins for blood sampling and drug administration, respectively. Arterial blood pressure was monitored from the cannulated femoral artery with a blood pressure transducer (Gould, Inc). Standard limb lead II of the ECG was recorded continuously and used to monitor the heart rate. A flow probe (model 2RB907, Transonic Systems Inc) was placed on the carotid artery, and blood flow was recorded continuously on a Grass polygraph interfaced with a MacLab data acquisition system and Macintosh PowerBook 140 (Apple Computer). The point of insertion of the intravascular electrode and positioning of the external mechanical constrictor were downstream with respect to the flow probe. The mechanical constrictor on the carotid artery was constructed of stainless steel, shaped to fit around the vessel. A nylon screw (2 mm in diameter) threaded through the C-shaped metal band was adjusted to decrease the circumference of the vessel and to produce a regional stenosis. The vessel was constricted to a point at which the pulsatile flow pattern was reduced by 50% without altering the mean carotid artery blood flow.
The intimal surface of the vessel was electrolytically injured with an intravascular electrode composed of a Teflon-insulated, silver-coated copper wire. Penetration of the vessel wall by the electrode was facilitated by attaching the tip of a 25-gauge hypodermic needle to the uninsulated part of the electrode. The intravascular electrode was connected to the positive pole (anode) of a dual-channel square-wave generator (Grass S88 stimulator and a Grass Constant Current Unit, model CCU1A, Grass Instrument Co). The cathode was connected to a distant subcutaneous site. The current delivered to each vessel was monitored continuously on separate ammeters and maintained at 300 μA for a maximum period of 3 hours. The intravascular anodal electrode was positioned to have the uninsulated portion (3 to 4 mm) in intimate contact with the endothelial surface of the vessel. Proper positioning of the electrode in the vessel was confirmed by visual inspection at the conclusion of each experiment.
Validation of the Experimental Model
The model used in this study is a modification of one developed by our laboratory for the study of experimentally induced coronary artery thrombosis. In this model, electrolytic injury to the intimal surface of the carotid artery invariably results in the formation of an occlusive, platelet-rich, intravascular thrombus.4 The carotid artery was selected for our experimental model, thereby allowing one vessel (the RCA) to be used as a control and the other (the LCA) to be used as a test vessel after the administration of SM-20302. We have previously validated the 2-vessel model of thrombosis, in which it was observed that the blood flow in the control and treated vessels was 122±6 and 135±14 mL/min, respectively. Similarly, the time to occlusion and thrombus weight were not different between right and left carotid arteries (time to occlusion: RCA, 145±4 minutes; LCA, 139±8 minutes. Thrombus weight: RCA, 67±7 mg; LCA, 56±14 mg).18 19 20 Our experience using this model indicates that the blood flow in the test vessel increases transiently but returns to control values after an adequate stabilization period. Therefore, in the present protocol, we provided a 60-minute stabilization period between the ligation of the control vessel and initiation of thrombosis in the test vessel.
The protocol used in the present investigation is outlined in detail in Figure 2⇓. Anodal current was first applied to the RCA (control vessel) and was terminated 30 minutes after the blood flow signal had remained stable at zero flow, indicating formation of an occlusive thrombus. At this point, the vessel segment was first ligated proximal to the thrombus and then distally without disturbing the thrombus. The vessel segment was opened longitudinally to allow removal of the intact thrombus. The extracted thrombus was quickly placed on a cotton gauze pad to absorb residual blood, and the weight of each thrombus was determined on an analytic balance. After removal of the occluded RCA segment, the LCA (test vessel) was instrumented as described above, and the animals were allowed to stabilize for an additional 30 minutes. SM-20302 (100, 300, 600, and 1000 μg/kg) was administered intravenously, after which the anodal current (300 μA) was applied to the intimal surface of the LCA. In all the experiments, the anodal current was applied for a maximum period of 3 hours or was discontinued 30 minutes after the formation of an occlusive and stable thrombus. Determinations of the tongue-template bleeding time and ex vivo platelet aggregation were made at baseline (presaline and predrug) and repeated at 60, 120, and 240 minutes after the administration of SM-20302.
Platelet Aggregation Studies
Blood (10 mL) was withdrawn from the right femoral vein cannula into a plastic syringe containing trisodium citrate solution (3.7%) as the anticoagulant (1:10 citrate to blood; final concentration, 0.37%) for the ex vivo platelet aggregation determinations. Heparinized blood (10 mL; 10 U/mL of blood) also was collected for platelet aggregation studies. The whole-blood cell count was determined with an H-10 cell counter (Texas International Laboratories, Inc). PRP, the supernatant present after centrifugation of anticoagulated whole blood at 1000 rpm for 5 minutes (140g), was diluted with PPP to achieve a platelet count of 200 000/mm3. PPP was prepared after the PRP was removed by centrifuging the remaining blood at 2000g for 10 minutes and discarding the bottom cellular layer. Ex vivo platelet aggregation was assessed by established spectrophotometric methods with a 4-channel aggregometer (BioData-PAP-4, Bio Data Corp) by recording the increase in light transmission through a stirred suspension of PRP maintained at 37°C. Aggregation was induced with ADP (20 μmol/L) or AA (0.65 mmol/L). A subaggregatory concentration of epinephrine (550 nmol/L) was used to prime the platelets before addition of the agonists. Values for platelet aggregation are expressed as percentage of light transmission standardized to PRP and PPP samples yielding 0% and 100% light transmission, respectively.
Template Bleeding Time Determination
Bleeding times were determined with a Simplate device (Simplate-R, Organon Teknika Corp) that made a uniform incision 5 mm long and 1 mm deep on the upper surface of the tongue. The tongue lesion was blotted with filter paper every 30 seconds until the transfer of blood to the filter paper ceased. In all the animals, bleeding time determinations were carried out for a maximum period of 30 minutes.
Concentration of Ionized Calcium in Citrated and Heparinized Plasma
The ionized calcium concentration in plasma was determined in blood samples collected from 3 anesthetized dogs. Whole-blood samples were collected in individual tubes containing either trisodium citrate (0.37%) or heparin (10 U/mL) as the anticoagulant. The blood samples were processed for the preparation of PPP and PRP as described above. After baseline blood samples had been obtained, a 1000-μg/kg dose of SM-20302 was administered intravenously to the dogs, and blood samples were collected at 1 hour. The predrug and postdrug PPP/PRP samples were analyzed for ionized calcium concentration with an ion analyzer (Nova-6, Nova Biomedical Instruments).
The data are expressed as mean±SEM. A one-way ANOVA (repeated measures) was used to assess differences over time within groups. One-way ANOVA (factorial) was used for group comparisons. Fisher’s protected least significant difference and Bonferroni-Dunn post hoc analysis were used to determine significance at P<0.05. A paired t test was used to assess the differences over time within a group, and values were determined to be statistically different at a level of P<0.05. The incidence of occlusion between groups was compared by use of Fisher’s exact test.
Animals to be included in the protocol satisfied the following preestablished criteria: (1) a circulating platelet count of ≥100 000/μL, (2) demonstrated ability for epinephrine-primed platelets to aggregate in response to AA and ADP before administration of saline, (3) thrombotic occlusion of the RCA (control vessel) within 3 hours from the onset of vessel wall injury with a 300-μA direct anodal current, and (4) absence of heartworms on final postmortem examination. One animal was excluded from the study because its carotid artery failed to occlude within 3 hours. The remaining animals satisfied the inclusion criteria.
Concentration of Ionized Calcium in Citrated and Heparinized Plasma
The concentration of ionized calcium was measured in predrug and postdrug PRP and PPP samples that were anticoagulated initially with either trisodium citrate or heparin (Table 1⇓). The concentration of ionized calcium in citrated samples was 18 to 21 times lower than that in heparinized samples. Identical results were obtained when postdrug PRP and PPP samples were analyzed, indicating that SM-20302 per se had no further effect on plasma ionized calcium concentrations.
Antiplatelet Effects of SM-20302
Comparison of the presaline and predrug aggregation profiles indicated that saline administration and thrombosis of the control vessel did not affect the aggregation status (data not shown). All platelet aggregation determinations were performed in cPRP and hPRP samples (Figures 3⇓ and 4⇓). The percent platelet aggregation induced by ADP was similar in cPRP and hPRP before the drug administration. Similar results were obtained with AA.
The aggregation profiles conducted in cPRP did not show a relationship to the drug dose. The maximal inhibition observed with the 300- to 1000-μg/kg was 88% to 90% and 83% to 98% for ADP and AA, respectively (Figures 3⇑ and 4⇑). In hPRP, however, SM-20302 produced a dose-dependent inhibition of ex vivo platelet aggregation (Figures 3⇑ and 4⇑). The maximum inhibition produced by 100 to 1000 μg/kg SM-20302 ranged from 18% to 80% for ADP and 44% to 88% for AA. Inhibition of platelets was reversed by 4 hours with the 100- and 300-μg/kg doses. However, reversal was not observed with the 600- and 1000-μg/kg doses, probably indicating an inhibitory threshold concentration of the drug that was exceeded throughout the protocol.
The template bleeding time was similar (2 to 3 minutes) when performed before saline and before drug administration. However, 1 hour after the intravenous administration of SM-20302, there was a significant increase in the bleeding time, which tended to normalize by 4 hours (Figure 5⇓).
Administration of a wide dose range of SM-20302 did not affect the platelet counts of the dogs under study (Table 2⇓).
Characteristics of the 2-Vessel Model of Thrombosis
Analysis of the hemodynamic and hematological parameters of the dogs indicated that these parameters did not change during thrombosis of either artery (Table 2⇑). The absolute blood flow values at baseline were similar in all the groups under study. The blood flow at baseline in the control and the 100-, 300-, 600-, and 1000-μg/kg groups was 101±4, 129±9, 128±17, and 128±20 mL/min, respectively (P=0.29). Electrolytic injury to the intimal surface of the carotid artery in the absence of antithrombotic intervention resulted in a progressive decline in the blood flow that culminated in an occlusive thrombus formation (Figure 6⇓). Compared with the control vessels, SM-20302 administered as a single intravenous dose showed a dose-related protection against the decrease in arterial blood flow. The blood flow at the end of the protocol was 7%, 30%, 62%, and 67% of the baseline value in the 100-, 300-, 600-, and 1000-μg/kg groups, respectively.
Vessel patency was defined as the presence of a 10-mL/min flow for >1 minute. In the saline-treated animals, all (100%) of the vessels occluded within 60 to 120 minutes after the initiation of anodal current injury to the vascular endothelium. In the drug-treated groups, however, a significant prolongation in the time to occlusion was observed (Table 3⇓). At the end of the protocol, there were more patent vessels in the drug-treated groups than in the control group.
At the conclusion of the experimental protocol, thrombi were removed from the carotid artery and weighed. A significant reduction in carotid artery thrombus weight was noted in the SM-20302–treated groups compared with the saline-treated group (Table 3⇑). In the 100-μg/kg group, however, an increase in the thrombus weight was observed. This discrepancy can be explained by the fact that these vessels occluded at a later time, although the flow progressively decreased. Incomplete inhibition of the platelets and the longer time required for total occlusion may have allowed the continued thrombus growth, thereby resulting in larger thrombi. The higher doses of SM-20302, by completely inhibiting platelet reactivity, may have more effectively prevented thrombus formation during the time of the experimental protocol.
In the present study, we examined the in vivo efficacy of SM-20302 in an electrolytic injury model of the canine carotid artery. The 2-vessel experimental model used in this study was validated earlier.18 19 20 In the absence of any drug intervention, the systemic hemodynamics, occlusion parameters, platelet counts, and hemostatic parameters do not change during the thrombosis of either vessel. The model therefore allows one to use the RCA as a control vessel and the LCA as a test vessel. This reduces the variability in response and allows one to examine the dose-response relationship of the drug under investigation.
In keeping with its ability to block the GP IIb/IIIa receptor, SM-20302 administration was associated with a dose-dependent maintenance of the carotid artery patency. The low dose (100 μg/kg) of SM-20302 used in this study had an initial beneficial effect in terms of slowing the rate of blood flow impairment in response to vessel wall injury. At the end of the observation period, the mean carotid artery blood flow in the low-dose group was <10% of the baseline value, suggesting that the dose of 100 μg/kg was ineffective in maintaining vessel patency. SM-20302 at a dose of 300 μg/kg was associated with a retardation in the rate of thrombus formation, as evidenced by a progressive decline in blood flow, which at the end of the experimental protocol averaged 30% of the initial baseline value. However, a more convincing antithrombotic response to SM-20302 was obtained with the next 2 dosing regimens of 600 and 1000 μg/kg. The data indicate that the latter 2 doses were equally effective in maintaining carotid artery patency. There was a marked decrease in the rate of thrombus development, so that blood flow at the end of the protocol was >60% of the baseline flow. The antithrombotic effects were accompanied by corresponding prolongation in the template bleeding time. The maximal increase in the bleeding times was observed at 1 hour after drug administration, after which there was a tendency for the tongue bleeding time to normalize. The minimal effect of the 100-μg/kg dose on the bleeding time corresponded with its marginal in vivo antithrombotic effect. Similarly, the time course of bleeding time prolongation observed with 300 and 600 μg/kg (or 1000 μg/kg) correlated with the time course of vessel patency.
The present study demonstrated a dichotomy between the SM-20302–associated antithrombotic effects and inhibition of the platelets, as assessed by the ex vivo platelet aggregation assays. This may be due to the nature of the aggregation assays. We used a range of doses with the expectation of obtaining a dose-response relationship with regard to the ex vivo platelet inhibition in cPRP. However, an all-or-none phenomenon was observed with cPRP. Conversely, when 2 different agonists were used, a similar dose-dependent inhibition of platelets was observed in hPRP. This discordance could be explained by the different concentrations of ionized calcium in the 2 assay media. It was noted that the ionized calcium concentration in cPRP was 18 to 21 times less than that in hPRP. A physiological concentration of ionized calcium is critical to maintain the integrity of the GP IIb/IIIa heterodimer complex on the platelet surface18 21 and fibrinogen binding to the activated integrin receptor.11 22 23 24 Phillips et al25 studied binding of PAC1, an antibody that simulates fibrinogen binding to activated GP IIb/IIIa receptors, after ADP stimulation. The concentration of Integrilin required to inhibit PAC1 binding in cPRP was lower than that required in PPACK-anticoagulated PRP, indicating that enhanced inhibition by Integrilin in cPRP was the result of enhanced inhibition of fibrinogen binding. In our study, the presence of SM-20302 in the cPRP may have acted in concert with the low [Ca2+] to reduce the platelet aggregation response and thereby yield a false indication of the in vivo antithrombotic potency of the drug.
The discrepancy in the aggregation profile in cPRP and hPRP was apparent only after the administration of SM-20302, an observation consistent with our previous report.16 The mean predrug percent aggregations for ADP or AA in cPRP and hPRP for different dose groups of SM-20302 were similar. Alteration of the extracellular calcium concentration does not appear to alter the interaction of ADP or thrombin with their respective platelet receptors or platelet activation.13 Moreover, von Willebrand factor can support ADP-induced platelet aggregation when the concentration of ionized calcium is low (20 μmol/L) but not in the presence of a physiological concentration (2 mmol/L) of ionized calcium.26 Lages and Weiss27 noted that the ADP-induced secretory response was less in hPRP than in cPRP, but the maximal extent of platelet aggregation was similar. It is important to note that the concentrations of ADP (20 μmol/L) and AA (0.65 mmol/L) used in the present study were not threshold but rather those that induced maximal platelet aggregation, which makes them relatively insensitive to the calcium concentration in the absence of SM-20302. The discrepancy between cPRP and hPRP does not appear to be due to the activation of platelets by heparin. Using PPACK as an anticoagulant, Phillips et al25 observed a 4- to 8-fold difference in the IC50 values of Integrilin to inhibit platelet aggregation in cPRP and PPACK-PRP. We found similar results with other GP IIb/IIIa receptor antagonists, such as c7E3, MK-383, and DMP-728 (unpublished data, 1997), adding further support to our present in vivo data.
Investigation of the current literature on antithrombotic drugs revealed that the discordance observed in platelet aggregation profiles is not unique to canine PRP or to SM-20302. The same phenomenon has been observed in human PRP with trisodium citrate and hirudin as anticoagulants. In human volunteers who were given dipyridamole, the inhibition of aggregation in response to collagen observed for hirudin-anticoagulated blood was 21%, but that for citrated blood was 66%.28 When 4 different dosing strategies of Integrilin were used in patients scheduled for elective percutaneous interventions, it was observed that administration of a bolus (90 to 180 μg/kg) followed by an infusion (0.5 to 1 μg · kg−1 · min−1 for 20 hours) did not result in a dose-related inhibition of ADP-induced platelet aggregation in cPRP. The maximum inhibition produced by all 4 dosing regimens was ≈90%.29 In the IMPACT-II study,30 it was noted that despite 70% to 78% inhibition of ADP-induced platelet aggregation in cPRP, the Integrilin treatment did not influence 6-month clinical outcomes after PTCA. Recently, Phillips et al25 demonstrated that the inhibitory activity of Integrilin was overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.
In conclusion, the present study revealed that SM-20302 is an effective GP IIb/IIIa receptor antagonist that can prevent primary thrombosis in the experimental animal subjected to deep vessel wall injury. Furthermore, the in vivo antithrombotic efficacy correlated well with the inhibition of platelets in PRP prepared from heparinized blood but not in blood anticoagulated with trisodium citrate. The observations suggest that the decrease in ionized calcium concentration renders the platelet highly susceptible to inhibition when challenged with either ADP or AA. The use of heparin (or hirudin or PPACK) as the anticoagulant during the collection of the blood sample maintains the ionized calcium concentration equal to that in the in vivo situation. The latter approach provides a more accurate ex vivo assessment of platelet reactivity in response to aggregating agents, thus permitting a better correlation with the observed in vivo response.
Selected Abbreviations and Acronyms
|cPRP||=||citrated platelet-rich plasma|
|hPRP||=||heparinized platelet-rich plasma|
|LCA||=||left carotid artery|
|RCA||=||right carotid artery|
This study was supported in part by the Cardiovascular Pharmacology Research Fund and a grant-in-aid from the Sumitomo Pharmaceutical Company. During the tenure of this study, Dr Rebello was the recipient of an Advanced Postdoctoral Fellowship from the American Heart Association, Michigan Affiliate, and a Merck Postdoctoral Fellowship.
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