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CARDIOVASCULAR
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan (T.-T.H., E.M.D., A.J.W., A.S.); and Research and Development Center of Cephalon France, Maisons Alfort, France (T.A.G.)
Received April 9, 2003; accepted May 2, 2003.
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Abstract |
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). Therefore, the concomitant use of adjunctive agents to
prevent rethrombosis after coronary angioplasty or successful thrombolysis is
needed to maintain vessel patency in patients undergoing revascularization.
Platelet adhesion to exposed subendothelial surfaces of injured vessels with
subsequent activation and platelet aggregation are known to be involved in
rethrombosis. Current clinical practice makes use of one or more
"antiplatelet" agents such as aspirin, ticlopidine, and
clopidogrel, used alone or in combination. Although having a beneficial
effect, the various treatment regimens are only partially effective due to
their limited platelet inhibitory effects.
The aggregation of platelets is mediated by activation of the platelet
membrane glycoprotein IIb/IIIa receptor (GPIIb/IIIa)
(Peerschke, 1985;
Plow and Ginsberg, 1989).
Platelet aggregation can be induced by activation of different membrane
receptors such as PAR1, PAR4, P2X1, and P2Y12. Aspirin is effective ex vivo in
preventing arachidonic acid-induced platelet aggregation, but lacks
significant antiaggregatory efficacy against other platelet agonists, for
example, 5-hydroxytryptamine, epinephrine, thrombin, and plasmin. However, the
final common pathway for platelet activation is the surface expression of
GPIIb/IIIa and subsequent cross-linking with soluble fibrinogen.
Pharmacological blockade of the GPIIb/IIIa will interfere with the final
common pathway for arterial thrombus formation, irrespective of the mode of
platelet activation. Agents directed at the platelet GPIIb/IIIa receptor
preserve vessel patency and adequate blood flow to maintain tissue viability
(Makkar et al., 1997). The
first platelet GPIIb/IIIa receptor antagonist was the chimeric 7E3 (ReoPro)
(Coller, 1985). Several
antiplatelet agents (abciximab, eptifibatide, tirofiban, ticlopidine, and
clopidogrel), having mechanisms of action differing from that of aspirin, show
efficacy in reducing the incidence of thrombotic occlusion after restoration
of arterial blood flow (Spinler et al.,
2001). The major drawback to current small molecule GPIIb/IIIa
receptor antagonists (tirofiban and eptifibatide) is the need to be
administered by the parenteral route and their relatively short
pharmacological half-life. Although orally effective GPIIb/IIIa receptor
antagonists have been developed and studied clinically, none have achieved the
status of approval due to problems related to bioavailability, an effective
oral dosing regimen, and potential for excessive bleeding.
Potential advantages of a new small molecule GPIIb/IIIa platelet receptor antagonist, CRL42796, are that it is effective by parenteral as well as oral administration. CRL42796 is a nonpeptide, bispiperidine, GPIIb/IIIa platelet receptor antagonist. In vitro studies indicate that CRL42796 exhibits a high potency (10–8 M) in preventing platelet reactivity in human, monkey, dog, and guinea pig platelets. Previous studies from our laboratory suggested that CRL42796 is effective in maintaining vessel patency with a minimum risk of bleeding when used as the singular antiplatelet agent. It is anticipated that the risk for bleeding may be reduced further by combining CRL42796 with one or more adjunctive agents that act at different sites in the coagulation cascade. Furthermore, the combined use of adjunctive therapies may serve to enhance the efficacy of thrombolysis while at the same time providing a high quality of arterial blood flow.
The potential for excessive bleeding after the administration of a GPIIb/IIIa platelet receptor antagonist, especially in the presence of a thrombolytic agent, presents an additional problem in patient management. Therefore, our recent efforts have focused on the concomitant use of a suboptimal dose of CRL42796, along with aspirin (current standard of care) in the absence or presence of low doses of a low molecular weight heparin, enoxaparin. It was hypothesized that the combined therapy would be more effective in terms of maintaining vessel patency while limiting the potential for excessive bleeding.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Reagents. CRL42796 was provided by Research and Development Center
of Cephalon France (Maisons-Alfort, France). 
-Thrombin was purchased
from Enzyme Research Laboratories Inc. (South Bend, IN). Sodium citrate, ADP,
arachidonic acid, epinephrine, and other standard reagents were purchased from
Sigma-Aldrich (St. Louis, MO).
Model of Vessel Occlusion. Healthy male and female purpose-bred beagle dogs (9–13 kg) were anesthetized with sodium pentobarbital (30 mg/kg i.v.), intubated, and ventilated with room air at a tidal volume of 30 ml/kg at a rate of 12 breaths/min (Harvard Apparatus Inc., Holliston, MA). Catheters were inserted into both the right and left femoral veins and the right jugular vein for blood sampling and drug administration, respectively. Arterial blood pressure was monitored from the cannulated femoral artery with a Millar Mikro-tip catheter (Millar Instruments Inc., Houston, TX). The standard limb lead II of the electrocardiograph was recorded continuously and used to monitor heart rate.
The model used in this study was a modification of one developed by our
laboratory for the study of experimentally induced coronary artery thrombosis
(Rote et al., 1994). The model
makes use of anodal current-induced electrolytic injury to the intimal surface
of the arterial wall that invariably results in the formation of an
intravascular thrombus. The heart was exposed by a left thoracotomy in the 5th
intercostal space and suspended in a pericardial cradle. A calibrated flow
probe (model 1.5RB907; Transonic Systems Inc., Ithaca, NY) was placed on the
left circumflex coronary artery to continuously monitor blood flow
(milliliters per minute). Securing a suture-ligature around the artery and an
adjacent 18-gauge hypodermic needle and then removing the needle produced an
external stenosis. An intracoronary electrode was fashioned from the tip of a
25-gauge (4-mm) hypodermic needle attached to a 30-gauge Teflon-insulated,
silver-coated copper wire. 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 CCUIA; Grass Instruments,
Quincy, MA). The cathode was connected to a distant subcutaneous site. The
current delivered to the artery was monitored continuously on with an ammeter
and maintained at 150 mA. Proper positioning of the electrode in the vessel
was confirmed by visual inspection at the conclusion of each experiment. The
arterial wall lesion and subsequent formation of an occlusive thrombus results
from direct application of an anodal direct current to the endothelial surface
of the vessel, leading to exposure of proaggregatory subendothelial elements
(Mickelson et al., 1990). The
resulting thrombus consists of a platelet-fibrin mass adherent to the vessel
wall at the site of intimal injury (Romson
et al., 1980) and possesses the morphological features of acutely
formed thrombi found in patients with acute myocardial infarction
(Falk, 1985).
Experimental Protocol. The present study was designed to assess the ability of CRL42796 to prevent rethrombosis after successful thrombolysis and to determine whether a subtherapeutic dose of CRL42796 (15 µg/kg i.v. loading dose followed by an intravenous infusion of 0.31 µg/kg/min) could prevent rethrombosis when combined with other adjunctive agents such as aspirin, enoxaparin, or both.
The proposed studies are outlined in Fig. 1 and were conducted in the anesthetized animal in which the coronary artery was subjected to electrolytic injury leading to the formation of a stable occlusive, platelet-rich thrombus. The occlusive thrombus was allowed to age for 60 min before initiating thrombolytic therapy. The thrombolytic agent rt-PA was administered as a local injection immediately proximal to the occlusive thrombus 15 min after the initiation of adjunctive therapy. The locally administered lytic agent was given as a slow intra-arterial injection of 0.2 mg/kg over 3 to 4 min followed by a continuous infusion of 0.72 mg/kg delivered over a period of 60 min. The experiment was terminated at 4 h after the initiation of intracoronary administration of rt-PA. The adjunctive therapy was maintained throughout the duration of the experimental protocol.
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Thirty-five dogs were randomized among six different groups as follows: group 0 (n = 6) received a placebo i.v. infusion as the adjunctive therapy; group 1 (n = 6) received CRL42796 i.v. loading-dose of 15 µg/kg followed by an intravenous infusion of 0.31 µg/kg/min; group 2 (n = 6) pretreatment with three doses of oral aspirin (7 mg/kg) at –47, –23, and –17 h and received placebo i.v. infusion on the day of the experiment; group 3 (n = 6) also received aspirin pretreatment and was given CRL42796 i.v. loading dose of 15 µg/kg followed by an infusion of 0.31 µg/kg/min i.v. infusion; group 4 (n = 6) received aspirin pretreatment and was administered enoxaparin 0.6 µg/kg i.v. loading dose followed by 6.0 µg/kg/min i.v. infusion; and group 5 (n = 5) was pretreated with aspirin and received CRL42796 and enoxaparin i.v. infusion simultaneously.
Blood samples were obtained at prescribed intervals to assess the coagulation parameters. The mean arterial blood pressure, heart rate, and LCX coronary artery blood flow were monitored throughout the experiment. Time to thrombolysis was determined as well as the duration of vessel patency. The thrombolytic agent rt-PA was administered via an indwelling cannula positioned immediately proximal to the occlusive thrombus. The lytic agent was administered as a loading dose followed by an infusion 15 min after the initiation of the adjunctive therapy. The local administration of rt-PA limits the total amount of lytic agent required to achieve clot lysis, thereby limiting the development of a systemic lytic effect. At the end of the protocol, all animals were euthanized by electrical induction of ventricular fibrillation and the injured vessel segment proximal and distal to the point of injury was removed without disturbing the intravascular thrombus. The vessel segments were opened longitudinally to allow removal of the intact thrombus. The weight of each thrombus was determined using an analytic balance.
Quantitive Analysis of Oscillatory Flow. Criteria were established
to quantify the quality of coronary artery blood flow and applied every 30 min
after application of the anodal injury current. The established criteria are
as follows: 1) A rating of 0 indicates the absence of blood flow. 2) A rating
of 1 is applied when flow declines to 0 and is restored spontaneously. 3) A
rating of 2 indicates a decline in blood flow that is restored spontaneously
before it reaches zero flow. 4) A rating of 3 is applied when the blood flow
is maintained relatively constant in a nonoscillatory manner
(Sudo and Lucchesi, 1996).
Inclusion Criteria. Animals included in the final protocol satisfied the following preestablished criteria: 1) A circulating platelet count of not less than 100,000/ml. 2) Demonstrated ability for epinephrine-primed platelets to aggregate in response to ADP or arachidonic acid before administration of saline or the candidate agonist. 3) Absence of heartworms and intestinal nematodes upon final post-mortem examination.
Hematological Measurements. Determinations of the aPTT,
tongue-template bleeding time, and ex vivo platelet aggregation were made at
baseline and repeated at 60 min after thrombotic occlusion, 15, 75, 195, and
255 min after i.v. administration of placebo, CRL42796, enoxaparin, or both of
the drugs. Blood (10 ml) was withdrawn from the right femoral vein cannula
into a plastic syringe containing 3.7% sodium citrate as the anticoagulant
[1:10 citrate to blood (v/v)] for the ex vivo platelet aggregation
determinations. An additional 10 ml of blood was withdrawn into another
syringe containing 50 IU heparin. The whole blood cell count was determined
with an H-2000 cell counter (Texas International Laboratories, Inc., Culver
City, CA). Platelet-rich plasma (PRP), the supernatant present after
centrifugation of whole blood at 660 rpm for 8 min, was diluted with saline to
achieve a platelet count of 
200,000/ml. Platelet-poor plasma (PPP) was
prepared by centrifuging the remaining blood at 12,000g for 10 min
and discarding the bottom cellular layer. Ex vivo platelet aggregation was
assessed by established spectrophotometric methods with the use of a
four-channel aggregometer (BioDataPAP-4; Bio/Data Corp., Horsham, PA) by
recording the increase in light transmission through a stirred suspension of
PRP maintained at 37°C. Aggregation was induced with ADP (20 or 5 µM),
AA (0.65 mM), or -thrombin (25 nM). A subaggregatory concentration of
epinephrine (550 nM) was used to prime the platelets before addition of the
agonists to induce platelet aggregation. Values for platelet aggregation are
expressed as percentage of light transmission standardized to PRP and PPP
samples yielding 0 and 100% light transmission, respectively.
The anticoagulation state of the animals was assessed by determining aPTT with a Hemochron analyzer (Technidyne, Edison, NJ) and reagents supplied by the manufacturer. Citrated whole blood (2 ml) was used for these determinations. The bleeding time was measured with a Simplate device (Simplate-R; Organon Teknika, Durham, NC) by making incisions of 5 mm in length and 1 mm in depth on the upper surface of the tongue. The tongue lesion was blotted with a filter paper every 15 s until the transfer of blood to the filter paper ceased. The time from the initial tongue incision to the cessation of active bleeding was recorded as the bleeding time.
Statistical Analysis. The data are expressed as mean ± S.E.M. The data were analyzed by one-way analysis of variance for group comparisons and for repeated measures followed by a Dunnett's post hoc t test to determine the level of significance. A paired t test is used to assess the differences over time within a group. Vessel flow patency is compared by a chi square test. Values are considered to be statistically different at a level of P < 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Blood Coagulation Parameters. Percentage of platelet aggregation
responses to AA, ADP, or -thrombin did not change throughout the
experiment in the controls (group 0). Platelet aggregation induced by AA was
inhibited significantly in all of the aspirin-pretreated animals, whereas
aspirin pretreatment alone did not affect ex vivo platelet aggregation in
response to ADP or -thrombin. CRL42796 alone caused significant
inhibition of ex vivo platelet aggregation responses to AA, ADP, and
-thrombin only in citrated blood, whereas the responses to the agonists
were partially decreased in PRP prepared from blood anticoagulated with
heparin. Enoxaparin treatment inhibited platelet aggregation in response to
-thrombin, but did not affect the aggregation responses to ADP or
arachidonic acid. However, in the presence of each of the three adjunctive,
platelet aggregation responses to each of the agonists was inhibited
significantly in both the citrate and heparin anticoagulated PRP preparations
(Tables 2 and
3).
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The aPTT determinations were not altered by any of the adjunctive interventions used alone or in combination when examined throughout the entire experimental protocol. Aspirin, CRL42796, or enoxaparin, used individually, did not increase the tongue bleeding time. In the groups that received aspirin pretreatment plus CRL42796 or enoxaparin, the bleeding time increased moderately when determined 1 h after the intracoronary artery administration of rt-PA. Despite the initial increase in the bleeding time, the values returned almost to baseline 2 h after discontinuation the rt-PA infusion. However, in the final group receiving rt-PA plus the three adjunctive agents concomitantly, the tongue bleeding time was prolonged significantly throughout the experimental protocol (Table 4). The increase in bleeding time represents the summation of the individual interventions upon the coagulation cascade and alteration of platelet reactivity and cannot be attributed exclusively to any one of the administered agents.
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Time to Thrombolysis, Total Reperfusion Time, and Thrombus Weight. The time to thrombolysis after local administration of rt-PA was similar in each of the six groups, suggesting that pretreatment with the adjunctive agents used alone or in combination did not influence the lytic action of rt-PA.
Aspirin alone decreased the thrombus weight, although the difference compared with the control group did not achieve statistical significance. CRL42796 alone, the combination of aspirin with CRL42796 or aspirin plus enoxaparin was associated with a significant decrease of thrombus weight. The combined administration of the three adjunctive agents caused a further decrease of thrombus weight (Fig. 2).
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The total reperfusion time or vessel patency, defined as blood flow >20% of baseline blood flow, is shown graphically in Fig. 3. Aspirin or CRL42796 alone, or aspirin plus enoxaparin increased the duration of reperfusion compared with the control group, although the difference was not statistically significant. However, aspirin plus CRL42796, in the absence or presence of enoxaparin, caused a significant increase in total reperfusion time, whereas the combination of the three adjunctive agents significantly prolonged the total reperfusion time.
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LCX Flow Patency and Reocclusion Incidence. A patency score of 3 was assigned to all of the vessels before application of the electrolytic injury current. In each case, the patency score achieved a value of 0 at the time of vessel occlusion due to formation of an occlusive arterial thrombus. Approximately 45 min after cessation of blood flow in the LCX coronary artery, each animal received the respective pretreatment regimen followed 15 min later by the local, intracoronary administration of rt-PA. Pretreatment with the selected adjunctive agents neither induced clot lysis nor influenced the efficacy of rt-PA, which brought about clot lysis in each of the animals.
In the control group, rt-PA administration resulting in a flow patency score of 1 that reverted to 0 within 60 min. In contrast, group 5 animals, which received aspirin + enoxaparin + CRL42796, achieved a coronary artery patency score of 3 within 60 min of having received rt-PA. The flow patency score of 3 was maintained for the duration of the study, although this was associated with an increase in the tongue bleeding time.
All the other groups that received different pretreatment regimens, the LCX flow patency score was intermediate between those of the control group and group 5 (Fig. 4). Whereas each of the vessels in the control group reoccluded by the end of the experimental protocol, none of the injured vessels reoccluded in group 5. The incidence of LCX reocclusion for each of the six groups is summarized in Table 5.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The final
common pathway in thrombus development involves platelets binding to
fibrinogen via the GPIIb/IIIa receptor. Therefore, platelet GPIIb/IIIa
receptor antagonists prevent platelet-dependent arterial thrombus formation
regardless of the inciting agonist. Currently approved GPIIb/IIIa antagonists
include the monoclonal antibody Fab fragment abciximab; cyclic peptides based
on RGD or related motifs, including eptifibatide; and the RGD-based
peptidomimetic tirofiban (Bennett,
2001). Several oral GPIIb/IIIa antagonists (xemilofiban,
sibrafiban, lamifiban, and roxifiban) were examined experimentally as well as
clinically but have not been approved for clinical use due to limited oral
bioavailability and the potential to induce serious bleeding.
The nonpeptide GPIIb/IIIa receptor antagonist CRL42796 was reported to
prevent primary thrombosis when administered intravenously
(Hennan et al., 2002) or after
oral dosing (Hennan et al.,
2003). The minimal effective intravenous dosing regimen for
preventing arterial thrombosis required a loading dose of 15 µg/kg and a
continuous infusion ranging between 0.31 and 0.69 µg/kg/min. Our previous
study demonstrated the in vivo efficacy of CRL42796 in an electrolytic injury
model of the canine carotid and coronary artery. With the lower dose, a
progressive decline in blood flow was observed over the duration of the
experimental protocol.
In the present study, we determined the minimal effective dose of CRL42796 for prevention of rethrombosis (secondary thrombosis) after successful thrombolysis. The results indicate that CRL42796, administered in a subtherapeutic dose, was of limited efficacy in preventing arterial rethrombosis after thrombolysis.
Ex vivo platelet responses from citrate anticoagulated whole blood did not
correlate with the inhibition of arterial thrombosis because citrate chelates
ionized calcium (Phillips et al.,
1997; Rebello et al.,
1998). Ionized calcium is essential for the formation of the
GPIIb/IIIa heterodimer complex (Lam,
1992) and for the interaction between the GPIIb/IIIa receptor and
fibrinogen (Steiner et al.,
1989). Binding of the GPIIb/IIIa receptor antagonist is enhanced
when the Ca2+ concentration of platelet-rich plasma is
decreased by citrate, thus overestimating the efficacy of an antiplatelet
agent. In animals treated with the low dose of CRL42796 (0.31 µg/kg/min),
rethrombosis was not prevented and the platelet response in PRP prepared from
heparin anticoagulated whole blood was reduced, but not inhibited
significantly. Thus, in the low dose CRL42769 treatment group, only two of six
vessels remained patent throughout the study protocol. Two-thirds of the
animals exhibited LCX coronary artery rethrombosis despite the fact that a
significant degree of ex vivo platelet aggregation was noted when the assay
was done in citrate anticoagulated samples.
Aspirin, an irreversible, noncompetitive inhibitor of platelet
cyclooxygenase and thromboxane A2 formation
(Smith and Willis, 1971), is
effective in secondary prevention of thrombosis in patients with
atherosclerosis (Hennekens,
1990; Koller,
1991) and is the standard therapy against which other agents are
judged. In the present study, 7 mg/kg aspirin was administered orally as a
pretreatment –47, –23, and –17 h before commencing the
experimental protocol. This dosing regimen of aspirin inhibits platelet
responses to arachidonic acid while allowing endothelial sources of
cycloxygenase-2 to regenerate and participate in the synthesis of prostacyclin
(prostaglandin I2) (Hennan et al.,
2001). Animals treated with aspirin were not protected from
rethrombosis as each of the coronary arteries reoccluded after successful
thrombolysis. The limited efficacy of aspirin may relate to its inability to
prevent platelet responses to ADP, thrombin, and collagen plus the added
prothrombotic actions of plasmin generated as a result of rt-PA
(Topol, 2000). Enhanced
protection against rethrombosis was observed among the animals administered
aspirin and CRL42796. Total reperfusion time in the aspirin + CRL42796-treated
group was longer compared with that of animals in which each drug was
administered separately. In animals treated with the lower infusion dose of
CRL42796 (0.31 µg/kg/min), ex vivo platelet responses in PRP prepared from
heparin anticoagulated whole blood were reduced, but not inhibited
significantly. Therefore, the addition of aspirin to the treatment regimen
with the subsequent inhibition of thromboxane A2 synthesis provides an added
degree of in vivo platelet inhibition.
Enoxaparin, a low molecular weight heparin, is approved for the management
of patients with unstable angina. Arterial thrombosis involves the activation
of both the coagulation cascade and circulating platelets and the combination
of an anticoagulant directed against the formation of thrombin along with an
antiplatelet agent may provide a means to maintain coronary artery patency
after thrombolysis. The enoxaparin-antithrombin III complex inhibits activated
factor X, thereby reducing the activation of the intrinsic coagulation pathway
and subsequent generation of thrombin. In the present study, the combination
of enoxaparin and aspirin prolonged total reperfusion time, but did not
prevent secondary occlusive thrombus formation after successful thrombolysis.
Whereas enoxaparin and unfractionated heparin can modulate thrombin formation,
their ability to inhibit platelet activation is limited. However the
combination of aspirin + enoxaparin + low dose CRL42796 was effective in
preventing secondary thrombosis in each of the animals studied. All the
vessels remained patent and the patency score for LCX coronary artery blood
flow was similar to that observed before the induction of vessel wall injury.
The findings in this study are consistent with clinical observations in which
the combined use of a lytic agent with abciximab or eptifibatide has yielded
high frequencies of infarct vessel patency
(Topol, 2000).
After fibrinolytic therapy, there remains a significant area of vessel wall
injury due to electrolytic injury (experimental model) or as in the case of a
clinical setting there is the continued presence of a fissured atherosclerotic
lesion. In both the experimental and clinical settings, a prothrombotic vessel
surface continues to exist. Added to this is the additional complicating fact
that plasmin dependent lytic agents are prothrombotic
(Ervin and Peerschke, 2001).
The mechanism of platelet activation or inhibition by plasmin is poorly
understood. Plasmin at a concentration in excess of 1 caseinolytic unit/ml
triggers platelet aggregation at 37°C through direct or indirect
activation of the integrin glycoprotein IIb/IIIa
(Loscalzo et al., 1995;
Rabhi-Sabile and Pidard,
1995). Plasmin may promote platelet activation by cleaving the
seven transmembrane thrombin receptor to expose the tethered ligand containing
the SFLLRN sequence, which directly activates platelets in the same manner as
thrombin (Kuliopulos et al.,
1999). Additional observations demonstrate a role for secreted
dense granule adenosine diphosphate in the potentiation of high-dose
plasmin-induced platelet aggregation via the P2Y12 receptor
(Ishii-Watabe et al.,
2000).
Bleeding times have been reported to be an unreliable measurement
(Channing-Rodgers and Levine,
1990). In the present study, tongue-bleeding times were well
correlated with the inhibition of platelet activity and thrombosis.
Pretreatment with aspirin caused a slight increase of baseline bleeding time
compared with the control group. At the current low dose of CRL42796, we did
not observe a significant increase in bleeding and rethrombosis was only
partially prevented. The infusion of enoxaparin also did not increase bleeding
time and rethrombosis was only partially prevented. In the last group, treated
with aspirin, CRL42796, and enoxaparin, rethrombosis was prevented, whereas a
significant increase in tongue bleeding time was also observed. The results
suggest that bleeding time may be a useful measure for the ability of a
platelet receptor antagonist or an anticoagulant to inhibit arterial
thrombosis. Inhibition of one or more factors in the intrinsic coagulation
cascade will cause an increase the aPTT. Enoxaparin inhibits factor X and
increased aPTT, whereas aspirin and CRL42796 were without effect on aPTT. Of
importance is the observation that plasmin-induced aggregation is not
inhibited by aspirin or ADP antagonists
(Ervin and Peerschke,
2001).
In conclusion, the use of a subtherapeutic dose of the platelet GPIIb/IIIa receptor antagonist CRL42796 was not effective in preventing reocclusive coronary arterial thrombosis or ex vivo platelet reactivity. However, the combination of aspirin, enoxaparin and CRL42796 prevented coronary artery rethrombosis after successful thrombolytic therapy. Deep vessel wall injury leads to the formation of a platelet rich white thrombus. The latter serves as a nidus that becomes surrounded by the fibrin rich red thrombus. Plasmin-dependent lytic agents are effective in bringing about dissolution of fibrin strands making up the red thrombus. However, the ability of plasmin to activate platelets facilitates local platelet aggregation along with exposure of thrombin previously embodied within the red thrombus. Thrombin is an effective activator of platelet reactivity, which in concert with plasmin-induced platelet activation promotes an intense prothrombotic environment. Combined use of several pharmacological interventions, each targeting a different component of the occlusive thrombus, may represent a means by which successful thrombolysis can be achieved while reducing the incidence of rethrombosis. A limitation of the approach is the potential for serious bleeding. The favorable pharmacodynamic properties of CRL42796 of rapid onset of platelet inhibition and relatively short duration of action may provide a greater margin of safety over currently available GPIIb/IIIa receptor antagonists. The present study results provide evidence for the ability of CRL42796 to maintain a favorable quality of blood flow when used in conjunction with aspirin and enoxaparin. Future clinical trails using combination therapy will be needed to determine the safety and efficacy of a combined dosing regimen.
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Footnotes |
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ABBREVIATIONS: GPIIb/IIIa, glycoprotein IIb/IIIa; CRL42796, (2S)-2-[(2-naphthyl-sulfonyl)amino]-3-{[2-({4-(4-piperidinyl)-2-[2-(4-piperidinyl) ethyl] butanoyl}amino)acetyl]amino}propanoic acid dihydrochloride; rt-PA, recombinant tissue plasminogen activator; LCX, canine left circumflex; aPTT, activated partial thromboplastin time; PRP, platelet-rich plasma; AA, arachidonic acid; MABP, mean arterial blood pressure; HR, heart rate; RPP, rate pressure product.
Address correspondence to: Dr. Benedict R. Lucchesi, Department of Pharmacology, 1301C Medical Science Research Bldg. III, University of Michigan Medical School, Ann Arbor, MI 48109-0632. E-mail: benluc{at}umich.edu
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