Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2005;112:2725-2734
doi: 10.1161/CIRCULATIONAHA.104.494468
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Freedman, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Freedman, J. E.
Related Collections
Right arrow Coagulation
Right arrow Aggregation
Right arrow Signal transduction
Right arrow Coagulation inhibitors
Right arrow Platelet function inhibitors
Right arrow Platelets
Right arrow Anticoagulant mechanisms

(Circulation. 2005;112:2725-2734.)
© 2005 American Heart Association, Inc.

Basic Science for Clinicians

Molecular Regulation of Platelet-Dependent Thrombosis

Jane E. Freedman, MD

From the Boston University School of Medicine, Whitaker Cardiovascular Institute, Boston, Mass.

Correspondence to Jane E. Freedman, MD, Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St, W507, Boston, MA 02118. E-mail freedmaj{at}bu.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowPlatelet Adhesion: Initiation of...
 down arrowConclusion
 down arrowReferences
 
Hemostasis is a normal process preventing the sequelae of uncontrolled hemorrhage. In certain settings, these same processes cause adverse clinical events due to thrombotic occlusion of a vessel. The majority of unstable coronary syndromes result from disruption of an atherosclerotic plaque, leading to the exposure of subintimal contents, which triggers coagulation and the formation of a platelet-rich thrombus. The central role of platelet activation in the events that lead to vessel occlusion is well known. However, this process is complex and influenced by a myriad of cellular and plasma-derived mediators that regulate the balance between occlusive and nonocclusive thrombosis.


Key Words: coagulation • platelets • thrombosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowPlatelet Adhesion: Initiation of...
 down arrowConclusion
 down arrowReferences
 
Thrombosis of either the venous or arterial circulation is a major cause of morbidity and mortality. Under normal circumstances, the hemostatic process is a delicate balance of prothrombotic and antithrombotic factors in the vasculature. Normal hemostasis prevents uncontrolled hemorrhage; however, these same pathways regulating hemostasis lead to pathological thrombosis and vessel occlusion. Advances in understanding the mechanisms of thrombosis, as well as the development of new techniques for studying its regulation, have led to a clearer understanding of thrombotic disease.

Thrombus formation within a vessel is the precipitating event in multiple vascular disease processes, including myocardial infarction, thrombotic cerebrovascular events, and venous thrombosis; however, the pathophysiological processes regulating these diseases are distinct. In venous thrombosis, primary hypercoagulable states reflecting defects of the proteins of coagulation and fibrinolysis or secondary hypercoagulable states involving abnormalities of blood vessels and blood flow lead to thrombosis (see the recent review by Andreotti and Becker1). Distinct from venous thrombosis, arterial thrombosis is highly dependent on the vessel wall and platelet. In the setting of coronary stenoses, rupture of atheromatous plaque in relatively mildly stenosed vessels and subsequent thrombus formation underlie the majority of acute coronary syndromes. Vessel injury caused by plaque rupture exposes collagen and von Willebrand factor to platelets.2 Platelets then adhere, and local platelet activation stimulates further thrombus formation and additional platelet recruitment by supporting cell-surface thrombin formation and releasing adenosine diphosphate (ADP), serotonin, and thromboxane A2 (TXA2).3 Thrombus forms as platelets aggregate via the binding of bivalent fibrinogen to the glycoprotein (GP) IIb/IIIa complex. The pathways regulating platelet adhesion, activation, aggregation, and recruitment will be described in further detail below.

Platelets share many of the same biological mechanisms as other cells, including cytoskeletal structure, housekeeping enzymes, and signal transduction components; however, unlike most cells, platelets lack a nucleus and are unable to adapt to changing biological settings by altered transcription. Although platelets maintain some protein synthetic capacity from megakaryocyte-derived mRNA, most molecules needed to respond to various physiological and pathological stimuli are present in storage granules and membranes. Because the primary function of platelets is regulation of hemostasis, the main receptors play a direct role in the processes regulating adhesion or thrombus formation.

Advances in understanding the mechanisms governing thrombosis have led to the availability of new classes of antithrombotic drugs. Initially, characterization of arachidonic acid metabolism in platelets furthered an understanding of the therapeutic utility of cyclooxygenase inhibitors in vascular disease, most notably aspirin. The discovery and characterization of platelet receptors, such as the ADP receptor and the GP IIb/IIIa complex, have been associated with the development of novel classes of antiplatelet drugs, including thienopyridine derivatives and GP IIb/IIIa receptor antagonists. Further knowledge of receptors, their inhibitors, and signaling pathways, as well as an understanding of the interaction of thrombosis, inflammation, and the vessel wall, will continue to advance our understanding of thrombosis and enhance the development of new therapeutic interventions.


*    Platelet Adhesion: Initiation of the Thrombotic Cascade
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Platelet Adhesion: Initiation of...
down arrowConclusion
 down arrowReferences
 
The process of platelet activation, aggregation, and subsequent thrombus formation is frequently initiated by platelet adhesion to the damaged vessel wall. The subendothelial components responsible for triggering platelet reactivity include different types of collagen, von Willebrand factor, fibronectin, and other adhesive proteins such as vitronectin and thrombospondin. The specific hemostatic response is dependent on the extent of damage, the specific matrix proteins exposed, and the determinants of blood flow. Several collagen-binding proteins that are expressed on the platelet surface also regulate collagen-induced platelet adhesion, specifically under flow conditions. These receptors include GP IV, GP VI, and integrin {alpha}2ß1. These receptors may interact in a synergistic way dependent on the flow conditions and relative exposure of distinct types of collagen or extracellular matrix proteins.4

The platelet-expressed GP Ib/IX/V complex adhesive receptor is central to both platelet adhesion and activation. Damage to the blood vessel wall exposes subendothelial von Willebrand factor and collagen to the circulating blood. This form of vascular injury occurs in the microcirculation and stenosed arteries, and the GP Ib/IX/V complex expressed on the platelet surface binds to the exposed von Willebrand factor, causing platelets to adhere to the subendothelium at the site of injury (Figure 1). In addition to anchoring the platelet to the injured vessel wall, engagement of the GP Ib/IX/V complex leads to the transduction of signals contributing to platelet activation. Von Willebrand factor–bound GP Ib/IX/V induces a conformational change in the GP IIb/IIIa receptor, transforming it from an inactive low-affinity state to an active receptor that binds additional von Willebrand factor or fibrinogen with high affinity.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Platelet adhesion and aggregation. Damage or vascular injury exposes subendothelial von Willebrand factor (VWF) or collagen to the circulating blood. Platelets adhere to the site of injury when GP Ib/IX/V complex expressed on the platelet surface binds to the subendothelial VWF. This event triggers the synthesis and release of TXA2, serotonin, and ADP and causes activation of various platelet receptors (R). These events cause a conformational change in GP IIb/IIIa, enabling the high-affinity binding of fibrinogen and resulting in thrombus formation. This process can be attenuated by endothelial or platelet release of NO.

Given the central role of the GP Ib/IX/V complex in platelet adhesion and aggregation, it has become an attractive target for the development of antiplatelet drugs. Therapeutics have been developed that target the interaction between the GP Ib/IX/V complex and von Willebrand factor as a way of preventing arterial thrombosis, including anti–von Willebrand factor monoclonal antibodies5 and the GP Ib/IX/V antagonists isolated from snake venoms.6,7 Another study examined the antithrombotic efficacy of the Fab fragments of the murine monoclonal antibody, 6B4, raised against the GP Ib/V/IX receptor of human platelets that significantly inhibited thrombus generation.8 The use of a monoclonal antibody to von Willebrand factor in a monkey model demonstrated sustained antiplatelet effect without extensive prolongation of the bleeding time.9 Currently, there are no published trials demonstrating clinical benefit with inhibition of GP Ib, and clinical success will depend on minimization of the presumed enhanced bleeding that may be seen as a result of inhibition of platelet adherence.

Platelet Receptors and Activation
Platelet activity is controlled by a variety of surface receptors that regulate various functions. Platelet receptors are affected by a wide variety of agonists (stimulants) and adhesive proteins. In general terms, the stimulation of platelet receptors triggers 2 distinct processes: (1) stimulation of various internal signaling pathways that lead to further platelet activation and granule release and (2) the capacity of the platelet to bind to other adhesive proteins/platelets, leading to the formation of a thrombus. Because both of these processes may cause thrombosis and vessel occlusion, many of these receptors have been and are being developed as targets for preventing clot formation. Because there are many families and subfamilies of receptors found on platelets that regulate a variety of platelet functions, only the main ones with current therapeutic relevance will be considered below.

Transmembrane Receptors
The 7-transmembrane receptor family is the main agonist-stimulated receptor family. There are several 7-transmembrane receptors found on platelets, including the thrombin receptor, ADP receptors, prostaglandin receptors, lipid receptors, and chemokine receptors.

Thrombin Receptor
Thrombin receptor is the major 7-transmembrane receptor found on platelets. The first identified was the protease activation receptor-1 (PAR1).10,11 The PAR class of receptors has a distinct mechanism of activation that involves specific cleavage of the N-terminus that acts as a ligand to the receptor. Other PAR receptors are present on platelets, including PAR2 (not activated by thrombin) and PAR4. Specific inhibitors of PAR1 and PAR4 are undergoing study for potential future clinical use (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Platelet Inhibitors and Their Mechanism of Action

ADP Receptors
With the success of the adenosine receptor inhibitors in the treatment of stroke and prevention of thrombosis after cardiac intervention, there has been an interest in these receptor-mediated pathways. Erythrocytes and endothelial cells secrete ADP, which contributes to hemostasis, thrombus formation, and vascular occlusion by stimulating platelet aggregation. In addition, the ADP secreted from the dense granules of stimulated platelets acts to potentiate platelet aggregation.12–14 In the platelet, ADP induces a rapid influx of external calcium, mobilizes calcium from intracellular stores, and attenuates the inhibition of adenylyl cyclase.

Transduction of ADP-induced signaling events involves the binding of ADP to purinergic receptors on the platelet surface. There are several distinct ADP receptors, classified as P2X1, P2Y1, and P2Y12 (Figure 2).13–17 The P2X1 receptor, a ligand-gated ion channel, mediates rapid, transient calcium influx and does not appear to play a role in platelet aggregation.18 ADP stimulation of the P2Y1 Gq-coupled receptor activates phospholipase C, causing internal calcium mobilization.17,18 Activation of the P2Y1 receptor is thought to be responsible for mediating ADP-induced platelet shape change18 in addition to ADP-induced platelet aggregation. Additional evidence to support the role of the P2Y1 receptor in platelet aggregation comes from studies of platelets from P2Y1-null mice that fail to aggregate in response to ADP.19,20



View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. Platelet activation by ADP. Three receptors that can be activated by ADP are present in the platelet. These are G protein–coupled receptors and mediate different responses. The P2Y12 receptor is the target for the antiplatelet thienopyridine derivatives ticlopidine and clopidogrel. AD indicates adenylyl cyclase, PLC, phospholipase C.

The activation of both the P2Y12 and P2Y1 receptors is essential for ADP-induced platelet aggregation.18,21 The activation of the platelet P2Y1 receptor mediates the initial rapid response to ADP, determining the maximal rate of ADP-induced platelet aggregation.21 The degree of sustained platelet aggregation, however, is believed to be due to ADP activation of the P2Y12 receptor.21 Selective antagonism of the P2Y12 receptor demonstrates that the P2Y12 receptor plays a role in sustaining and amplifying ADP-induced aggregation.22

The thienopyridine derivatives ticlopidine and clopidogrel are clinically utilized inhibitors of ADP-induced platelet aggregation (Table 1). The antiplatelet effect of thienopyridine derivatives is due to irreversible inhibition of ADP binding to platelet purinergic receptors.23,24 Metabolism of clopidogrel by the hepatic cytochrome P450-1A enzyme system to an active metabolite23,24 is essential for its in vivo antiplatelet effects.23,24 This metabolite of clopidogrel displays the same in vivo effects as seen in vitro, such as inhibition of adenylyl cyclase and inhibition of ADP-induced aggregation.23,24 Both drugs are mechanistically and structurally similar; however, owing to a higher incidence of severe neutropenia and other adverse effects associated with ticlopidine, clopidogrel has become the primary thienopyridine derivative used in clinical settings.25

Integrins
Integrins are adhesive and signaling molecules that consist of noncovalently associated heterodimers of {alpha}- and ß-subunits. They normally exist in either a low- or high-affinity state that is altered by cytoplasmic signaling and phosphorylation of their cytoplasmic domains. Platelets possess 3 families of integrins, and only those present in abundant amounts and with current therapeutic relevance will be discussed.

Glycoprotein IIb/IIIa ({alpha}IIbß3)
Although platelets lack a nucleus, they possess many of the signaling pathways found in nucleated cells. The activation of platelets results in a rapid series of a variety of signal transduction events, including tyrosine kinase activation, serine/threonine kinase activation, and lipid kinase activities. In unstimulated platelets, the major platelet integrin GP IIb/IIIa is maintained in an inactive conformation and functions as a low-affinity adhesion receptor for fibrinogen.26 This integrin is unique in that it is only expressed on platelets. After stimulation, the interaction between fibrinogen and GP IIb/IIIa forms intracellular bridges between platelets, leading to platelet aggregation (Figure 1). A conformational change in the extracellular domain of GP IIb/IIIa enables the high-affinity binding of soluble plasma fibrinogen as a result of a complex network of inside-out signaling events.27 This primary, reversible phase of platelet aggregation is precipitated by a series of extremely rapid and complex signaling pathways.28

The GP IIb/IIIa receptor serves as a bidirectional conduit, with GP IIb/IIIa–mediated signaling (outside-in signaling) occurring immediately after the binding of fibrinogen and initiating intracellular signaling that further stabilizes the aggregate (Figure 3). The initial phase of outside-in signaling contributes to further activate the integrin GP IIb/IIIa via integrin clustering and the formation of a complex network of signaling and structural cytoskeletal proteins. Calcium mobilization, tyrosine phosphorylation, activation of phosphoinositide metabolism, and cytoskeletal reorganization result from the activation of the GP IIb/IIIa complex. This series of events transforms platelet aggregation from a reversible to an irreversible process.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Platelet activation by thrombin and inside-out signaling. PAR1 is coupled to G-mediated effector pathways. This results in calcium flux, platelet shape change, and activation of many platelet-signaling pathways. This eventually leads to activation of the GP IIb/IIIa receptor complex and inside-out signaling. PKC indicates protein kinase C. TRAP indicates thrombin receptor activated peptide.

Although aspirin and clopidogrel are effective antiplatelet agents, they are relatively weak antiaggregatory drugs. Drugs that specifically target the GP IIb/IIIa receptor and prevent the binding of fibrinogen inhibit this final common pathway for platelet aggregation.29 Platelet aggregation is completely inhibited by blockade of 80% of the surface GP IIb/IIIa receptors,30 and antagonism of the GP IIb/IIIa receptor inhibits platelet aggregation irrespective of the platelet activator. Multiple clinical trials have shown inhibition of GP IIb/IIIa to be a highly effective antithrombotic strategy.31 Currently, there are several GP IIb/IIIa inhibitors in clinical use (Table 1). Abciximab is a Fab fragment of the 7E3 antibody that has high affinity and a slow rate of dissociation from GP IIb/IIIa. In contrast, the small molecules eptifibatide and tirofiban have a much more rapid rate of dissociation.

Several ligands of integrins, including fibrinogen and von Willebrand factor, possess 1 of the 2 amino acid sequences 95 to 97 (Arg-Gly-Asp or RGD) and 572 to 575 (Arg-Gly-Asp-Ser or RGDS) that enable them to bind to the activated GP IIb/IIIa receptor and inhibit platelet aggregation.32 These nonpeptide mimetics are unique in that they have the potential for oral administration that initially was thought to be useful for long-term antiplatelet therapy; however, several oral GP IIb/IIIa inhibitors have been evaluated, and their use has not been associated with clinical benefit.33–36 Reasons for this discrepancy are unclear, and potential explanations have included hypothetical partial agonist activity, a prothrombotic effect of the oral compounds, or a direct toxic effect that is unrelated to platelet activation, such as apoptosis.37–39 Additionally, it has been observed that some oral GP IIb/IIIa inhibitors augment small platelet microaggregate formation after stimulation.38,39

Platelet Secretion and Recruitment
After stimulation and aggregation, platelets release various substances that lead to activation of additional circulating platelets. These platelets also become activated, and, by fibrinogen bridging with the adherent platelets, a thrombus is formed. This process of attracting additional platelets is termed recruitment. As with activation and aggregation, the process of recruitment is a balance between prothrombotic and antithrombotic forces (Figure 1). Platelets release the contents of their intracellular granules in response to activation. Impairment of this process affects not only normal hemostasis and thrombosis but vascular remodeling. The secretory granules contain coagulation proteins, adhesion molecules, cytokines, integrins, growth factors, and inflammatory modulators.

One of the best-characterized substances released by platelets that lead to recruitment is thromboxane. Not preformed in a granule, it is generated on platelet stimulation. After platelet activation, arachidonic acid is liberated from membrane phospholipids by phospholipase A2 and C. The catalytic activities of prostaglandin (PG)H-synthase sequentially metabolize arachidonic acid to generate prostoglandins.40 Aspirin inhibits the cyclooxygenase activity of PGH-synthase, which in turn blocks the metabolism of arachidonic acid to prostaglandin H2 (PGH2), the precursor of TXA2 and other cyclic prostanoids (prostacyclin and other prostaglandins).

Platelets synthesize and release TXA2 on stimulation with the agonists collagen, thrombin, or ADP.41,42 The TXA2 released by platelets then binds to platelet thromboxane receptors.41,42 Engagement of the platelet TXA2 receptor activates phospholipase C and liberates intracellular calcium. The increase in intracellular calcium amplifies platelet stimulation and results in the synthesis and release of additional ADP and TXA2 from activated platelets. Both ADP and TXA2 participate in a positive feedback loop that leads to platelet aggregation and recruitment. In both normal subjects and patients with atherosclerotic vascular disease, aspirin produces a dose-dependent inhibition of platelet cyclooxygenase activity after the administration of a single oral dose. Owing to the irreversible inhibition of cyclooxygenase by aspirin and the inability of platelets to synthesize new protein, the effect of aspirin is maintained for the life span of the platelet (7 to 10 days). Cyclooxygenase activity returns only as new platelets are formed and released into the circulation. The inhibitory effects of aspirin are pronounced when relatively weak platelet agonists are used but are less pronounced against stronger agonists, such as thrombin, that can induce platelet activation in the absence of TXA2. Importantly, the majority of platelet responses remain unaffected by aspirin treatment. Aspirin does not inhibit shear stress–induced platelet activation and platelet adhesion.43

Clinical trials have demonstrated the efficacy of aspirin in both primary and secondary prevention of myocardial infarction, stroke, and cardiovascular death. Despite proven benefit, the absolute risk of recurrent vascular events among patients taking aspirin remains relatively high, an estimated 8% to 18% after 2 years. Therapeutic resistance to aspirin might explain a portion of this risk,44 although the mechanism for aspirin resistance is uncertain. The answer is likely a combination of clinical, biological, and genetic properties affecting platelet function. The redundancy of platelet activation pathways and receptors may contribute to the problem of aspirin resistance,44 as may compliance or effects of increased shear stress.43,45 In addition, epinephrine is known to enhance platelet activation, and elevated catecholamines found in patients with acute coronary syndromes may render them resistant to the effects of aspirin.45

Substances such as thromboxane and ADP may recruit additional platelets to a growing thrombus; this process is self-limited in most clinical situations. As each platelet activates and adheres, it releases these prothrombotic substances; however, mechanisms must exist to limit or prevent the process of thrombus growth before vessel occlusion. Although rheological forces within the vessel are important, platelets also release antithrombotic substances that may provide negative feedback for thrombus formation.

One such substance is nitric oxide (NO). NO is known to be released by the intact endothelium preventing adherence of platelets to the vessel wall under normal conditions (Figure 1). NO inhibits platelet activation,46,47 prevents thrombosis,48 and inhibits the normal activation-dependent increase in the expression of platelet surface GPs, including P-selectin and the integrin GP IIb/IIIa complex.49 NO inhibits platelet function by stimulating soluble guanylyl cyclase to produce cGMP. This action results in the stimulation of cGMP-dependent protein kinase, leading to a reduction in fibrinogen binding to GP IIb/IIIa and modulation of phospholipase A2– and C–mediated responses.50 NO decreases the oxidation of arachidonate51 and inhibits the agonist-dependent increase in platelet cytosolic-free calcium in a cGMP-dependent52 and -independent manner.53 NO released specifically from platelets inhibits platelet recruitment to a growing thrombus54 (Figure 1), and activated platelets from patients with acute coronary syndromes produce significantly less NO than that in patients with stable coronary artery disease.55

Activation and recruitment of platelets are tightly regulated. Normally, adhesion of platelets to the endothelium and platelet activation are prevented by several mechanisms, including endothelial cell production of prostacyclin, NO, and ecto-ADPase.56,57 Prostacyclin, released by the vessel wall, inhibits platelet function via cAMP, and its effects are also mediated by high-affinity prostanoid receptors found on the platelet.58 CD39 is a major ecto-ADPase (also known as nucleoside triphosphate diphosphohydrolase [NTPDase]-1) and is the main vascular NTPDase that converts ATP and ADP to AMP. By converting ADP to AMP, CD39 suppresses platelet activation and platelet–endothelial cell interactions.59

Thrombus Stability and Disaggregation
The final phase of thrombus formation occurs after the rapid signals arising from G protein–coupled receptors have occurred and the receptors have been desensitized. This phase serves to stabilize the platelet thrombus and prevent premature disaggregation. Although less is known about this phase, it appears that outside-in signaling events regulated through cell-surface integrins and receptor tyrosine kinase signals are primary mediators.

Although much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. The use of fibrinolytic strategies is clearly established for the dissolution of fibrin thrombus, but the disengagement of fibrinogen from the platelet has not been a common therapeutic target. GP IIb/IIIa inhibitors have been shown to promote dissolution of platelet-rich thrombi,60 but these in vitro observations required relatively high concentrations of GP IIb/IIIa inhibitors, suggesting that such treatment may only be feasible with the use of local delivery. Although phosphoinositide kinase (PI3-kinase) does not appear to be essential in the initial activation of GP IIb/IIIa, the sustained activation and thus maintenance of aggregation may be due to the generation of the PI3-kinase product PtdIns(3,4)P2 after GP IIb/IIIa engagement.61,62 Therefore, the activation of PI3-kinase is critical for strengthening platelet aggregation.

The effects of NO on platelet function are similar to the effects of PI3-kinase inhibition. Moreover, NO donors are able to reverse GP IIb/IIIa activation,63 suggesting that NO plays a consequential role in mediating platelet aggregation. Inhibition of PI3-kinase and the addition of exogenous NO donors appear to have additive platelet inhibitory effects64; however, the relevance of these findings to endogenous NO release is unclear. Superoxide generated by stimulated platelets can increase both platelet adhesion and aggregation,65 presumably as a consequence of its reaction with NO and the attenuation of NO bioactivity. PI3-kinase plays a role in regulating NADPH oxidase–generated superoxide in platelets, altering the bioactivity of platelet NO that contributes to platelet disaggregation.66 PI3-kinase inhibition causes progressive reversal of aggregation after initial aggregation.62 The ability of NO donors to induce platelet disaggregation has also been linked to the diminished activation of GP IIb/IIIa and fibrinogen binding.49 The prolonged activation of GP IIb/IIIa activation appears essential for irreversible aggregation.

Genetic Variants and Arterial Thrombosis
The best-characterized genetic variants associated with thrombosis have been those effecting the coagulation and fibrinolytic cascades. In these settings, genetic alteration of blood components may lead to enhanced thrombosis, specifically related to deep venous thrombosis and pulmonary embolism. However, many reports of polymorphisms have been association studies that are prone to error and have had inconstant outcome. Unlike venous thrombosis, the search for relevant genetic variants in arterial thrombosis has been less successful. Likely, this is because atherothrombosis is a complex and highly prevalent polygenic disease, and the phenotype is rarely due to an isolated genetic abnormality or variant.

Through the recognition of familial tendencies in venous thrombosis, genetic abnormalities in the coagulation system have been found. Those initially described were mutations that led to a loss of function of endogenous anticoagulants, including antithrombin III, protein C, or protein S (Table 2). Other mutations have been associated with enhanced thrombotic function such as factor V Leiden or increased coagulation factors (prothrombin G20210A). Alterations of the fibrinolytic cascade have also been linked with changes in thrombosis but have been complicated by the presence of numerous mutations, which has made genetic testing difficult. These variants have included those of tissue plasminogen activator.67


View this table:
[in this window]
[in a new window]
  		TABLE 2. Genetic Variants Associated With Thrombosis

Although the vast number of polymorphisms studied have been factors in the coagulation cascade and are associated with venous thrombotic disease, numerous other associations have been reported that may potentially alter arterial thrombosis. This has included abnormalities in homocysteine metabolism and platelet surface GPs (Table 2). The GP VI gene is polymorphic with several identified variants, and the T13254C polymorphism specifically leads to an amino acid substitution (serine to proline) and has been associated with increased risk of myocardial infarction68; however, the functional effects of many of these polymorphisms have not been clearly ascertained. A large, population-based study suggested that heritable factors play a major role in determining platelet aggregation compared with environmental covariates.69 The study also reported that the platelet GP IIIa PlA2 and fibrinogen HindIII polymorphisms contributed <1% to the overall variance. This study concluded that there is a paucity of data with regard to the genetic epidemiology of abnormal platelet aggregability and that additional studies are needed to identify key genetic variants regulating platelet function.69 Polymorphisms in platelet surface GPs have been identified, including the (A1/2) polymorphism resulting in conformational change at the amino terminus of the ß-3 chain of the platelet fibrinogen receptor GP IIb/IIIa and polymorphisms in the platelet collagen (GP Ia/IIa and GP VI) and von Willebrand receptors (GP Ib/IX). Mutations have also been identified in other platelet surface receptors including G(i)-linked platelet ADP receptor P2Y.13 The relevance of these polymorphisms to their relevant class of antithrombotic drug is not fully known at this time.

Other polymorphisms in thrombosis have been examined, including those of the endothelial NO synthase (eNOS) gene. Several polymorphic variants of the eNOS gene have been described, and the most extensively characterized variant is the 894-G/T polymorphism in exon 7 of the gene, resulting in a glutamate or aspartate at position 298. The 894T allele is associated with higher plasma levels of NOs in healthy individuals.70 Epidemiological studies have shown an increased risk of hypertension, myocardial infarction, and stroke in patients homozygous for the Glu298Asp variant.71–73 Another eNOS polymorphism, designated ecNOS4a, has been identified on intron 4 and has been associated with premature coronary artery disease and also with a history of myocardial infarction.74 Polymorphisms in the promoter region and exon 7 have been associated with lower levels of platelet-derived NO.75

Other genetic abnormalities that may regulate thrombosis are polymorphisms of glutathione peroxidase. Glutathione peroxidase is a selenium-containing enzyme, and selenium deficiency has been reported in patients with acute myocardial infarction and coronary atherothrombotic disease.76 Glutathione peroxidase potentiates the inhibition of platelet function by NO by decreasing LOOH concentrations.77 Impairment of this process can lead to a clinical thrombotic disorder,78 and a similar deficiency has been reported in 5 other families with childhood stroke.79

Contribution of Inflammation to Thrombosis
Accumulating evidence suggests that inflammation plays an important role during the acute thrombotic phase of unstable coronary syndromes. Recent data demonstrate that patients with acute coronary syndromes not only have increased interactions between platelets (homotypic aggregates) but also have increased interactions between platelets and leukocytes (heterotypic aggregates) detectable in circulating blood. These aggregates form when platelets are activated and undergo degranulation, after which they adhere to circulating leukocytes; accumulating information suggests that these events contribute to the atherothrombotic process. Platelets bind via P-selectin (CD62P) expressed on the surface of activated platelets to the leukocyte receptor, P-selectin GP ligand-1 (PSGL-1).40 This association leads to increased expression of CD11b/CD18 (Mac-1) on leukocytes,80 which itself supports interactions with platelets, partially via bivalent fibrinogen linking this integrin with its platelet surface counterpart, GP IIb/IIIa.81,82

The binding of platelets and leukocytes in acute coronary syndromes highlights the interaction between inflammation and thrombosis in atherothrombotic disease. Plaque rupture promotes activation of the inflammatory response, and increased expression of tissue factor initiates extrinsic coagulation. The expression of tissue factor on both endothelial cells and monocytes is partially regulated by proinflammatory cytokines, including tumor necrosis factor and interleukin-1.83 In addition to initiating coagulation, tissue factor interacts with P-selectin, accelerating fibrin formation and deposition.83 Platelet surface P-selectin also induces the expression of tissue factor on monocytes,84 enhances monocyte cytokine expression,85 and promotes CD11b/CD18 expression.80 This prothrombotic process is regulated by production of endothelium-derived NO, which reduces endotoxin- and cytokine-induced expression of tissue factor.86

In addition to the formation of platelet-monocyte aggregates, measurement of the cytokine, soluble CD40 ligand, also reflects the interface between thrombosis and inflammation. The CD40 ligand is a trimeric, transmembrane protein of the tumor necrosis factor family and, with its receptor CD40, is an important contributor to the inflammatory processes leading to atherosclerosis and thrombosis.87 Many immunologic and vascular cells have been found to express CD40 and/or CD40 ligand, and both have been clearly shown to be present in human atheroma.88 In platelets, CD40 ligand is rapidly translocated to the surface after stimulation and is upregulated in fresh thrombus.87 The surface-expressed CD40 ligand is then cleaved from the platelet over a period of minutes to hours, subsequently generating a soluble fragment (soluble CD40 ligand).89 Although also shed from stimulated lymphocytes, it is estimated that >95% of the circulating CD40 ligand is derived from platelets.89 Soluble CD40 ligand has been shown to be associated with increased cardiovascular risk in apparently healthy women.88 Soluble CD40 ligand can identify patients at high risk of acute coronary syndromes,90 and this increased risk associated with elevated soluble CD40 ligand levels is reduced by abciximab treatment. These observations suggest that soluble CD40 ligand can be used to identify patients with enhanced thrombotic risk.

Soluble CD40 ligand has also been implicated in the acute thrombotic process. On endothelial cells or monocytes, the engagement of CD40 with CD40 ligand leads to the synthesis of adhesion molecules, chemokines, and tissue factor and causes the activation of matrix metalloproteinases that are known to contribute to atherothrombotic pathophysiology. Soluble CD40 ligand has a lysine-arginine-glutamic acid (KGD) motif that also allows for its binding to the platelet GP IIb/IIIa complex. It is possible that such binding is blocked in the setting of GP IIb/IIIa inhibitors, potentially altering the clot-stabilizing properties of soluble CD40 ligand.89

There is also evidence that inflammation leads the activation of coagulation with subsequent fibrin deposition. Expression of procoagulant mediators such as tissue factor can initiate activation of the coagulation cascade, and the thrombin subsequently generated activates platelets and fibrinogen. The effect of inflammation may also occur as a result of physiological anticoagulant pathways. The interaction between inflammation, coagulation, and fibrinolysis has been recently reviewed by Levi and colleagues.91


*    Conclusion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowPlatelet Adhesion: Initiation of...
 *Conclusion
down arrowReferences
 
It is essential to further our understanding of the thrombotic pathway to continue to develop novel ways of controlling these processes in vivo. An important lesson that has emerged from numerous trials is that increased antithrombotic potency may not guarantee enhanced clinical benefit. Whether this is due to problems with the specific antithrombotic agents or the inherent limitations of our current means of measuring thrombosis is not known. Novel therapies are also being developed to target the redundant pathways of platelet adhesion, activation, and aggregation. These include (1) new inhibitors of the P2Y1 and P2Y12 receptors92; (2) the CD40-CD40 ligand system; (3) RANTES; (4) P-selectin; (5) CD39/NTPDase; (6) the GP Ib/V/IX complex–von Willebrand factor; and (7) protease-activated receptors. Other specific targets are listed in Table 1, including anti–GP VI therapy93 and NO-releasing aspirin derivatives.94 Efforts are also ongoing to enhance implementation of existent therapy, to target therapy selectively to high-risk patients and to those likely to respond (pharmacogenomics), and to study the incremental benefits and safety of various antiplatelet combinations and their interaction with other medications. Continuing advances in understanding the mechanisms of thrombosis, as well as the development of new techniques for studying its regulation, have led to a clearer understanding of thrombotic disease as well as the availability of new classes of antithrombotic drugs. Because the coagulation and platelet-dependent pathways are also intimately tied to the atherosclerotic process, future understanding may also affect the global and chronic state of the vasculature and further protect vessel patency.


*    Acknowledgments
 
This study was supported by National Institutes of Health grants RO1AG08226 and NIH RO1HL62267 to Dr Freedman and an Established Investigator Award from the American Heart Association to Dr Freedman.

Disclosure

Dr Freedman has received research funding from Boehringer-Ingelheim.


*    Footnotes
 
Guest editor for this article was Donald D. Heistad, MD.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowPlatelet Adhesion: Initiation of...
 up arrowConclusion
 *References
 

  1. Andreotti F, Becker RC. Atherothrombotic disorders: new insights from hematology. Circulation. 2005; 111: 1855–1863.[Free Full Text]
  2. Davies MJ, Thomas AC. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic death. N Engl J Med. 1984; 310: 1137–1140.[Abstract]
  3. Santos MT, Valles J, Marcus AJ, Safier M, Broekman J, Islam N, Ullman HL, Eiroa AM, Aznar J. Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes: a new approach to platelet activation and recruitment. J Clin Invest. 1991; 87: 571–580.[Medline] [Order article via Infotrieve]
  4. Hugel B, Socie G, Vu T, Toti F, Gluckman E, Freyssinet JM, Scrobohaci ML. Elevated levels of circulating procoagulant microparticles in patients with paroxysmal nocturnal hemoglobinuria and aplastic anemia. Blood. 1999; 93: 3451–3456.[Abstract/Free Full Text]
  5. Yamamoto H, Vreys I, Stassen JM, Yoshimoto R, Vermylen J, Hoylaerts MF. Antagonism of vWF inhibits both injury induced arterial and venous thrombosis in the hamster. Thromb Haemost. 1998; 79: 202–210.[Medline] [Order article via Infotrieve]
  6. Chang MC, Lin HK, Peng HC, Huang TF. Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox. Blood. 1998; 91: 1582–1589.[Abstract/Free Full Text]
  7. Yeh CH, Chang MC, Peng HC, Huang TF. Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist. Br J Pharmacol. 2001; 132: 843–850.[CrossRef][Medline] [Order article via Infotrieve]
  8. Cauwenberghs N, Meiring M, Vauterin S, van Wyk V, Lamprecht S, Roodt JP, Novak L, Harsfalvi J, Deckmyn H, Kotze HF. Antithrombotic effect of platelet glycoprotein Ib–blocking monoclonal antibody Fab fragments in nonhuman primates. Arterioscler Thromb Vasc Biol. 2000; 20: 1347–1353.[Abstract/Free Full Text]
  9. Kageyama S, Yamamoto H, Nakazawa H, Matsushita J, Kouyama T, Gonsho A, Ikeda Y, Yoshimoto R. Pharmacokinetics and pharmacodynamics of AJW200, a humanized monoclonal antibody to von Willebrand factor, in monkeys. Arterioscler Thromb Vasc Biol. 2002; 22: 187–192.[Abstract/Free Full Text]
  10. Hung DT, Vu TK, Wheaton VI, Ishii K, Coughlin SR. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest. 1992; 89: 1350–1353.[Medline] [Order article via Infotrieve]
  11. Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057–1068.[CrossRef][Medline] [Order article via Infotrieve]
  12. Clutton P, Folts JD, Freedman JE. Pharmacological control of platelet function. Pharmacol Res. 2001; 44: 255–264.[CrossRef][Medline] [Order article via Infotrieve]
  13. Cattaneo M, Lombardi R, Zighetti ML, Gachet C, Ohlmann P, Cazenave JP, Mannucci PM. Deficiency of (33P)2MeS-ADP binding sites on platelets with secretion defect, normal granule stores and normal thromboxane A2 production: evidence that ADP potentiates platelet secretion independently of the formation of large platelet aggregates and thromboxane A2 production. Thromb Haemost. 1997; 77: 986–990.[Medline] [Order article via Infotrieve]
  14. Gachet C, Cattaneo M, Ohlmann P, Hechler B, Lecchi A, Chevalier J, Cassel D, Mannucci PM, Cazenave JP. Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors. Br J Haematol. 1995; 91: 434–444.[Medline] [Order article via Infotrieve]
  15. Daniel JL, Dangelmaier C, Jin J, Kim YB, Kunapuli SP. Role of intracellular signaling events in ADP-induced platelet aggregation. Thromb Haemost. 1999; 82: 1322–1326.[Medline] [Order article via Infotrieve]
  16. Daniel JL, Dangelmaier C, Jin J, Ashby B, Smith JB, Kunapuli SP. Molecular basis for ADP-induced platelet activation, I: evidence for three distinct ADP receptors on human platelets. J Biol Chem. 1998; 273: 2024–2029.[Abstract/Free Full Text]
  17. Fagura MS, Dainty IA, McKay GD, Kirk IP, Humphries RG, Robertson MJ, Dougall IG, Leff P. P2Y1-receptors in human platelets which are pharmacologically distinct from P2Y(ADP)-receptors. Br J Pharmacol. 1998; 124: 157–164.[CrossRef][Medline] [Order article via Infotrieve]
  18. Jin J, Kunapuli SP. Coactivation of two different G protein–coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A. 1998; 95: 8070–8074.[Abstract/Free Full Text]
  19. Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dierich A, LeMeur M, Cazenave JP, Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor–null mice. J Clin Invest. 1999; 104: 1731–1737.[Medline] [Order article via Infotrieve]
  20. Leon C, Vial C, Gachet C, Ohlmann P, Hechler B, Cazenave JP, Lecchi A, Cattaneo M. The P2Y1 receptor is normal in a patient presenting a severe deficiency of ADP-induced platelet aggregation. Thromb Haemost. 1999; 81: 775–781.[Medline] [Order article via Infotrieve]
  21. Jarvis GE, Humphries RG, Robertson MJ, Leff P. ADP can induce aggregation of human platelets via both P2Y(1) and P(2T) receptors. Br J Pharmacol. 2000; 129: 275–282.[CrossRef][Medline] [Order article via Infotrieve]
  22. Storey RF, Sanderson HM, White AE, May JA, Cameron KE, Heptinstall S. The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity. Br J Haematol. 2000; 110: 925–934.[CrossRef][Medline] [Order article via Infotrieve]
  23. Savi P, Pereillo JM, Uzabiaga MF, Combalbert J, Picard C, Maffrand JP, Pascal M, Herbert JM. Identification and biological activity of the active metabolite of clopidogrel. Thromb Haemost. 2000; 84: 891–896.[Medline] [Order article via Infotrieve]
  24. Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y, Herbert JM. The antiaggregating activity of clopidogrel is due to a metabolic activation by the hepatic cytochrome P450–1A. Thromb Haemost. 1994; 72: 313–317.[Medline] [Order article via Infotrieve]
  25. CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet. 1996; 348: 1329–1339.[CrossRef][Medline] [Order article via Infotrieve]
  26. Savage B, Ruggeri ZM. Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets. J Biol Chem. 1991; 266: 11227–11233.[Abstract/Free Full Text]
  27. Naik UP, Patel PM, Parise LV. Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain. J Biol Chem. 1997; 272: 4651–4654.[Abstract/Free Full Text]
  28. Clemetson KJ. Platelet activation: signal transduction via membrane receptors. Thromb Haemost. 1995; 74: 111–116.[Medline] [Order article via Infotrieve]
  29. Lefkovits J, Plow EF, Topol EJ. Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med. 1995; 332: 1553–1539.[Free Full Text]
  30. Coller BS, Scudder LE, Beer J, Gold HK, Folts JD, Cavagnaro J, Jordan R, Wagner C, Iuliucci J, Knight D, et al. Monoclonal antibodies to platelet glycoprotein IIb/IIIa as antithrombotic agents. Ann N Y Acad Sci. 1991; 614: 193–213.[Medline] [Order article via Infotrieve]
  31. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty: the EPIC Investigation. N Engl J Med. 1994; 330: 956–961.[Abstract/Free Full Text]
  32. Dennis MS, Henzel WJ, Pitti RM, Lipari MT, Napier MA, Deisher TA, Bunting S, Lazarus RA. Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci U S A. 1990; 87: 2471–2475.[Abstract/Free Full Text]
  33. O’Neill WW, Serruys P, Knudtson M, van Es GA, Timmis GC, van der Zwaan C, Kleiman J, Gong J, Roecker EB, Dreiling R, Alexander J, Anders R, for the EXCITE Trial Investigators. Evaluation of oral xemilofiban in controlling thrombotic events: long-term treatment with a platelet glycoprotein-receptor antagonist after percutaneous coronary revascularization. N Engl J Med. 2000; 342: 1316–1324.[Abstract/Free Full Text]
  34. Cannon CP, McCabe CH, Wilcox RG, Langer A, Caspi A, Berink P, Lopez-Sendon J, Toman J, Charlesworth A, Anders RJ, Alexander JC, Skene A, Braunwald E. Oral glycoprotein IIb/IIIa inhibition with orbofiban in patients with unstable coronary syndromes (OPUS-TIMI 16) trial. Circulation. 2000; 102: 149–156.[Abstract/Free Full Text]
  35. Curtin R, Fitzgerald DJ. A cold start for oral glycoprotein IIb/IIIa antagonists. Eur Heart J. 2000; 21: 1992–1994.[Free Full Text]
  36. Chew DP, Bhatt DL, Sapp S, Topol EJ. Increased mortality with oral platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase iii multicenter randomized trials. Circulation. 2001; 103: 201–206.[Abstract/Free Full Text]
  37. Peter K, Schwarz M, Nordt T, Bode C. Intrinsic activating properties of GP IIb/IIIa blockers. Thromb Res. 2001; 103 (suppl 1): S21–S27.[Medline] [Order article via Infotrieve]
  38. Cox D, Smith R, Quinn M, Theroux P, Crean P, Fitzgerald DJ. Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes. J Am Coll Cardiol. 2000; 36: 1514–1519.[Abstract/Free Full Text]
  39. Quinn MJ, Cox D, Foley JB, Fitzgerald DJ. Glycoprotein IIb/IIIa receptor number and occupancy during chronic administration of an oral antagonist. J Pharmacol Exp Ther. 2000; 295: 670–676.[Abstract/Free Full Text]
  40. Rinder HM, Bonan JL, Rinder CS, Ault KA, Smith BR. Dynamics of leukocyte-platelet adhesion in whole blood. Blood. 1991; 78: 1730–1737.[Abstract/Free Full Text]
  41. Smith WL. Prostanoid biosynthesis and mechanisms of action. Am J Physiol. 1992; 263: F181–F191.[Medline] [Order article via Infotrieve]
  42. Smith WL, DeWitt DL, Shimokawa T, Kraemer SA, Meade EA. Molecular basis for the inhibition of prostanoid biosynthesis by nonsteroidal anti-inflammatory agents. Stroke. 1990; 21: IV24–IV28.[Medline] [Order article via Infotrieve]
  43. Maalej N, Folts JD. Increased shear stress overcomes the antithrombotic platelet inhibitory effect of aspirin in stenosed dog coronary arteries. Circulation. 1996; 93: 1201–1205.[Abstract/Free Full Text]
  44. Mason PJ, Freedman JE, Jacobs AK. Aspirin resistance: current concepts. Rev Cardiovasc Med. 2004; 5: 156–163.[CrossRef][Medline] [Order article via Infotrieve]
  45. Folts JD, Rowe GG. Epinephrine potentiation of in vivo stimuli reverses aspirin inhibition of platelet thrombus formation in stenosed canine coronary arteries. Thromb Res. 1988; 50: 507–516.[CrossRef][Medline] [Order article via Infotrieve]
  46. Stamler J, Mendelsohn ME, Amarante P, Smick D, Andon N, Davies PF, Cooke JP, Loscalzo J. N-Acetylcysteine potentiates platelet inhibition by endothelium-derived relaxing factor. Circ Res. 1989; 65: 789–795.[Abstract/Free Full Text]
  47. Cooke JP, Stamler J, Andon N, Davies PF, McKinley G, Loscalzo J. Flow stimulates endothelial cells to release a nitrovasodilator that is potentiated by reduced thiol. Am J Physiol. 1990; 259: H804–H812.[Medline] [Order article via Infotrieve]
  48. Shultz PJ, Raij L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. J Clin Invest. 1992; 90: 1718–1725.[Medline] [Order article via Infotrieve]
  49. Michelson AD, Benoit SE, Furman MI, Breckwoldt WL, Rohrer MJ, Barnard MR, Loscalzo J. Effects of nitric oxide/endothelium-derived relaxing factor on platelet surface glycoproteins. Am J Physiol. 1996; 270: H1640–H1648.[Medline] [Order article via Infotrieve]
  50. Radomski M, Moncada S. Regulation of vascular homeostasis by nitric oxide. Thromb Haemost. 1993; 70: 36–41.[Medline] [Order article via Infotrieve]
  51. Wirthumer-Hocke C, Silberbauer K, Sinzinger H. Effect of nitroglycerin and other organic nitrates on the in-vitro biosynthesis of arachidonic acid-metabolites in washed human platelets. Prostaglandins Leuko Med. 1984; 15: 317–323.[CrossRef]
  52. Negrescu E, Sazonova L, Baldenkov G, Mazaev AV, Tkachuk V. Relationship between the inhibition of receptor-induced increase in cytosolic free calcium concentration and the vasodilator effects of nitrates in patients with congestive heart failure. Int J Cardiol. 1990; 26: 175–184.[CrossRef][Medline] [Order article via Infotrieve]
  53. Trepakova ES, Cohen RA, Bolotina VM. Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase–dependent refilling of Ca2+ stores. Circ Res. 1999; 84: 201–209.[Abstract/Free Full Text]
  54. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF Jr, Michelson A. Nitric oxide released from activated platelets inhibits platelet recruitment. J Clin Invest. 1997; 100: 350–356.[Medline] [Order article via Infotrieve]
  55. Freedman JE, Ting B, Hankin B, Loscalzo J, Keaney JFJ, Vita JA. Impaired platelet production of nitric oxide in patients with unstable angina. Circulation. 1998; 98: 1481–1486.[Abstract/Free Full Text]
  56. de Graaf JC, Banga JD, Moncada S, Palmer RM, de Groot PG, Sixma JJ. Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation. 1992; 85: 2284–2290.[Abstract/Free Full Text]
  57. Radomski MW, Palmer MJ, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun. 1987; 148: 1482–1489.[CrossRef][Medline] [Order article via Infotrieve]
  58. Kahn NN, Bauman WA, Sinha AK. Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury. Proc Natl Acad Sci U S A. 1996; 93: 245–249.[Abstract/Free Full Text]
  59. Stafford NP, Pink AE, White AE, Glenn JR, Heptinstall S. Mechanisms involved in adenosine triphosphate–induced platelet aggregation in whole blood. Arterioscler Thromb Vasc Biol. 2003; 23: 1928–1933.[Abstract/Free Full Text]
  60. Goto S, Tamura N, Ishida H. Ability of anti-glycoprotein IIb/IIIa agents to dissolve platelet thrombi formed on a collagen surface under blood flow conditions. J Am Coll Cardiol. 2004; 44: 316–323.[Abstract/Free Full Text]
  61. Sorisky A, King W, Rittenhouse S. Accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in thrombin-stimulated platelets: different sensitivities to Ca2+ or functional integrin. Biochem J. 1992; 286: 581–584.[Medline] [Order article via Infotrieve]
  62. Kovacsovics T, Bachelot C, Toker A, Vlahos C, Duckworth B, Cantley L, Hartwig J. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem. 1995; 270: 11358–11366.[Abstract/Free Full Text]
  63. Keh D, Gerlach M, Kurer I, Seiler S, Kerner T, Falke KJ, Gerlach H. The effects of nitric oxide (NO) on platelet membrane receptor expression during activation with human alpha-thrombin. Blood Coagul Fibrinolysis. 1996; 7: 615–624.[Medline] [Order article via Infotrieve]
  64. Pigazzi A, Heydrick S, Folli F, Benoit S, Michelson A, Loscalzo J. Nitric oxide inhibits thrombin receptor–activating peptide-induced phosphoinositide 3-kinase activity in human platelets. J Biol Chem. 1999; 274: 14368–14375.[Abstract/Free Full Text]
  65. Salvemini D, de Nucci G, Sneddon JM, Vane JR. Superoxide anions enhance platelet adhesion and aggregation. Br J Pharmacol. 1989; 97: 1145–1150.[Medline] [Order article via Infotrieve]
  66. Clutton P, Miermont A, Freedman JE. Regulation of endogenous reactive oxygen species in platelets can reverse aggregation. Arterioscler Thromb Vasc Biol. 2004; 24: 187–192.[Abstract/Free Full Text]
  67. Jannes J, Hamilton-Bruce MA, Pilotto L, Smith BJ, Mullighan CG, Bardy PG, Koblar SA. Tissue plasminogen activator –7351C/T enhancer polymorphism is a risk factor for lacunar stroke. Stroke. 2004; 35: 1090–1094.[Abstract/Free Full Text]
  68. Ollikainen E, Mikkelsson J, Perola M, Penttila A, Karhunen PJ. Platelet membrane collagen receptor glycoprotein VI polymorphism is associated with coronary thrombosis and fatal myocardial infarction in middle-aged men. Atherosclerosis. 2004; 176: 95–99.[CrossRef][Medline] [Order article via Infotrieve]
  69. O’Donnell CJ, Larson MG, Feng D, Sutherland PA, Lindpaintner K, Myers RH, D’Agostino RA, Levy D, Tofler GH. Genetic and environmental contributions to platelet aggregation: the Framingham Heart Study. Circulation. 2001; 103: 3051–3056.[Abstract/Free Full Text]
  70. Lacolley P, Gautier S, Poirier O, Pannier B, Cambien F, Benetos A. Nitric oxide synthase gene polymorphisms, blood pressure and aortic stiffness in normotensive and hypertensive subjects. J Hypertens. 1998; 16: 31–35.[CrossRef][Medline] [Order article via Infotrieve]
  71. Shimasaki Y, Yasue H, Yoshimura M, Nakayama M, Kugiyama K, Ogawa H, Harada E, Masuda T, Koyama W, Saito Y, Miyamoto Y, Ogawa Y, Nakao K. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. J Am Coll Cardiol. 1998; 31: 1506–1510.[Abstract/Free Full Text]
  72. Elbaz A, Poirier O, Moulin T, Chedru F, Cambien F, Amarenco P, for the GENIC Investigators. Association between the Glu298Asp polymorphism in the endothelial constitutive nitric oxide synthase gene and brain infarction. Stroke. 2000; 31: 1634–1639.[Abstract/Free Full Text]
  73. Hingorani AD, Liang CF, Fatibene J, Lyon A, Monteith S, Parsons A, Haydock S, Hopper RV, Stephens NG, O’Shaughnessy KM, Brown MJ. A common variant of the endothelial nitric oxide synthase (Glu298->Asp) is a major risk factor for coronary artery disease in the UK. Circulation. 1999; 100: 1515–1520.[Abstract/Free Full Text]
  74. Wang XL, Sim AS, Badenhop RF, McCredie RM, Wilcken DE. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med. 1996; 2: 41–45.[CrossRef][Medline] [Order article via Infotrieve]
  75. Tanus-Santos JE, Desai M, Deak LR, Pezzullo JC, Abernethy DR, Flockhart DA, Freedman JE. Effects of endothelial nitric oxide synthase gene polymorphisms on platelet function, nitric oxide release, and interactions with estradiol. Pharmacogenetics. 2002; 12: 407–413.[CrossRef][Medline] [Order article via Infotrieve]
  76. Keaney J, Stamler J, J, Folts J, Loscalzo J. NO forms a stable adduct with serum albumin that has potent antiplatelet properties in vivo. Clin Res. 1992; 40: 194A.
  77. Freedman JE, Frei B, Welch GN, Loscalzo J. Glutathione peroxidase potentiates the inhibition of platelet function by S-nitrosothiols. J Clin Invest. 1995; 96: 394–400.[Medline] [Order article via Infotrieve]
  78. Freedman JE, Loscalzo J, Benoit SE, Valeri CR, Barnard MR, Michelson AD. Decreased platelet inhibition by nitric oxide in two brothers with a history of arterial thrombosis. J Clin Invest. 1996; 97: 979–987.[Medline] [Order article via Infotrieve]
  79. Inbal A, Kenet G, Freedman J, Michelson A, Loscalzo J. Impaired NO-mediated inhibition of platelet aggregation and P-selectin expression by plasma from patients with childhood stroke. Thromb Haemost. 1997; suppl: 304.
  80. Neumann FJ, Zohlnhofer D, Fakhoury L, Ott I, Gawaz M, Schomig A. Effect of glycoprotein IIb/IIIa receptor blockade on platelet-leukocyte interaction and surface expression of the leukocyte integrin Mac-1 in acute myocardial infarction. J Am Coll Cardiol. 1999; 34: 1420–1426.[Abstract/Free Full Text]
  81. Gawaz MP, Loftus JC, Bajt ML, Frojmovic MM, Plow EF, Ginsberg MH. Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions. J Clin Invest. 1991; 88: 1128–1134.[Medline] [Order article via Infotrieve]