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Prepublished online as a Blood First Edition Paper on March 20, 2003; DOI 10.1182/blood-2002-12-3882.
Blood, 15 July 2003, Vol. 102, No. 2, pp. 449-461 Platelet-collagen interaction: is GPVI the central receptor?From the Department of Vascular Biology, Rudolf Virchow Center for Experimental Biomedicine Versbacher, Würzburg, Germany; and the Division of Medical Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
At sites of vascular injury, platelets come into contact with subendothelial collagen, which triggers their activation and the formation of a hemostatic plug. Besides glycoprotein Ib (GPIb) and  IIb 3 integrin, which indirectly interact with collagen via von Willebrand factor (VWF), several collagen receptors have been identified on platelets, most notably 21 integrin and the immunoglobulin (Ig) superfamily member GPVI. Within the last few years, major advances have been made in understanding platelet-collagen interactions including the molecular cloning of GPVI, the generation of mouse strains lacking individual collagen receptors, and the development of collagen receptor–specific antibodies and synthetic peptides. It is now recognized that platelet adhesion to collagen requires prior activation of integrins through "inside-out" signals generated by GPVI and reinforced by released second-wave mediators adenosine diphosphate (ADP) and thromboxane A2. These developments have led to revision of the original "2-site, 2-step" model, which now places GPVI in a central position in the complex processes of platelet tethering, activation, adhesion, aggregation, degranulation, and procoagulant activity on collagen. This review discusses these recent developments and proposes possible mechanisms for how GPVI acts in concert with other receptors and signaling pathways to initiate hemostasis and arterial thrombosis.
Vessel wall injury triggers sudden platelet activation and platelet plug formation, followed by coagulant activity and the formation of fibrin-containing thrombi that occlude the site of injury. These events are crucial to limit blood loss at sites of tissue trauma but may also block diseased vessels, leading to ischemia and infarction of vital organs. One of the major clinical problems in the developed world is arterial thrombosis caused by rupture or erosion of an atherosclerotic plaque, leading to platelet adhesion and subsequent thrombus formation in coronary and cerebral arteries causing myocardial infarction and stroke, respectively. Therefore, a detailed understanding of the mechanisms underlying the formation of the atherosclerotic plaque as well as (arterial) thrombosis is required in order to control ischemic cardiovascular diseases while retaining hemostasis. The first step in the hemostatic cascade is platelet interaction with the exposed extracellular matrix (ECM) at sites of injury. Among the macromolecular constituents of the ECM, collagen is considered to play a major role in this process, as in vitro it not only supports platelet adhesion through direct and indirect pathways but it also directly activates the cells initiating aggregation and coagulant activity.1 Platelet adhesion and aggregation on collagen is an integrated process that involves several platelet agonists that act through a variety of surface receptors, including integrins, immunoglobulin (Ig)–like receptors, and G-protein–coupled receptors. Over the last 20 years, immense effort has been spent on the identification of these receptors and their individual contribution to the complex processes of platelet tethering, adhesion, secretion, aggregation, and coagulant activity on collagen. However, the multiplicity of candidate collagen receptors and lack of detailed knowledge of the molecular events that underlie these responses have severely hampered developments in this field.
Platelet-collagen interactions are believed to have the greatest significance at the medium and high shear rates found in arteries and diseased vessels. At the very high shear rates found in small arteries and arterioles, the rapid onset of interaction between glycoprotein Ib-V-IX (GPIb-V-IX) and von Willebrand factor (VWF) immobilized on collagen is crucial for the initial tethering (or capture) of flowing platelets.2,3 The interaction between VWF and GPIb-IX-V, however, is rapidly reversible and insufficient for stable adhesion. This can be illustrated by the rolling of GPIb-IX–expressing cells or platelets on a VWF monolayer at high shear. Rapid conversion to stable adhesion requires additional contacts between the platelet and the ECM. Integrins are recognized as the major class of surface receptor mediating stable adhesion at high shear in hematopoietic cells. Integrins are heterodimeric proteins consisting of This new appreciation of the role of GPVI in mediating integrin activation has led to revision of the so-called "2-site, 2-step" model, which now places a critical role for GPVI in the initial interaction with collagen and upstream of adhesion. This review will discuss this new role of GPVI in platelet activation and define outstanding questions and future directions in this rapidly progressing field.
A thorough understanding of the molecular events that underlie platelet activation by collagen can be achieved only by consideration of the available experimental tools. This includes many different types, preparations, and species forms of collagen, receptor-specific synthetic collagens, snake toxins, and a rapidly increasing number of antibodies with the ability to inhibit or mimic collagen-receptor interactions. Each of these tools has important advantages and disadvantages that must be appreciated in using them to evaluate the role of collagen receptors in platelet-collagen interaction. There are genes for more than 20 forms of collagen in the human genome of which 9 have been identified to be expressed in the vessel wall, namely types I, III, IV, V, VI, VIII, XII, XIII, and XIV. Fibrillar types I and III are the major constituents of the ECM of blood vessels and have been the focus of most attention. In addition, the network-forming type IV collagen is the major form in the subendothelial basement membrane. Collagens consist of repeat GXY motifs where G is glycine and X and Y are frequently proline (amino acid code, P) and hydroxyproline (amino acid code, O). The sequence GPO makes up approximately 10% of collagens I and III. The GXY repeat sequence forms a single left-handed helix that associates with 2 other chains to form a right-handed super-helix. In collagens I and III, the chains have approximately 1000 amino acids flanked by short nonhelical N- and C-terminal telopeptide extensions. The cross-linking of these monomeric collagen structures forms fibrillar collagen, the predominant structure that platelets come into contact with in the ECM. Fibrillar collagen usually consists of more than one collagen type along with other matrix components. The utility of preparations of fibrillar collagen to simulate the behavior of subendothelial matrix collagen is limited because of this imprecise composition. Collagen fibers can be broken down to their monomeric forms by peptidases such as pepsin that target the nonhelical sequences in collagens, whereas specific collagenases are required to solubilize the parent molecule. The pepsin-generated monomers can be cross-linked to form collagens that lack the nonhelical regions, although it is important to recognize that the degree of cross-linking can have a marked influence on the biologic activity. Collagens are isolated from tissues and therefore represent a mixture of various types, although they are usually enriched in 1 or 2 particular forms as determined by the tissue source. Collagens are also prepared from several species, usually bovine, equine, and rat (but seldom human), and the same type may therefore have important differences in sequence that could influence activity. The most commonly used preparation of collagen for platelet studies, "Horm" collagen, is a suspension of fibrils made up of equine collagen type I and a small amount of equine type III, along with low levels of other ECM proteins.
The differences between the various preparations of collagens can have important experimental implications. In addition, the mode of presentation of collagen can also influence the biologic activity. For example, monomeric collagen interacts selectively with the integrin
There is considerable interest in the development of synthetic collagens of defined composition and in the synthesis of receptor-selective peptides. This is not a trivial matter in that it is generally recognized that the peptides need to be present in helical form to maintain activity at collagen receptors. A breakthrough in this area came from the observation that GPP or GPO repeats of 5 or more spontaneously form the helical structures that are present in collagen chains. The synthesis of peptides based on these sequences by Morton et al in Cambridge led to the unexpected observation that a peptide with a repeat GPO motif, cross-linked by N- and C-terminal cysteine or lysine residues, was a powerful platelet agonist, whereas cross-linked GPP was inactive.4 The GPO-containing peptide was termed collagen-related peptide (CRP) with CRP-XL being recommended for the cross-linked form. Significantly, CRP-XL is unable to support adhesion under the same stringent conditions used to show adhesion to collagen, but can induce platelet activation in the presence of
Knight et al later synthesized an A number of snake venom peptides that mediate their actions through GPVI have been identified in recent years and have proved to be powerful tools in the study of the Ig receptor. The snake C-type lectin convulxin was the first identified member of this group and was instrumental in the cloning of the protein.12,13 A number of additional C-type lectins from snake venoms have since been shown to activate GPVI, along with the metalloproteinase alborhagin, which binds to a distinct site on the glycoprotein.14 The snake venom toxins are multimeric and induce platelet activation by clustering the receptor. Several of these snake toxins bind to a second surface glycoprotein in addition to GPVI. For example, alboaggregin A binds to GPVI and GPIb, inducing powerful activation.15,16 The interaction with GPIb, however, is not essential for activation, as alboaggregin A activates Bernard-Soulier platelets and cell lines transfected with GPVI.16 Nevertheless, it may be the norm that snake venom toxins mediate their effects through binding to more than one platelet surface glycoprotein. The reader is referred to a recent review for further information on the action of snake venom toxins on GPVI and other platelet glycoproteins (Andrews et al17). A number of antibodies to GPVI have been raised within the last few years. GPVI-specific antibodies are powerful platelet agonists when cross-linked by secondary antibodies, but on their own several of them can serve as receptor antagonists. The antibodies are powerful tools used to identify the sequences within the extracellular domain of GPVI that confer binding to collagen.
GPVI was first identified as a 60- to 65-kDa platelet glycoprotein by 2-D gel electrophoresis more than 20 years ago.18 The first indication that GPVI may be an important platelet receptor for collagen, however, came from studies on a patient who presented to the clinic with an autoimmune thrombocytopenia caused by autoantibodies to a 65-kDa protein that was present in healthy individuals but absent in the patient.19 Gel electrophoresis (2-D) was used to demonstrate that the antiserum recognized GPVI. Platelets from this patient were unresponsive to collagen, whereas activation by other stimuli was normal. A F(ab)2 preparation of the IgG fraction from the patient was found to strongly activate platelets from healthy individuals, whereas monovalent F(ab) fragments inhibited collagen-induced activation. A small number of additional patients with low levels of GPVI have been described.6,20,21 In most cases, the patients display a mild bleeding phenotype and their platelets exhibit defective aggregation to collagen. The early studies on the GPVI-deficient patients provided compelling evidence for a key role of the glycoprotein in platelet activation by collagen, but this was not initially recognized because of the multiplicity of other candidates for this role, most notably 21.
A new era on collagen receptors followed the discovery of the GPO-based CRP-XL and the demonstration that the peptide and also collagen induce platelet activation through a tyrosine kinase–based signaling pathway that involves the kinase Syk and phospholipase C
The first antimouse GPVI monoclonal antibody (mAb), JAQ1, was reported in 2000.32 JAQ1 was detected during the screening of a panel of rat mAbs that had been generated against murine platelet membranes. The antibodies were screened against FcR
Several groups have now confirmed the inactivity of Horm collagen on FcR The phosphorylation events evoked by CRPs, snake toxins, and cross-linked GPVI antibodies are qualitatively similar to those induced by collagen, although the intensity of response is considerably greater. The GPVI-specific stimuli are all multimeric in nature and have the capacity to induce the formation of clusters of GPVI on the platelet surface, thereby generating a powerful intracellular signal. On the other hand, the GPVI-specific motif, GPO, is present at a level of approximately 10% in collagens and may not be appropriately spaced to induce an equivalent degree of clustering. In this context, it is noteworthy that F(ab')2 fragments of JAQ1, which can induce only receptor dimerization, stimulate a weak intracellular signal that is sufficient to synergize with Gi-based signaling pathways but alone is unable to induce shape change, aggregation, or release.40 These findings question the extent to which these GPVI-specific stimuli can be used to mimic signaling by collagen, an issue that is discussed later in the review.
In October 1999, Clemetson et al reported the cloning of human GPVI, revealing it to be a member of the Ig receptor superfamily.41 The sequence of GPVI is most closely related to human Fc R and natural killer (NK) cell receptors as well as to polymorphic mouse receptors known as paired Ig-like receptors (PIRs).41 Human GPVI is composed of 339 amino acids but displays an apparent molecular weight of 62 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis due to glycosylation. Human GPVI contains 2 Ig-C2–like extracellular domains formed by disulfide bonds, a mucinlike stalk, a transmembrane region, and a short 51–amino acid cytoplasmic tail (Figure 1). In a single publication, a splice variant, GPVI-II, has been described in human erythroleukemic megakaryocyte-like cells (HEL cells) that lack 18 amino acids in the mucin stalk.42 A further splice variant, GPVI-III, that lacks the transmembrane domain was also reported in this study. A number of polymorphic variations at the human GPVI locus have been identified (Figure 1),43 but it is unclear whether these variations significantly influence structure and function of the receptor, although there is evidence that at least one polymorphic form influences susceptibility to myocardial infarction as discussed in "The clinical significance of GPVI and 21 in hemostasis and thrombosis." Mouse GPVI has 319 amino acids and shares 64.4% and 67.3% identity with human GPVI at the protein and nucleotide levels, respectively.44 Human GPVI has an intracellular tail of 51 amino acids. Murine GPVI has an intracellular tail of 27 amino acids that lacks the 24 amino acids that lie C-terminal to the proline-rich region in human GPVI (Figure 1).44
The transmembrane and cytoplasmic tail of human GPVI has been shown to contain distinct regions that mediate association with other proteins. GPVI has a positively charged arginine in its transmembrane region that is essential for association with the FcR
Signaling through GPVI/FcR
Direct confirmation of GPVI as a collagen receptor has come from studies on transfected cell lines. Clemetson et al first showed that DAMI cells, a megakaryocytic cell line with a low level of GPVI expression, become responsive to collagen when transfected with GPVI and show enhanced responses to the more powerful convulxin.41 Interestingly, however, several other groups were unable to show activation of GPVI by collagen in megakaryocytic cells and in other transfected lines despite obtaining robust responses to convulxin.45,46,58 This was partially due to a low level of expression of GPVI, as RBL-2H3 cells, which express a comparable level of GPVI to that found in platelets, were subsequently shown to be responsive to collagen.59 The ability to obtain activation by convulxin in cells that express a low level of the receptor can be explained by its much greater affinity for GPVI and its multimeric nature. It is important to stress that RBL-2H3 cells do not express
There is evidence that collagen interacts with GPVI at 2 distinct sites.61 JAQ1 completely blocks activation of platelets by the GPO-rich peptide CRP, suggesting that the antibody occupies the "CRP binding site," which is very likely identical to the major collagen binding site on the receptor. However, the blocking action of JAQ1 can be overcome at high collagen concentrations indicating a second site of interaction. When one considers that FcR
Convulxin, CRP-XL, and cross-linked antibodies such as JAQ1 elicit rapid, powerful aggregation of platelets and are able to induce full aggregation in the presence of inhibitors of cyclo-oxygenase and antagonists of the 2 major ADP receptors, P2Y1 and P2Y12.40,62,63 In contrast, collagen induces aggregation after a delay of at least 20 to 30 seconds through a pathway that is primarily mediated by release of ADP and TxA2.40,63 This is illustrated by the inhibitory effects of cyclo-oxygenase inhibitors such as aspirin64 and the ADP receptor antagonists clopidogrel65 and the AR-C series of compounds.66 Furthermore, a major characteristic of patients with storage pool disease, in which ADP is lacking in the dense granules, is an impaired aggregation to collagen.67 Similarly, Pearl mice, which are deficient in dense granules, also exhibit a marked inhibition of aggregation to collagen.63
The receptors for ADP and TxA2 couple to several heterotrimeric G-proteins, namely Gi,Gq,G12, and G13.68 The importance of Gq-coupled receptors for collagen-induced platelet aggregation is highlighted by the finding that platelets from G
It is important to consider why collagen but not GPVI-specific agonists are dependent on the release of these mediators to induce aggregation. There are likely to be several factors that contribute to this: (1) Collagen is unable to induce the same strength of intracellular signal as that induced by powerful GPVI-specific agonists such as CRP-XL, convulxin, and cross-linked antibodies,5,61,73 most likely because of the infrequent spacing of GPO throughout its sequence. On the other hand, it stimulates a similar level of tyrosine phosphorylation to that induced by concentrations of the GPVI-selective agonists that are sufficient to induce aggregation, suggesting that this alone cannot account for the differential reliance on secondary mediators. (2) Fibrillar collagen is a relatively bulky material that is presented to platelets in vivo as a monolayer. When presented in suspension, platelets do not gain uniform access to the fibrillar collagen in contrast to soluble released mediators or the GPVI-specific agonists. This can be revealed by flow cytometric analysis of collagen-stimulated platelets, which allows examination of the activation state of single cells in diluted suspensions while excluding the accumulation of released mediators. Under these conditions, only 10% to 15% of normal platelets are directly activated (as shown by
Together, these observations suggest that the dependency of aggregation by collagen on secondary mediators reflects 2 independent processes. First, both ADP and TxA2 activate platelets that do not come into direct contact with the collagen in suspension. This process mimics the situation of in vivo thrombus formation, in which only the first layer of platelets is activated by collagen, whereas thrombus growth is predominantly mediated by soluble agonists in combination with VWF and fibrinogen. Second, the mediators may potentiate the activation of integrins
It has been accepted for many years that GPVI is essential for platelet activation by collagen but that 21 is required for adhesion. In support of this, platelets adhere extremely weakly to CRP-XL under flow but adhere strongly to collagen through an integrin-dependent pathway.79 It was therefore an unexpected finding that FcR  -chain–deficient and GPVI-depleted mouse platelets show virtually no adhesion to collagen under static and flow conditions even though they express normal levels of the major adhesion receptors 21, GPIb-V-IX, and IIb3.35 Defective adhesion of GPVI/FcR-deficient platelets to collagen under static conditions through 21 and IIb3/VWF was restored in the presence of Mn2+, which is known to directly activate integrins,35 and by agonists that activate integrins, such as ADP, via inside-out signaling (B.N. et al, unpublished observations, May 2002; and Inoue et al80). This demonstrates that the role of GPVI is to generate intracellular signals that promote integrin activation rather than to serve as an adhesion receptor. This is supported by studies in mice in which adhesion to collagen under static conditions is maintained in platelets that express 50% or 20% of the control level of GPVI.60 Low levels of adhesion are also observed in platelets in which GPVI has been substantially (> 98%), but not completely, depleted from the surface by in vivo administration of JAQ1.39 These platelets retain only trace amounts of GPVI on their surface, and yet this is sufficient to mediate adhesion under static conditions, demonstrating that even a very low copy number of the glycoprotein is sufficient to induce integrin activation, most likely through release of ADP and thromboxanes. Importantly, this residual adhesion is blocked in the presence of JAQ1.35 Under moderate to high shear flow conditions, however, GPVI-depleted platelets are unable to adhere to collagen, demonstrating that strong integrin activation is essential for shear-resistant adhesion.35 Initial studies with human platelets did not identify a critical role for GPVI in platelet adhesion to collagen, reporting only moderate adhesion defects of GPVI-deficient platelets.20,81 Goto et al, however, have recently reported complete abolition of adhesion of platelets from 2 GPVI-deficient patients to collagen at high shear.82 A possible explanation for these discrepancies is that the GPVI-deficient platelets used in the former studies expressed sufficient levels of the receptor to support adhesion. In support of this, we see substantial adhesion of murine platelets that express 20% of the normal level of GPVI at an intermediate rate of shear (800 s–1) (D. Best and S. P. W. unpublished data, June 2002). Together, these observations provide compelling evidence that cellular activation is a prerequisite for platelet adhesion to collagen and that GPVI plays the central role in this process, but that GPVI-mediated activation is reinforced by ADP and thromboxanes in vivo.
Integrin 21 (also known as platelet GPIa/IIa or lymphocyte VLA-2) was the first collagen receptor to be identified on platelets83,84 and is known to mediate adhesion in a Mg2+-dependent manner.85 For a long time, 21 was considered to be the major receptor for collagen on the platelet surface supporting adhesion and activation, and considered to play a key role in hemostasis. This was largely the result of studies on 2 patients with reduced levels of 21 who suffered from posttraumatic bleeding and excessive menorrhagia and whose platelets failed to respond to collagen.83,86 However, aggregation to collagen in one patient could be restored by the addition of thrombospondin-1,86 and the second was later shown to have defective adhesion to a number of adhesion molecules, demonstrating that the defect was not specific to collagen.87 Although it is unclear why addition of thrombospondin-1 was able to restore collagen responses, this observation demonstrated that 2 deficiency was not responsible for the loss of collagen responses.
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Abstract
Introduction
RI, Fc