To facilitate the discussion of therapeutic advances, a brief review of the mechanisms responsible for the fundamental aggregation response of platelets is provided, as is a review of mechanisms responsible for thrombin-induced fibrin generation activated by the coagulation cascade and the role of the inflammatory process in thrombosis. These three areas pose both challenges and opportunities in the treatment of acute coronary syndromes.

The cellular components of the normal vasculature and of atherosclerotic plaques synthesize and secrete a number of proteins into the subendothelial space, including von Willebrand factor, collagens, fibronectin, vitronectin, thrombospondin, and laminin.5 These proteins have within their sequences stretches of amino acids that make them sticky, or adhesive, and provide a mechanism for cells to interact with the matrix.

In addition, smooth muscle cells, endothelial cells, and macrophages synthesize tissue factor, a powerful membrane-bound procoagulant protein that stimulates thrombin production and thereby induces fibrin formation to contribute to the development of the fibrin clot.7–11

Normally, the endothelium does not react with either circulating platelets or coagulation factors. In fact, the endothelium produces substances, such as prostacyclin, thrombomodulin, heparan sulfate, tissue plasminogen activator (t-PA), and nitric oxide, that actually inhibit thrombosis and hemostasis.12–14

However, injury to a blood vessel or disruption of an atherosclerotic plaque exposes blood elements to thrombogenic components of the subendothelium, such as adhesion and extracellular matrix proteins, and cell-associated tissue factor.

Highly reactive platelets respond to and interact with the damaged vascular wall by a succession of rapid biochemical reactions and cellular changes leading to the formation of a platelet-rich thrombus on the damaged vascular wall.15, 16

Platelets possess a number of specific membrane-bound glycoprotein (GP) receptors that enable them to bind to adhesive proteins and mediate their own adherence and aggregation. 17 Platelet adherence is primarily achieved via simultaneous interaction of the multimeric subendothelial von Willebrand factor protein with the GP receptor Ib-IX on the platelet membrane and collagen within the subendothelium.

The cytosolic portion of the transmembrane GP Ib-IX receptor is coupled in the platelet to actin-binding proteins. Binding of von Willebrand factor to the GP Ib-IX receptor results in actin-mediated activation of the platelet that leads to the platelet changing its shape from a smooth disc to a tiny sphere and to spreading of the platelet via multiple contact sites in the damaged vessel wall.

The change in platelet morphology has three major consequences:

(1) The platelets release their granular contents, including adenosine diphosphate (ADP), serotonin, thromboglobulin, platelet-activating factor (PAF), fibrinogen, von Willebrand factor, platelet-derived growth factor (PDGF), and thromboxane A2; (2) the multimeric GP IIb/IIIa receptor con-formation is altered, facilitating platelet-platelet aggregation through GP IIb/IIIa interaction with fibrinogen, fibronectin, and endothelial thrombospondin; and (3) the displacement of phosphatidylserine from the inside to the outside of the platelet membrane provides a surface for binding and steric arrangement of the clotting factors.18

Another consequence of the change in platelet shape following activation is that clotting factors become associated with the platelet membrane, which then serves as a procoagulant surface for the generation of thrombin, which further strengthens the clot.11, 12, 16, 19–21 Thrombin generates fibrin from fibrinogen for inclusion in the thrombus (Fig. 1).22

Presently, there is much interest in the role of inflammation as a precursor of plaque rupture.23 The presence of inflammatory cells within complex plaque and detection of a wide variety of inflammatory mediators in and around unstable lesions support the hypothesis that inflammation correlates positively to plaque instability. Indeed, histological analysis at sites of plaque fissure or ulceration indicates that disruption correlates with the presence of inflammatory cells (macrophages, mast cells, and activated T lymphocytes) and suggests that plaque rupture generally occurs at the shoulder of the lesion, where the majority of the macrophages and mast cells reside.24–28

Enzymes expressed by these cells, which digest extracellular matrix proteins, weaken the fibrous cap and precipitate cap disruption.10, 23, 27, 28

Interaction between the component cells of the plaque may accentuate this effect. For example, secretion of interferon-and/or histamine releasing factor by activated T lymphocytes can stimulate the release of these degradative enzymes from mast cells and macrophages.10, 27 Furthermore, the reduced density of vascular smooth muscle cells within advanced atherosclerotic lesions predisposes the plaque cap to disruption on two levels: (1) The decreased density of the principal

Coagulation Cascade

FIG. 1 The coagulation cascade illustrating the role of the tissue factor pathway in clot initiation, interactions between the pathways, and the role of thrombin in sustaining the cascade by feedback activation of coagulation factors.

HK = high–molecular-weight kininogen,

PK = prekallikrein,

PL = phospholipid,

PT = prothrombin,

TF = tissue factor,

TH = thrombin.

Reproduced from Ref. No. 22 by permission of Blackwell Science, Inc.

cell type within the cap directly lessens the cap’s structural integrity, and (2) vascular smooth muscle cells are the major synthetic source of the extracellular matrix (e.g., collagen) component of the fibrous cap. Accordingly, a reduction in the number of smooth muscle cells will lead to decreased synthesis of the matrix proteins on which the mechanical strength of the plaque cap ultimately depends.7

It is interesting to note that the reduction in smooth muscle cell numbers may itself result from the chronic inflammatory nature of the plaque in that interferon-inhibits smooth muscle proliferation.10 Alternatively, or in addition, reduced smooth muscle cell density may result from apoptotic cell death induced by cytokines (elaborated by macrophages), aperforins (expressed by T lymphocytes), or free radicals.7, 29

Further supporting a role for inflammation in plaque rupture is the determination that the plasma C-reactive protein level is a long-term predictor of MI risk and that aspirin is associated with reduced C-reactive protein levels and reduced occurrences of infarcts, hypothetically because of its anti-inflammatory mechanism.23, 30

As detailed above, platelet adhesion, activation, and aggregation play pivotal roles in thrombus generation following vascular injury.

[Karl Note:  This author seems to be saying that there are three major items in the coagulation cascade:  First is the adhesion among platelets, then the "activation" and the "aggregation."  ]

Thus, it stands to reason that pharmacologic intervention at any one of these steps may effectively reduce the incidence of thrombosis. Determination of the biological and molecular mechanisms involved in vascular-wall–platelet interactions, platelet-platelet interactions, and coagulation has identified multiple targets for drug discovery and has led to the development of numerous antithrombotic agents.

Because of inherent redundancies in mechanisms leading to clot formation, interfering with any single aspect of platelet function fails to provide complete protection against the development of intravascular thrombosis, especially when the stimulus for thrombosis is strong, such as occurs with the disruption of an atherosclerotic plaque.31

Accordingly, successful prevention of thrombosis will likely require a multidimensional pharmacologic approach (Table I).

It is not within the scope of this article to review all antithrombotic options; several comprehensive reviews on this subject have been published recently.  12, 21, 32–38 Rather, the focus of this article is on platelet membrane GP receptor inhibitors (oral GP IIb/IIIa and GPIb antagonists), ADP inhibitors (ticlopidine and clopidogrel), antithrombins (hirudin, argatroban, and hirulog), and anti-thrombotic and anti-inflammatory therapy (inhibition of P-selectin and cyclo-oxygenase type II). In addition, the application of adenovirus-mediated gene expression (gene therapy) to treat thrombotic disease is introduced.

The platelet GP IIb/IIIa receptor has been identified as the pivotal mediator in the final common pathway of platelet aggregation. 35 The critical role of the GP IIb/IIIa receptor is exemplified in persons with Clansman's thrombasthenia, in whom there is a congenital defect or deficiency in intact GPIIb/IIIa on the platelets.32, 39 Platelets from these persons fail to bind fibrinogen and do not aggregate at all.

The GP IIb/IIIa site is a member of a large family of receptors called integrins.32 All of these receptors are composed of two chains, an subunit and a subunit, which are held together by noncovalent bonds. Both and subunits are trans-membrane proteins. The cloning of the cDNAs for GP IIb and GP IIIa 40, 41 led to the identification of GP IIb as a typical integrin and GP IIIa as a typical subunit. The GP IIb/IIIa receptor is specific to platelets. It is activated and exteriorized when the platelets are stimulated.

The change in conformation of the GP IIb/IIIa receptor upon platelet activation is an absolute requirement for interaction with macromolecules.39 Fibrinogen and von Willebrand factor are the principal macromolecules that bind GP IIb/IIIa.

These multivalent molecules can simultaneously bind more than one GP IIb/IIIa receptor and, as such, can link adjacent platelets together. As a result of multiple reactions of this type, the platelets become aggregated into a hemostatic plug.35 The conformational change and activation of the GP IIb/IIIa receptor occur regardless of the particular individual stimulus that activates the platelets, and therefore, GP IIb/IIIa is the final common pathway for platelet aggregation.32, 35, 39 This, together with the specificity of the GP IIb/IIIa receptor, renders the receptor an ideal target for development of a drug that will selectively inhibit platelet aggregation.

TABLE I New developments in antithrombotic therapy

GP = glycoprotein,

VCL = von Willebrand factor comprising residues Leu-504 to Lys-728,

ATA = aurintricarboxylic-acid,

ADP = adenosine diphosphate,

COX = cyclo-oxygenase.

Intense effort over the past decade has resulted in the development of a number of GP IIb/IIIa receptor antagonists, and to date, three of these agents are approved for use in the Unit-ed States: abciximab, eptifibatide, and tirofiban; the fourth, lamifiban, is not approved for use. As reviewed in depth recently,37, 42 10 large clinical trials involving more than 32,000 patients have been performed with the intravenous formulations of monoclonal antibody (abciximab) or small-molecule (eptifibatide, tirofiban, and lamifiban) GP IIb/IIIa receptor inhibitors.

Five of these trials were in patients undergoing percutaneous coronary interventions: Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC),43–45 Evaluation in PTCA to Improve Long-Term Outcome with Abciximab GPIIb/IIIa Blockade (EPILOG),46 c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE),47 Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT),48 and Randomized Efficacy Study of Tirofiban for Outcomes and Restenosis (RESTORE).49

Five trials were conducted in patients with unstable angina or non–Q-wave MI: Platelet IIb/IIIa in Unstable Angina: Receptor Suppression Using Integrilin Therapy (PURSUIT),50

(PRISM),51 PRISM in Patients Limited by Unstable Signs and Symptoms (PRISM-PLUS),52 Canadian Lamifiban Study,53 and Platelet IIb/IIIa Antagonist for the Reduction of Acute Coronary Syndrome Events in a Global Organization Network (PARAGON).54

All 10 trials included standard background therapy with aspirin and heparin. Viewed in aggregate, these trials have demonstrated significant and consistent benefits in the reduction of death or nonfatal MI for the GP IIb/IIIa receptor blockers compared with placebo. Overall, there was a highly significant 20% reduction in death or MI.37

Orally active GP IIb/IIIa receptor blockers are currently under development, although initial reports have been discouraging.

Oral forms allow more sustained receptor antagonism, thus offering the potential for greater long-term benefit and the secondary prevention of recurrent ischemic events.55, 56 Large-scale clinical trials are now under way; however, development of two agents, xemilofiban and orbofiban, has been terminated.

Results of the Oral Glycoprotein IIb/IIIa Receptor Blockade to Inhibit Thrombosis (ORBIT) trial, a randomized, 549-patient, multicenter, double-blind study of xemilofiban in the setting of percutaneous revascularization, were published recently.

55 Patients were randomized to one of three groups:  placebo, 15 mg of xemilofiban, or 20 mg of xemilofiban. Following percutaneous intervention, the study medication was administered three times daily for 2 weeks and then twice daily for 2 weeks. All patients received concomitant aspirin and were followed clinically for 3 months. Xemilofiban therapy was well tolerated and did not increase serious bleeding events compared with placebo. Mild bleeding (particularly epistaxis) was observed more frequently in subjects who received xemilofiban and correlated with the degree of platelet inhibition on Day 1. Although this study was not powered for the definitive evaluation of differences in clinical outcomes between the treatment groups, there was a trend at 3 months toward a reduction in cardiovascular events in patients who received 20 mg of xemilofiban.

The larger scale phase III Evaluation of Oral Xemilofiban in Controlling Thrombotic Events (EXCITE) study demonstrated no clinical benefit for the oral agent, and enrollment was halted in the Orbofiban Post Unstable Coronary Syndromes (OPUS) trial after an interim analysis found an excess of mortality in the treatment group. Results from the Sibrafiban Versus Aspirin to Yield Maximum Protection from Ischemic Heart Events Post Acute Coronary Syndromes (SYMPHONY) trial are awaited. Oral GP IIb/IIIa receptor inhibitors present dosing, efficacy, and safety challenges because their bioavailabilities are unpredictable from patient to patient and it is difficult to know what percentage of receptor blockade has been achieved and then to maintain receptor blockade levels.

Potential limitations of the GP IIb/IIIa receptor inhibitors include the facts that (1) there is generally no reduction in platelet adhesion; (2) there is no prevention of platelet activation and secretion; (3) there may not be effective inhibition of tissue factor or of the thrombus-bound thrombin, which may not be accessible to these inhibitors; and (4) the GP IIb/IIIa receptor inhibitors do not preclude inflammation. There also is some nuisance bleeding associated with the orally administered GP IIb/IIIa receptor antagonists, including bleeding from the gums, epistaxis, bleeding associated with shaving, and so forth. Thus, despite the optimism regarding the oral GPIIb/IIIa receptor antagonists, efforts are under way to inhibit the GP Ib integrin, to reduce platelet adhesion, and to inhibit tissue factor and thrombin. Clinical studies are under way to determine the effect of combinations of GP IIb/IIIa receptor inhibitors and other antithrombotic therapies, such as fibrin-olyticagents.

The GP Ib receptor (a nonintegrin) exists in complex with GP V on the platelet surface and binds von Willebrand factor.

GP Ib is the principal GP involved in the initial contact between platelets and the vessel wall, particularly in cases of high shear stress as exists in the coronary arteries.35 The do-main of the von Willebrand factor that interacts with the GP Ib receptor is composed of residues Val-449 to Lys-728. The same region contains the peptide-binding domains for heparin and collagen.57 A recombinant peptide fragment of human von Willebrand factor comprising residues Leu-504 to Lys-728, named VCL, has been developed. Studies have demonstrated in canine and baboon models of arterial injury that VCL effectively inhibits cyclic flow variations, prolongs the time to development of intracoronary thrombus, enhances t-PA–induced thrombolysis, and delays coronary artery re-occlusion.  57, 58 More recently, in a rabbit model of arterial in-jury, aurintricarboxylic acid (ATA), a substance that inhibits platelet GP Ib–von Willebrand factor interaction, reduced the extent of neointimal hyperplasia measured 21 days after injury.59 These studies confirm that inhibition of platelet adhesion by blocking the GP Ib receptor is an attractive possibility for the prevention and treatment of acute coronary syndromes.

In particular, the VCL peptide fragment may provide an effective and relatively specific inhibitor of thrombosis and may represent a new class of antithrombotic compound.

Adenosine Diphosphate Inhibitors:  Ticlopidine and Clopidogrel Ticlopidine and clopidogrel, less potent than GP IIb/IIIa receptor inhibitors as broad inhibitors of thrombosis, are thienopyridine derivatives that inhibit platelet aggregation via selective noncompetitive inhibition of platelet membrane ADP receptors.60, 61 This interference with a specific ADP-de-pendent step of GP IIb/IIIa complex activation results in less platelet aggregation and, thus, ultimately impairs thrombus formation.61 Both compounds are prodrugs that require break-down in the liver to unidentified active metabolite(s).38, 61 Exvivo, the antiaggregation effect is concentration-dependent and recovery is linked to platelet survival, suggesting a permanent platelet effect 21 consistent with a reduction in functional platelet membrane ADP receptors.60 Clopidogrel is approximately 40 times as active as ticlopidine in inhibiting ADP-induced platelet aggregation in animal models and about 6 times as potent as ticlopidine in inhibition of ADP-induced aggregation of human platelets.38

These agents differ from aspirin in that they do not inhibit the cyclo-oxygenase pathway, and they have no effect on phospholipase A activity or thromboxane A2 and prostacyclin synthesis, as aspirin does.61 In addition, these compounds provide broader inhibition of platelet aggregation than aspirin. Ticlopidine inhibits most of the known stimuli to platelet activation when tested at physiologic concentrations,62, 63 and clopidogrel inhibits thromboxane-, serotonin-, and arachidonic acid-mediated platelet aggregation as well as ADP-induced platelet aggregation.64, 65 These two agents have no effect on platelet-collagen adhesion. Neither drug has an effect on coagulation or fibrinolysis, but they may reduce myointimal thickening of the arterial wall, presumably by the inhibition of platelet aggregation and therefore diminished liberation of -granule contents, particularly the mitogenic PDGF.38, 62

Ticlopidine has been available for many years; it was initially approved for the secondary prevention of stroke and also has been shown to be beneficial for the prevention of MI secondary to unstable angina and the treatment of peripheral arterial obliterative disease. Food and Drug Administration approval for ticlopidine was obtained principally on the basis of two clinical trials: the Ticlopidine Aspirin Stroke Study (TASS)63 and the Canadian American Ticlopidine Study (CATS).66

TASS was a blinded, multicenter trial involving 3,069 patients with recent transient ischemic attack or minor stroke. Patients were randomly assigned to receive ticlopidine (250 mg, twice daily) or high-dose aspirin (625 mg, twice daily). At 3 years, the ticlopidine group had a slight but significant (p =0.048) reduction in the primary endpoints of nonfatal stroke or death compared with the aspirin group.

CATS was a double-blinded study of 1,072 patients with re-cent thromboembolic stroke randomly assigned to receive ticlopidine (250 mg, twice daily) or placebo. At 2-year follow-up, patients who received ticlopidine had a 23.3% reduction in the combined endpoint of stroke, MI, or vascular death com-pared with the placebo group (p = 0.02). Subsequent trials of ticlopidine for various indications, including transient ischemic attacks and stroke, peripheral arterial disease and stenting, or is-chemic heart disease, are summarized in a recent review by Sharis et al.61

Adverse effects of ticlopidine administration include diarrhea, pruritis and urticaria, and epistaxis and ecchymosis. The most potentially serious problem is bone marrow suppression, particularly leukopenia, which necessitates careful monitoring for the first 12 weeks of ticlopidine therapy.21 Because of this adverse event profile, current recommendations suggest the use of ticlopidine in place of aspirin for secondary prevention of transient ischemic attack or stroke only when the patient cannot tolerate aspirin.17

Clopidogrel, which lacks the bone marrow suppression side effect of ticlopidine, is better tolerated by most patients and was recently approved for use in the prevention of ischemic events.61 In the large Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events (CAPRIE) trial,67 the incidence of severe neutropenia with clopidogrel was only 0.1%; this was similar to the rate seen with aspirin. CAPRIE was a phase III study conducted to assess the relative efficacy, safety, and tolerability of clopidogrel and aspirin in reducing the incidence of the composite outcome of ischemic stroke, MI, or vascular death among patients who had survived a recent ischemic stroke or MI, or who had symptomatic peripheral arterial disease. This trial was a randomized, stratified, multicenter, double-blind, parallel group design study, with 19,185 patients randomly assigned to receive either clopidogrel (75 mg/day) or aspirin (325 mg/day) for a maximum of 3 years. At least 5,000 patients were assigned to one of three clinical subgroups (recent stroke, recent MI, and peripheral arterial disease).

CAPRIE was powered to detect a realistic treatment effect in the whole study cohort but not in each of the three clinical subgroups.

The intent-to-treat analysis showed an overall relative risk reduction of 8.7% (p = 0.043). It was concluded that long-term administration of clopidogrel to patients with atherosclerotic vascular disease is more effective than aspirin in reducing the combined risk of ischemic stroke, MI, or vascular death. Future studies are needed to determine additional indications for clopidogrel (e.g., prevention of restenosis with stenting) and to confirm the safety of using clopidogrel in combination with aspirin.

Ticlopidine and clopidogrel each cost significantly more than aspirin ($1 to $3 compared with < $0.01/tablet). Nevertheless, the safer profile of clopidogrel and its promising clinical efficacy render this new drug an exciting new option in antithrombotic therapy, particularly for patients who are un-able to tolerate aspirin.

Thrombin Inhibitors: Hirudin, Argatroban, and Hirulog Fibrinolytic agents such as alteplase (t-PA), reteplase (r-PA), and streptokinase are used routinely to resolve occlusive thrombi and reperfuse occluded coronary arteries.

However, the ability of thrombolytic agents to induce early reperfusion may be offset by an attendant increase in procoagulant activity.68 The addition of heparin and aspirin to thrombolytic regimens attenuates this activity and improves overall potency rates. However, the most effective regimens using heparin and aspirin achieve adequate reperfusion in only 55% of patients at 90 min, and acute arterial thrombotic reocclusion subsequently develops in 6% of patients.6 This failure of standard anticoagulant therapy to afford full protection from thrombotic phenomena is largely a result of heparin’s inability to inhibit thrombin that is bound to a fibrin clot and factor Xa that is complexed to activated platelets. This and other limitations of heparin and low–molecular-weight heparins (e.g., narrow therapeutic window, endogenous modulation by proteins and platelet factor 4, heparin-induced thrombocytopenia, and bleeding) prompted the search for and development of new classes of antithrombotic drugs to improve vessel potency in patients treated with thrombolytic agents.6, 69

Hirudin is substantially different from other anticoagulants because it does not interfere with the biosynthesis of clotting factors and it is not inhibited by endogenous proteins such as platelet factor 4. 72 Unlike heparin, which requires endogenous cofactors for activity (antithrombin III, heparin cofactor II), hirudin does not require the presence of cofactors or an inter-mediate enzyme to elicit its anticoagulant effect.68, 72 Hirudin is an effective anticoagulant in patients who are antithrombin III–deficient.72 The thrombin-hirudin complex inhibits all proteolytic functions of thrombin and thereby prevents (1) fibrinogen clotting, (2) further thrombin-catalyzed hemostatic reactions (i.e., activation of clotting factors V, VIII, and XIII), and (3) thrombin-induced platelet activation with subsequent accelerated generation of additional thrombin.68 Hirudin is a highly specific inhibitor of -thrombin. Accordingly, apart from its anticoagulant activity, it is pharmacologically inert.72

Hirudin binds tightly to multiple sites on thrombin. De-tailed structure-function studies determined that the carboxyterminus of hirudin binds to the substrate recognition site of thrombin, whereas the aminoterminus binds and inactivates the catalytic domain of the enzyme.6 These findings led to the rational design of a new class of synthetic bivalent thrombin inhibitors, the hirulogs, which consist of these two binding domains separated by a short linker sequence that is sized to mimic the interatomic distance between thrombin’s recognition site and catalytic site.6

Results from two large trials, Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIb 73 and Thrombolysis in Myocardial Infarction (TIMI) 9, 74 and one pilot study, Organization to Assess Strategies for Ischemic Syndromes (OASIS),75 detail the relative efficacy of hirudin com-pared with heparin in patients with acute coronary syndromes.

GUSTO IIb was a multicenter trial with 12,142 patients randomly assigned to 72 h of therapy with either intravenous hirudin (0.1 mg/kg bolus, 0.1 mg/kg/h infusion) or heparin (5,000 U bolus, 1,000 U/h infusion). At 24 h, the incidence of death or MI was significantly reduced in the hirudin group (1.3 vs. 2.1%; p = 0.001). However, at 30 days, the incidence of the primary composite endpoint of death, MI, or reinfarction was 9.8% in the heparin group and 8.9% in the hirudin group (p = 0.06). There was no significant difference between the serious or life-threatening bleeding event rates, but hirudin was associated with more frequent moderate bleeding (8.8 vs. 7.7%; p = 0.03).

The TIMI-9 trial, which involved 3,002 patients with acute MI, found no statistically significant benefit of hirudin com-pared with heparin at 30 days. Thus, although the GUSTO-IIb trial was marginally positive for hirudin, the cumulative out-come of these two trials failed to demonstrate a superior effect of hirudin over heparin on the combined endpoint.38

The OASIS pilot trial did demonstrate a significant benefit for medium-dose hirudin infused over 72 h compared with heparin or a low-dose hirudin infusion in patients with unstable angina or suspected MI without ST elevation. Despite the initial disappointment of the GUSTO-IIb and TIMI-9 trials, this result suggests that direct thrombin inhibitors may still prove useful for the treatment of acute coronary syndromes.

In the PTCA setting, however, the Hirudin in a European Trial vs. Heparin in the Prevention of Restenosis After PTCA (HELVETICA) trial 76 and the Hirulog Angioplasty Study 77 did not show any durable benefit of hirudin or hirulog over heparin.

The recombinant hirudin variant and the trial design used may be critical to successful demonstration of a benefit with this class of drugs. Indeed, a number of recent reports indicate that direct thrombin inhibition by hirulog may improve early clinical outcome in patients undergoing angioplasty 77, 78 or receiving fibrinolytics.69, 79 There continues to be potential for this class of drugs as an adjunct to fibrinolytics in the treatment of thrombotic disorders.

Over the past 2 decades, the proinflammatory properties of platelets have gradually been elucidated.80 Platelet involvement in inflammation was previously thought to be restricted to a passive role, as a target for inflammatory mediators re-leased by leukocytes, in particular PAF. It has become apparent, however, that platelets also play an active role in inflammation, releasing their own intracellular platelet factor 4,-thromboglobulin, PDGF, and histamine-releasing growth factor, which are potent amplifiers of basophil, mast cell, and neutrophil activity. As such, anti-inflammatory agents may prove useful in the prevention of cardiovascular disease, for example, infection as a possible source of atherosclerosis. In-deed, one recent prospective study of 1,086 apparently healthy men, the Physician’s Health Study, showed that the baseline plasma concentration of C-reactive protein (an acute-phase reactant that is a marker for underlying systemic inflammation) predicts the risk of future MI and stroke.30 Moreover, the reduction in the risk of a first MI associated with the use of aspirin correlated directly with the C-reactive protein level, suggesting that part of the benefits of aspirin is mediated through its anti-inflammatory effects and not strictly through its anti-platelet properties.

This supposition that inflammation plays a key role in the atherosclerotic processes is confirmed by observations that the inducible inflammatory COX-II enzyme is present in atherosclerotic plaques. The development and recent approval of COX-II–specific inhibitors 81, 82 affords the opportunity to test these compounds in animal models to determine their potential for cardiovascular indications. The outcome of such studies may not be as straightforward as initially hoped, given the recent evidence that COX-II induction in vascular cells (endothelial cells and platelets) may actually be desirable following endothelial injury because it may replace the protective effect of the constitutively expressed COX-I–mediated prostaglandin production lost upon endothelial damage.83

P-selectin is a 140-kd GP member of the selectin family of vascular adhesion molecules.84 P-selectin is typically stored in the granules of platelets and the Weibel-Palade bodies of endothelial cells until cellular activation (e.g., treatment with thrombin, histamine, or oxygen-derived free radicals; ischemia and reperfusion) induces rapid (within minutes) mobilization of P-selectin to the cell surface. Because of the central role of thrombin in thrombosis and coagulation, subsequent induction of P-selectin is involved in the thrombotic process associated with acute coronary events.84 Demonstration of antibody blockade of P-selectin–attenuated fibrin deposition and clot formation in an in vivo animal model confirmed the role of P-selectin in the thrombotic process.85

Willerson et al. have shown that a low–molecular-weight molecule that has combined inhibitory activity for P-, E-, and L-selectins inhibited thrombosis and inflammation in animal models with interventional injury.86 These authors also have demonstrated both in vitro (clot lysis assay) and in vivo (rat mesenteric artery cyclic flow protocol) that a chimeric P-selectin/ t-PA fusion protein is an effective thrombolytic agent.86

In vitro cell adhesion assays indicated that the chimeric proteins retain P-selectin binding activity. The chimeric protein targeted t-PA to the thrombotic region where the t-PA actively lysed the clot and, perhaps, the P-selectin binding properties attenuated nascent fibrin deposition and clot formation as well.

The much-anticipated future of antithrombotic therapy is likely to include transferring genes to the site of vascular injury to reduce the incidence of coronary thrombus associated with acute or recurrent ischemic episodes. Great optimism exists that gene therapy will prove effective in the management of thrombotic disorders. This optimism is based upon combined emerging successes with gene therapy in the treatment of other diseases and from laboratory data indicating this as a viable approach for inhibiting thrombosis. For example, the Zold-helyi-Willerson laboratory has demonstrated that adenovirus-mediated delivery of the gene for human cyclo-oxygenase I enzyme to porcine carotid arteries restored prostacyclin production and abolished cyclic flow variations for at least 10 days following arterial injury (crush or balloon).87 Histological examination of the arteries verified that the vessels that overexpressed COX-I were devoid of thrombus. It is hoped that human trials using gene therapy to prevent thrombosis in humans with arterial injury will begin in late 1999.

Alternatively, one may inhibit tissue factor, which accumulates in the media and intima of injured arteries, with an endogenous inhibitor of tissue factor pathway inhibitor (TFPI).

The Zoldhelyi-Willerson laboratory has used similar gene therapy with an adenoviral vector and the human cDNA for TFPI to prevent thrombosis after vascular injury in experimental animal arteries.88 This laboratory plans future studies in humans in which COX I and TFPI are overexpressed together in the hope of preventing or markedly attenuating vascular thrombosis after injury.

Acute coronary syndromes result from the onset of platelet activation, thrombosis, and fibrin generation after disruption of vulnerable atherosclerotic plaques or vascular injury following interventional revascularization. Given the substantial knowledge gained over the past several years regarding cellular and molecular biology of thrombosis/hemostasis and coagulation, the mechanisms through which selected pharmacologic interventions reduce new and recurrent coronary events are better appreciated.

In 1995, a workshop was held by the National Heart, Lung, and Blood Institute to survey what is known about mechanisms precipitating acute cardiac events and to assess the potential of advances in this field leading to new preventive methods.

23 Among the recommendations made at the workshop were the following: (1) It is important to identify methods that inhibit the multiple mediators of acute thrombosis, and (2) it is important to develop means to express antithrombotic sub-stances at local sites of injury. The development of and continued research to find effective and safe GP IIb/IIIa receptor antagonists, GP Ib receptor antagonists, ADP inhibitors, thrombin inhibitors, and anti-inflammatory agents as described herein have contributed significantly to fulfilling the first recommendation. The application of gene therapy to treat thrombosis will hopefully meet the second recommendation. Thus, despite the remarkable progress made in antithrombotic therapy in the past decade, there is still much to be done and additional, exciting therapies remain undiscovered.

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