Platelets, February 2008; 19(1): 1–8
REVIEW
Platelet receptor redox regulation JANE F. ARTHUR1, ELIZABETH E. GARDINER1, DERMOT KENNY2, ROBERT K. ANDREWS1, & MICHAEL C. BERNDT1 1
Department of Immunology, Monash University, Alfred Medical Research & Education Precinct, Melbourne 3004, Victoria, Australia, and 2Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland
(Received 29 October 2007; accepted 30 October 2007)
Abstract Several recent findings point to an important role for redox regulation of platelet responses to collagen involving the receptor, glycoprotein (GP)VI. First, the antioxidant dietary compound, quercetin, was shown to inhibit GPVI-dependent platelet activation and signaling responses to collagen. Second, collagen increased platelet production of the oxygen radical, superoxide anion (O 2 ), mediated by the multi-subunit enzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase. In that case, O 2 was implicated in regulating not initial aggregation, but collagen-induced thrombus stabilization involving release of ADP. Third, our laboratory showed that an unpaired thiol in the GPVI cytoplasmic tail undergoes rapid oxidation to form GPVI homodimers following ligand binding, preceding GPVI signaling and ectodomain metalloproteolysis, and indicating formation of an oxidative submembranous environment in activated platelets. This review examines receptor/redox regulation in other cells, and relevance to the pathophysiological function of GPVI and other platelet receptors initiating thrombus formation in haemostasis or thrombotic diseases such as heart attack and stroke.
Keywords: Redox, platelets, GPVI
Abbreviations GP, glycoprotein; NAD, nicotinamide adenine dinucleotide; O 2 , superoxide anion; FcRg, Fc receptor g-chain; ITAM, immunoreceptor tyrosine-based activation motif; CRP, collagen-related peptide; Nox, NAD(P)H oxidase; PI3-kinase, phosphoinositide 3-kinase; PKA/C, protein kinase A/C; DPI, diphenylene iodonium; ROS, reactive oxygen species.
Introduction The ability to produce oxygen radicals (the ‘respiratory burst’) in a rapid and controlled manner is a signature defence mechanism against invading microbes in phagocytic cells, and the molecular nature of oxygen radical chemistry in biological systems has essentially been defined through examination of redox systems in professional phagocytes [1]. The ability of non-phagocytic cells to also generate oxygen radicals implies alternative roles for these reactive species, although less is known about intracellular redox regulation of cellular functions.
A role for redox regulation of human blood platelets has long been known, and antioxidants provide potential agents for prevention/treatment of cardiovascular disease or thrombotic diseases such as myocardial infarction or stroke [2–6]. Reactive oxygen species (ROS) influence platelet function and coagulation, and are associated with the pathogenesis of cardiovascular disease [7, 8]. However, mechanisms for redox regulation of platelet adhesion receptors are still not fully resolved. Here, we review recent findings on the redox regulation of the platelet collagen receptor, glycoprotein (GP)VI, focusing on the effect of redox changes on GPVIdependent platelet activation [9, 10], and how
Correspondence: Prof. Michael C. Berndt, Monash University, Department of Immunology, Alfred Medical Research and Education Precinct (AMREP), Commercial Road, Melbourne, Australia. Tel: 61 3 9903 0713. Fax: 61 3 9903 0038. E-mail:
[email protected] ISSN 0953–7104 print/ISSN 1369–1635 online ß 2008 Informa UK Ltd. DOI: 10.1080/09537100701817224
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disulfide-dependent dimerization following ligand binding involving the ultimate Cys residue of the GPVI cytoplasmic tail reports on oxidative changes occurring inside the platelet after activation [11]. The significance of these findings is what they reveal about potential pathways for regulating pathological platelet thrombus formation, especially in terms of ischaemia-reperfusion injury [12]. Platelet thrombus formation In response to vessel wall injury exposing subendothelial matrix (including collagen and von Willebrand factor) or activating endothelial cells (leading to expression of P-selectin and high molecular weight von Willebrand factor multimers), circulating platelets in flowing blood interact with the vessel wall, primarily mediated by surface receptors GPVI and GPIb-IX-V, which form an adhesionsignaling complex on platelets [13, 14]. GPVI, which binds collagen, and GPIb (the major ligand-binding subunit of GPIb-IX-V) which binds von Willebrand factor, initiate platelet signaling, secretion of agonists such as ADP, and activation of integrin IIb 3dependent platelet aggregation. ADP acting in an autocrine manner at platelet purinergic receptors, P2Y1 and P2Y12, acts to elevate cytosolic Ca2þ levels (via P2Y1/G q) and decrease cAMP (via P2Y12/ G i), together leading to activation of IIb 3 and increasing thrombus stability [15–19]. GPVI, GPIbIX-V and IIb 3 are essentially platelet-specific, and deficiency or dysfunction of any of these receptors results in a bleeding disorder that can be severe [20–22]. GPVI, GPIb-IX-V and IIb 3 are also critical in regulating thrombus formation at high (arterial) shear rates in ex vivo or in vivo experimental models [23–27], and GPVI and GPIb-IX-V initiate thrombosis in experimental models of cerebral vascular stroke [28]. Based on experimental systems under shear, in atherosclerotic arteries, plaque rupture (exposing subendothelial matrix) or vascular occlusion (causing pathological shear stress) could also induce thrombus formation involving GPVI/GPIb-IX-V [23, 29]. One mechanism by which both GPVI and GPIbIX-V are regulated involves metalloproteinasemediated ectodomain shedding induced by ligand binding and signaling. Alternatively, calmodulin inhibitors that dissociate calmodulin from cytoplasmic binding sites in GPVI, GPIb or GPV cause shedding without platelet activation, with release of soluble ectodomain fragments and production of membrane-associated remnants [30–33]. The sheddase, ADAM10, cleaves a membrane-proximal sequence of GPVI, and shedding from platelets releases an 55-kDa soluble extracellular fragment and generates an 10-kDa platelet-associated remnant [34]. GPVI may also be internalized (without proteolysis) [35]. Depletion of GPVI (and other
adhesion receptors) post-adhesion is postulated to control platelet-subendothelial matrix interactions involved in platelet spreading and platelet accumulation at the site of thrombus growth, limiting platelet signaling responses involved in activation and secretion, and/or destabilizing thrombus strength leading to detachment of parts of the thrombus or preventing indefinite propagation of a thrombus [36]. Platelet surface expression levels of GPVI measured in clinical studies have also been linked to risk of acute coronary syndrome [37]. Redox changes are recognized as critical in platelet function and the development of vascular disease [2–6], and recent findings reveal pathways for how redox changes may be involved in regulation of GPVI and other platelet adhesion receptors such as GPIb-IX-V.
GPVI Glycoprotein (GP)VI is a member of the immunoreceptor family, with two extracellular immunoglobulin domains, a mucin-like domain, a transmembrane domain, and a cytoplasmic tail of 51 residues [38, 39]. The cytoplasmic domain of GPVI contains binding sites for calmodulin, and Src family kinases, Fyn and Lyn [40–42]. GPVI is also associated with the Fc receptor g-chain (FcRg), which contains an immunoreceptor tyrosine-based activation motif (ITAM) within the cytoplasmic tail [43–46], allowing ligand-induced cross-linking of GPVI to promote Src-dependent phosphorylation of the FcRg ITAM and activation of Syk signaling pathways. This suggests that ITAM-dependent signaling via GPVI is facilitated by increased ligand valency and the capacity of GPVI to form dimers or higher oligomers [11, 47]. Collagen-induced ITAM signaling is pivotal in the development of myocardial ischaemia-reperfusion injury in mice [12]. GPVI signals by ITAM-dependent and -independent pathways [42, 44, 48]. The major physiological ligand of GPVI is collagen, which can bind GPVI and other platelet collagen-binding receptors such as 2 1, but additional ligands include laminin (that also binds platelet 6 1), snake venom toxins such as convulxin (C-type lectin-like family) and alborhagin or crotarhagin (metalloproteinase-disintegrin family), and cross-linked collagen-related peptide (CRP) [44–46, 49–52]. Divergent ligand-induced signals emanating from GPVI/FcRg can lead to metalloproteinase-dependent GPVI shedding, internalization and/or ADP secretion/ IIb 3-dependent platelet aggregation (Figure 1A). We recently showed that ITAM-dependent signals from GPVI/FcRg can also lead to intracellular calpain-mediated proteolysis of the platelet Fc receptor, FcgRIIa, resulting in loss of the cytoplasmic ITAM domain and irreversibly inhibiting signaling via FcgRIIa by anti-platelet antibodies [53].
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response to collagen, and early events in GPVI signaling, including phosphorylation/activation of FcRg, Syk and phosphoinositide 3-kinase (PI3kinase) [9]. These events are upstream of GPVI shedding and IIb 3-dependent platelet aggregation, since inhibitors of Syk (piceatannol) and PI3-kinase (wortmannin) block these processes (Figure 1A) [31]. In a pre-clinical pilot study, platelets isolated from healthy volunteers ingesting quercetin aggregated less well to collagen, and GPVI-dependent phosphorylation was inhibited at 0.5–2 h after oral administration [54, 55], consistent with other epidemiological data [56]. How quercetin inhibits GPVI function was not directly demonstrated, but polyphenolic compounds act by depleting free radicals and ROS [3–8]. A single report noted lower ADP production in platelets treated with quercetin [57] and other studies suggest that platelet production of ROS promotes collagen responsiveness, and this is mediated by NAD(P)H oxidase [58]. Recently, it was reported that the combined effect of quercetin and another polyphenol, catechin, inhibited the platelet NAD(P)H oxidase-dependent production of ROS [59]. Figure 1. GPVI signaling pathways. (A) Engagement of platelet GPVI leads to secretion of ADP that acts on purinergic receptors, P2Y1 and P2Y12, and activation of the integrin, IIb 3, which binds fibrinogen and von Willebrand factor and mediates platelet aggregation. Ligand binding to GPVI can also lead to internalization (not shown), or activation of platelet sheddase (ADAM10) resulting in an 55-kDa soluble fragment and an 10-kDa membrane-associated remnant, and activation of a parallel proteolytic pathway leading to calpainmediated intracellular proteolysis of FcgRIIa (removing the ITAM domain). Calpain also inactivates IIb 3. Inhibitors of Src kinase (PP1/PP2), Syk kinase (piceatannol) and PI3-kinase (wortmannin) inhibit both metalloproteinase-dependent shedding and IIb 3mediated shedding. Superoxide anion (O 2 ) inhibits ADPdependent recruitment of platelets to a developing thrombus following collagen stimulation. See the text for details and references. (B) Ligand binding to GPVI induces rapid formation of disulfide-dependent homodimers involving the penultimate unpaired Cys residue in the cytoplasmic domain, and is disulfide bond formation is unaffected by inhibitors of GPVI signaling that block GPVI shedding and GPVI-dependent platelet aggregation.
Redox changes and GPVI Effect of the antioxidant, quercetin, on GPVI function One line of evidence linking redox changes to GPVI function involves a report by Hubbard and colleagues showing the antioxidant dietary compound, quercetin, inhibits GPVI-dependent platelet activation and signaling responses to collagen acting at GPVI [9]. Quercetin is a polyphenolic compound found in fruit (apples, berries), vegetables (onions, broccoli) and beverages (wine, tea) [5, 54, 55]. Interestingly, quercetin treatment of washed platelets for five minutes inhibited platelet aggregation in
Platelet NAD(P)H oxidase One of the major sources of ROS in the vasculature is NAD(P)H oxidase, primarily described in phagocytic cells as part of the innate immunity system. There is now strong evidence that generation of ROS by NAD(P)H oxidases in non-phagocytic cells is coupled to specific signal transduction pathways [60]. NAD(P)H oxidase is a multi-component enzyme that consists of a major catalytic subunit gp91phox and a regulatory subunit p22phox, both membrane-bound, in addition to the cytosolic regulatory components p47phox, p67phox, p40phox and the GTP-binding protein Rac1/2 (Figure 2). Following discovery of six homologs (Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2) of the catalytic subunit gp91phox (now known as Nox2), these enzymes are collectively referred to as the Nox family of NAD(P)H oxidases [61]. The expression of a platelet NAD(P)H oxidase was first alluded to more than 25 years ago when platelet aggregation induced by ADP or thrombin was inhibited using a non-specific inhibitor of NAD(P)H oxidase, diphenylene iodonium (DPI) [62]. Platelets have subsequently been shown to express a Nox2-containing NADPH oxidase, similar to the classic Nox in neutrophils [63–65]. Activated Nox produces superoxide anion (O 2 ), which is a precursor of other ROS (e.g. hydrogen peroxide [H2O2] and the hydroxyl radical [OH.]), and a negative regulator of nitric oxide (NO), that downregulates platelet function. (The dual oxidases (Duox 1 and 2) produce H2O2 rather than O 2 [66].) Translocation of cytoplasmic subunits and assembly of active Nox is regulated by
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Figure 2. Platelet NAD(P)H oxidase complex (Nox2). Activation of p47phox by phosphorylation leads to recruitment of the p47phox/p67phox complex (cytosolic components) to the Nox2/ p22phox subunits (membrane associated) to form an active complex that converts NADPH to NADPþ and generates O 2 . The p47phox subunit interacts with PI(3,4)P2, p22phox via Nterminal PX and SH3 domains, and p67phox which, in turn, interacts with Rac1/2. See the text for references.
phosphorylation of p47phox, which can be mediated by protein kinase C (PKC). Activated p47phox then interacts with both p22phox (via a Src homology-3 [SH3] domain), and the membrane-associated PI3-kinase product PI(3,4)P2 (via a phox homology [PX] domain) (reviewed in [67]; Figure 2). Platelet Nox generates little, if any [10, 58], extracellular ROS compared to neutrophils—being important for intracellular signaling mechanisms rather than host defence responses—although the respiratory burst of neutrophils is enhanced in the presence of platelets [63, 68]. Similarly, platelets and other vascular cells potentially contribute to superoxide-induced damage to cardiomyocytes in ischaemia-reperfusion injury [12], or the effects of ROS on the function of different vascular cells could involve common pathways. While the extent of these interactions is unclear, recent studies provide evidence for ROS-dependent regulation of platelet function. Krotz et al. reported that collagen, but not other agonists (thrombin or ADP), increased platelet production of the oxygen radical, superoxide anion (O 2 ), and that this was mediated by Nox [10]. Rather than initiating aggregation, O 2 was implicated in regulating collagen-induced thrombus stabilization involving secretion of ADP (see Figure 1A). Scavenging O 2 with superoxide dismutase also attenuated secondary phases of aggregation, while exogenous O 2 increased ADP release from collagen-stimulated platelets. Of interest, scavenging O 2 is a very effective way for limiting myocardial injury [69]. Studies using gp91phoxknockout mice also support a pro-thrombogenic role for platelet Nox in the context of vascular dysfunction in hypercholesterolemia [70]. Another important pathological role for Nox in platelets is in
complement-independent platelet destruction and thrombocytopenia induced by anti-platelet (integrin 3 subunit) antibodies in HIV-1 infection; p47phoxdeficient mice showed protection from anti- 3 antibody-induced thrombocytopenia [71]. Adhesion of platelets at high shear (1000 s1) involves GPIb-IX-V and GPVI [26, 72]. Shear stress stimulates Nox-dependent ROS production in endothelial cells [73, 74], suggesting i) endothelialderived ROS could regulate platelet adhesion to the vessel wall, and/or ii) as in endothelial cells, shear stress on platelets could be involved in ROS regulation of GPIb-IX-V/GPVI-mediated platelet adhesion and activation on von Willebrand factor/ collagen. Thrombus formation on collagen under high shear was blocked by inhibitors of NAD(P)H oxidase, DPI and apocynin, whereas secretion and shape change were not affected [58]. This suggests a role for intracellular production of ROS, mainly O 2 , in platelet activation leading to activation of IIb 3. The mechanism by which O 2 exerts its effects on collagen-dependent platelet recruitment, for example, altering ADP interaction with P2Y1/P2Y12, or inactivating a platelet ADP-destroying ectonucleotidase [10], is unknown. This also raises the question of how this effect is induced by collagen but not other agonists. An additional observation is that platelets from diabetic patients release more O 2 and exhibit higher ADP-induced Ca2þ signals [75]. In this respect, P2Y1 (G q-linked) triggers activation of Ca2þ signals in platelets, and, like GPVI, the cytoplasmic domain directly associates with the cytosolic regulatory protein, calmodulin [15, 16]. In both P2Y1 and GPVI, the calmodulin-binding sequence regulates Ca2þ signaling [15, 42], and in GPVI, dissociation of calmodulin following platelet activation regulates GPVI ectodomain shedding [31]. It is interesting to speculate on how redox changes would influence calmodulin-dependent GPVI shedding which presumably decreases platelet reactivity towards collagen. A further consideration is that Nox2 itself is predicted to have at least two possible calmodulin-binding sites (calmodulin target database, http://calcium.uhnres.utoronto.ca/ctdb/ ctdb/home.html [76]), and there is evidence that calmodulin inhibitors block NAD(P)H oxidase activity [77]. On the other hand, platelet nitric oxide synthase (NOS), which produces nitric oxide (NO) in both resting and activated platelets [78, 79], is also dependent on Ca2þ/calmodulin and NADPH [80]. NO generation in platelets has been proposed to act as a negative feedback mechanism regulating platelet activation in an autocrine fashion [81]. The activational state of IIb 3 has been recently shown to be directly regulated by NO [82]. Both GPVI and GPIb-IX-V have been shown to regulate platelet NO synthesis via activation of phospholipase C (PLC), protein kinase C (PKC) and the PI3-kinase pathway [83, 84]. In contrast to the effects of Nox-dependent
Platelet receptor redox regulation release of platelet O 2 [10], NO release inhibits the recruitment of platelets to a growing thrombus [85]. Due to the free radical nature of NO and O 2 , reaction of platelet NO with Nox-derived O 2 can ablate their individual effects [7]. Further, if platelet NOS becomes ‘uncoupled’ due to a shortage of cofactors, it can contribute to platelet-derived O 2 production [7]. More work is needed to understand the relationships between potential pro- and antithrombotic effects of redox changes, and this may help explain the clinical risk of arterial thrombosis and increased reactive oxygen species (e.g., diabetes) and/or therapeutic applications of antioxidant agents [2–10, 54–56].
Disulfide-dependent homodimers of GPVI Our laboratory recently found that GPVI undergoes rapid oxidation to form a disulfide-dependent homodimer following ligand binding, detected by western blotting GPVI on platelets separated on SDS-polyacrylamide gels under non-reducing conditions [11]. There was little detectable dimer on resting unstimulated platelets, but the timescale for disulfide-bond formation preceded GPVI signaling and ectodomain metalloproteolysis, and was independent of these processes. The extent of disulfide cross-linking was maximal with more potent agonists, such as convulxin where more than half of total GPVI could be dimerized, and with collagen or convulxin the amount of detectable homodimer was preserved by inclusion of metalloproteinase inhibitor (GM6001) to prevent GPVI shedding. Together, this suggests at least a subpopulation of GPVI exists as constitutive noncovalent dimer on resting platelets, consistent with other evidence [11, 47, 86]. Dimerization involved an apparently unpaired thiol in the GPVI cytoplasmic tail, since mutation of this residue, but not the other unpaired thiol in the transmembrane domain, blocked dimer formation in transfected cells. Together, the results highlight an oxidative submembranous environment in activated platelets, conducive to GPVI-GPVI disulfide bond formation. This occurs very rapidly upon ligand binding (within 10 seconds), and in advance of downstream signaling events [11] (Figure 1B). Certain protein tyrosine phosphatases have similarly been shown to form brief disulfidedependent dimers upon treatment with hydrogen peroxide and this intracellular oxidation caused transient enzyme inactivation [87]. Although disulfide cross-linking appears to be independent of ensuing GPVI signaling or shedding, it is possible that formation of dimer and its detection by western blot may provide a unique read-out of ligand-induced oxidative changes occurring in human platelets.
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Discussion and perspectives As outlined above, several lines of evidence suggest a role for redox regulation in the function of platelet GPVI, and platelet responsiveness to collagen. For example, the antioxidant agent, quercetin, inhibits GPVI-dependent platelet aggregation and signaling in vitro, and platelets from subjects administered quercetin are less reactive towards collagen [9, 54, 55]. In addition, superoxide anion production by platelet Nox increases platelet recruitment to collagen-stimulated platelet aggregates [10]. Ligand-binding to GPVI also leads to rapid oxidation of the cytoplasmic Cys residue to form disulfidedependent homodimers, whereas there is little detectable disulfide dimer on resting platelets, showing how rapidly redox changes occur, independently of GPVI signaling and other outcomes of GPVI-dependent platelet activation (Figure 1). Together, these results suggest that manipulating redox conditions may provide a way to control pathological thrombosis involving collagen/GPVI [2–10, 54–56]. Three key questions regarding the mechanism for GPVI and intraplatelet redox regulation are: is GPVI involved in submembranous localization/activation of subunits of the NAD(P)H oxidase complex; how is the balance between oxidation/reduction (and pro/anti-thrombotic consequences) affected; and how is the timecourse for redox changes related to GPVI-dependent dimerization, signaling, proteolysis and platelet aggregation? Interaction between GPVI and NAD(P)H oxidase Although platelets express functional NAD(P)H oxidase [63, 65, 88], it is not known how this complex is physically and functionally linked to GPVI and other platelet receptors (including GPIbIX-V, FcRg, and FcgRIIa which are physically and functionally linked to GPVI) (reviewed in [13, 14]). Defining these interactions is critical for understanding how ligand binding to GPVI results in rapid redox changes (disulfide dimer formation), and Nox-generated O 2 required for the thrombus development. Missing from the picture is a link between the receptor and one or more subunits of Nox, or GPVI signaling events leading to activation of p47phox (Figure 2). In platelets as well as other cells, translocation of cytoplasmic subunits and assembly of active Nox is regulated by phosphorylation downstream of PI3-kinase pathways [67, 89]. In platelets, PI3-kinase is activated downstream of GPVI/GPIb-IX-V [44–46], and PI3-kinase inhibitors block GPVI-dependent shedding [31], but a direct interaction between these platelet receptors and Nox subunits is not known. The GPVI cytoplasmic tail contains a proline-rich sequence that binds the SH3 domains of Lyn/Fyn [41], and a direct interaction with SH3 domains of p47phox is not ruled out.
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In endothelial cells, localized NAD(P)H-dependent production of ROS is achieved by the direct binding of p47phox to an adaptor protein called TRAF4 [90]. The tumour necrosis factor (TNF) -receptor associated factor (TRAF) family of proteins (TRAF1–6) act as signaling assemblage proteins linking receptors to downstream signaling pathways in various systems. Following binding to TRAF4, p47phox in turn binds a number of cytoskeletal regulatory proteins (including Hic-5, MYH9 and the focal adhesion kinase [Fak] analogue, protein tyrosine kinase 2 [Pyk2] found in platelets) localizing Nox to focal complexes where ROS production is required for cytoskeletal rearrangements associated with lamellipodia formation and cell migration [90–92]. The Nox subunit p47phox is localized intracellularly by the binding partner, Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein-1 (WAVE-1) [92]. WAVE-1 is also activated downstream of GPVI in platelets, and required for platelet spreading on collagen [93]. Future studies should determine how subunits of the NAD(P)H oxidase complex are physically and functionally linked to platelet adhesion receptors such as GPVI and GPIb-IX-V (direct or indirect), and the mechanisms for how adhesion to collagen, von Willebrand factor and other ligands controls redox regulation of platelet function. The precise localization and perhaps temporal staggering of proand anti-thrombotic signals is likely to be key to therapeutic attenuation of thrombotic diseases involving GPVI/GPIb-IX-V. Acknowledgments This work was supported in part by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and Monash University and Science Foundation Ireland. References 1. Segal AW. How neutrophils kill microbes. Annu Rev Immunol 2005;23:197–223. 2. Essex DW, Li M. Redox modification of platelet glycoproteins. Curr Drug Targets 2006;7:1233–1241. 3. Blomhoff R. Dietary antioxidants and cardiovascular disease. Curr Opin Lipidol 2005;16:47–54. 4. Diaz MN, Frei B, Vita JA, Keaney JF Jr.. Antioxidants and atherosclerotic heart disease. N Engl J Med 1997;337:408–416. 5. Hubbard GP, Wolffram S, Lovegrove JA, Gibbins JM. The role of polyphenolic compounds in the diet as inhibitors of platelet function. Proc Nutr Soc 2003;62:469–478. 6. Calzada C, Bruckdorfer KR, Rice-Evans CA. The influence of antioxidant nutrients on platelet function in healthy volunteers. Atherosclerosis 1997;128:97–105. 7. Krotz F, Sohn HY, Pohl U. Reactive oxygen species: players in the platelet game. Arterioscler Thromb Vasc Biol 2004;24:1988–1996. 8. Gorlach A. Redox regulation of the coagulation cascade. Antioxid Redox Signal 2005;7:1398–1404.
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