• GPIb-IX-V;
  • GPVI;
  • metalloproteineses;
  • platelets;
  • thrombosis.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References

Summary.  Platelet adhesion receptors play a critical role in vascular pathophysiology, and control platelet adhesion, activation and aggregation in hemostasis, thrombotic disease and atherogenesis. One of the key emerging mechanisms for regulating platelet function is the programmed autologous cleavage of platelet receptors. Induced by ligand binding or platelet activation, proteolysis at extracellular (ectodomain shedding) or intracellular (cytoplasmic domain deactivation) sites down-regulates the adheso-signaling function of receptors, thereby controlling not only platelet responsiveness, but in the case of ectodomain shedding, liberating soluble ectodomain fragments into plasma where they constitute potential modulators or markers. This review discusses the underlying mechanisms for dual proteolytic pathways of receptor regulation, and the impact of these pathways on thrombus formation and stability in vivo.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References

One of the most fundamental homeostatic mechanisms is hemostasis, the ability to arrest blood loss after traumatic injury. In mammals, this is initiated by the adhesion of circulating blood platelets to the damaged vessel wall, culminating in platelet plug formation. Ironically, however, when triggered under pathological conditions, this normally protective cascade of events results in arterial thrombosis, responsible for major clinical sequelae such as heart attack and ischemic stroke. Two platelet receptors, the glycoprotein (GP) Ib-IX-V complex and the GPVI/FcRγ-chain (a complex of GPVI and Fc receptor γ-chain), are pivotal in initiating and propagating both hemostasis and thrombosis [1–8]. In the arterial circulation when an atherosclerotic plaque ruptures, these receptors initiate platelet adhesion in response to exposed thrombogenic materials by binding vessel wall von Willebrand factor (VWF) and collagen, respectively. The adherent platelets are activated, spread, release the contents of their storage organelles, and become cohesive toward circulating platelets by activating the platelet integrin, αIIbβ3, resulting in occlusive thrombus [7,8].

While these adhesive processes and subsequent events in thrombus formation have been the subject of intense investigation, the mechanisms that negatively regulate the function of these and other receptors in activated platelets, and thus act to limit thrombus formation and stability, are poorly understood. One common mechanism for regulating receptor function upon cell activation is proteolysis, by which receptor function is modified by either metalloproteinase-induced removal of the ligand-binding domain or by cleavage within the receptor cytoplasmic tail by intracellular proteases such as calpain. There is now convincing evidence that both of these mechanisms play a significant role in regulating platelet function mediated by GPVI/FcRγ [9–12], GPIb–IX–V [13–15] or other platelet receptors [16–19].

Ectodomain shedding as a mechanism of receptor down-regulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References

Cell receptors can be proteolytically cleaved at their juxta-membrane region, resulting in detachment of their extracellular region (the ectodomain). Shedding can release cytokines and growth factors from their membrane-bound precursors or, conversely, down-regulate receptor function [20–22]. Membrane protein receptor shedding is mediated almost exclusively by two ubiquitously expressed members of the ADAM family of metalloproteinases, ADAM10 and ADAM17 (also termed TACE). Knock-out of ADAM10 in mice is embryonic lethal, while that of ADAM17 is perinatal lethal, suggesting important roles for both in normal development. ADAM10 and ADAM17 consist of an N-terminal pro-domain, a zinc-binding metalloproteinase domain, a disintegrin domain, a Cys-rich domain, a transmembrane domain and a cytoplasmic tail (Fig. 1). Other surface-expressed ADAMs also include an epidermal growth factor-like domain (between the Cys-rich and transmembrane domains). ADAMs are expressed on the cell membrane in an inactive precursor form involving a ‘cysteine-switch’ mechanism, in which the presence of the unpaired cysteine residue and pro-domain maintain the metalloproteinase in a catalytically inactive form [22–26]. Blockade of the pro-domain cysteine with thiol-modifying reagents such as p-chloromercuribenzoate or N-ethylmaleimide (NEM) results in activation of the ADAM and induction of receptor shedding [22–26]. Mammalian ADAMs are structurally related to snake venom metalloproteinase-disintegrins (Fig. 1). The crystal structure of the rattlesnake metalloproteinase, VAP-1, showing physical proximity of the catalytic and Cys-rich domains, suggests the latter may act as a regulatory domain [27]. In this regard, ADAM10-mediated proteolysis of the ephrin receptor complex, occurring in trans [28], is regulated by the isolated disintegrin/Cys-rich (dc) domain (Fig. 1).


Figure 1.  Metalloproteinase-disintegrin structure and regulation. (A) Mammalian members of the ADAM family, ADAM10 and ADAM 17, consist of a pro-peptide domain, catalytic domain, disintegrin domain, regulatory Cys-rich domain, transmembrane region, and cytoplasmic tail, and may be regulated by (a) cysteine-switch, where a free sulfhydryl in the pro-peptide domain interacts with the active-site metal ion inhibiting the enzyme (removal of the pro-peptide or thiol-modification of the cysteine activates the ADAM), or (b) intracellular signals, required for ADAM-mediated ectodomain shedding. (B) Snake venom metalloproteinase-disintegrins are structurally related to mammalian ADAM family metalloproteinases, and exist as multiple processed forms, including distintegrin/Cys-rich (dc) forms.

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In a cellular context, ADAMs are normally activated by ligation of the potentially shed receptor, or by cell activation, although the precise mechanism by which this occurs is poorly understood. One of the earliest examples of receptor shedding is the ADAM17-mediated cleavage of L-selectin, one of the selectin adhesion receptors mediating leukocyte rolling on endothelium. Kahn et al. [29,30] demonstrated that the positively-charged, amphipathic, juxtamembrane cytoplasmic sequence of L-selectin bound calmodulin, and that activation-dependent dissociation of calmodulin triggered ADAM17-dependent shedding of the L-selectin ectodomain. Treatment of neutrophils with calmodulin antagonists, such as trifluoperazine or W7, also triggers shedding, as these antagonists compete for the same binding site on calmodulin as the amphipathic peptide sequence, and thus cause calmodulin dissociation from the receptor. Similarly, mutagenic disruption of the L-selectin calmodulin-binding site leads to enhanced ectodomain shedding of the receptor (in the absence of inhibitors) [28]. There are now multiple examples of shed receptors where shedding is regulated by a membrane-proximal cytoplasmic calmodulin-binding sequence [10,12,29,31].

The GPIb–IX–V complex

The GPIb–IX–V complex is a pivotal mucin adhesion receptor at the interface between thrombosis and inflammation [1–3]. At high arterial flow rates, GPIb–IX–V facilitates initial platelet adhesion, as well as platelet deposition on the developing thrombus, which involves an interaction with VWF and/or other ligands [1–4,8,32–35]. In addition to this, it is a key receptor mediating the interaction of platelets with activated endothelium and with leukocytes, through binding P-selectin and Mac-1 (αMβ2), respectively [36,37]. Recent evidence indicates that the interaction of GPIb with endothelial P-selectin is critical for early development of atherosclerosis, and that the interaction of GPIb with Mac-1 is essential for the transmigration of monocyte/macrophages through mural thrombus [36–40]. The GPIb–IX–V complex also is intimately involved with coagulation, through binding of kininogen, factors (F) XI and XII, and α-thrombin [41–51]. GPIb–IX–V is a complex of glycoproteins of the leucine-rich repeat family: GPIbα (∼130 kDa) and GPIbβ (∼25 kDa) are disulfide-linked and non-covalently associated with GPIX (∼22 kDa) and GPV (∼82 kDa) as a 2:2:2:1 (or higher order) complex [1–3,52]. The N-terminal globular domain of GPIbα (residues 1–282) is the major ligand-binding region of GPIb–IX–V and contains non-identical but partially overlapping binding sites for VWF, α-thrombin, FXI and XII, Mac-1, and P-selectin [1–4,36,37,41–51]. Immediately C-terminal to the N-terminal globular domain, is a mucin domain, rich in O-linked carbohydrate, followed by a short peptide linker sequence containing a membrane-proximal disulfide link(s) to GPIbβ, a transmembrane domain, and a cytoplasmic tail (Fig. 2) [1–3,52].


Figure 2.  Structure of the glycoprotein Ib–IX–V complex, illustrating ectodomain shedding. GPIb–IX–V complex consists of GPIbα (the major ligand-binding subunit) disulfide-linked to GPIbβ and non-covalently associated with GPIX and GPV. GPIbα binds von Willebrand factor, the leukocyte integrin Mac-1 (αMβ2), P-selectin and other ligands. The cytoplasmic domains of GPIbβ and GPV bind calmodulin. Metalloproteinase-mediated ectodomain shedding generates a soluble ectodomain fragment of GPIbα (glycocalicin) and GPV (not shown).

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The GPVI/FcRγ-chain complex

GPVI is a collagen receptor of the immunoglobulin superfamily [4,5,53,54]. There are two extracellular immunoglobulin-like domains, a mucin-like core, a short peptide linker sequence, a transmembrane domain, and a short cytoplasmic tail of ∼51 amino acids. It forms a non-covalent complex with the Fc receptor γ-chain (FcRγ) dimer (Fig. 3) [55,56], and is the major collagen signaling receptor on platelets, leading to activation of platelet aggregation, through the fibrinogen- and VWF-binding integrin, αIIbβ3, and to activation of the collagen-binding integrin, α2β1, which stabilizes the interaction of platelets with fibrillar collagen [5,6]. Platelet levels of GPVI not only reflect platelet responsiveness to collagen [57–59], but may also act as a marker of thrombotic risk [60]. We have previously shown that the cytoplasmic tail of GPVI has constitutively bound Src kinases, Fyn and Lyn [61]. When the GPVI receptor is cross-linked by binding collagen, or a GPVI-specific activating collagen-related peptide (CRP), or the snake venom GPVI agonists, such as convulxin (CVX) or alborhagin, the constitutively-bound Src kinases phosphorylate the ITAM (immunoreceptor tyrosine-based activation motif) sequence in FcRγ, allowing the assemblage of Syk and default activation of a downstream signaling pathway [5,6,61–63].


Figure 3.  Structure of the GPVI/FcRγ complex, illustrating ectodomain shedding. Binding of collagen, collagen-related peptide or the snake toxin convulxin to GPVI/FcRγ leads to activation of ITAM-dependent signaling pathways, dissociation of calmodulin from the cytoplasmic domain of GPVI, and metalloproteinase-mediated ectodomain shedding generating an ∼55-kDa soluble ectodomain fragment and an ∼10-kDa remnant that remains membrane-associated. GPVI shedding is also induced by the calmodulin inhibitor, W7.

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Ectodomain shedding of GPIbα, GPV and GPVI

In recent years we, and others, have demonstrated that calmodulin is associated with membrane-proximal, positively-charged, amphipathic sequences in GPIbβ, GPV and GPVI in resting human platelets (Figs 2 and 3) [56,64,65]. Activation of platelets through GPIb–IX–V by thrombin or by ristocetin/VWF leads to the rapid dissociation of calmodulin from GPIbβ and GPV [64]. Similarly, activation of platelets with the GPVI-specific agonist, CRP, led to the rapid dissociation of calmodulin from GPVI [65]. We therefore investigated whether calmodulin dissociation acted as a trigger for ectodomain shedding in these receptors [10]. In this regard, it has long been recognized that an ectodomain fragment of GPIbα (termed glycocalicin) is continuously and constitutively shed from platelets, and that glycocalicin circulates at high concentrations in plasma (∼3 μm) [2,34,66]. GPV has also been demonstrated to be shed from activated platelets, by a metalloproteinase-dependent mechanism [13]. Calmodulin inhibitors such as W7 induce shedding of GPIbα and GPV, as well as GPVI [10, 67; unpublished observations]. Analysis with a rabbit polyclonal antibody directed against the GPVI cytoplasmic tail indicated that loss of intact GPVI correlated with the formation of a membrane-bound GPVI stump of ∼10 kDa molecular weight. The loss of intact receptor was blocked by EDTA, and by the generic metalloproteinase inhibitor, GM6001 [10]. Shedding was also induced by treating intact platelets with the thiol-modifying reagent, NEM, suggesting shedding was ADAM dependent [67]. Indeed, in mouse platelets, Nieswandt, Wagner et al. [13–15] have demonstrated using mice where ADAM17 is expressed in an inactive form in the hematopoietic compartment (the ADAM17-knockout mouse is perinatal lethal), that shedding of GPIbα and GPV are mediated almost exclusively by ADAM17, although it is unclear whether this also applies to human platelets. Treating platelets with the mitochondrial-targeting reagent, CCCP, mimicking platelet aging also induces ADAM17-mediated GPIbα shedding, and there is decreased GPIbα expression on aged platelets [14]. Aspirin also promoted ADAM17-mediated shedding of GPIbα and GPV from human or mouse platelets, with increased levels of the respective ectodomain fragments occurring in plasma [15]. The mechanism for this metalloproteinase-mediated shedding may involve acylation of ADAM17 and/or substrate(s), rather than the classical antithrombotic target for aspirin, cyclooxygenase-1 (COX-1), as shedding was normal in COX-1-deficient mice [15].

Consistent with the GPVI agonist-dependent loss of GPVI-associated calmodulin [65], GPVI agonists such as collagen, CRP and CVX induce a rapid loss of GPVI from intact platelets, and the appearance of a ∼55-kDa soluble fragment in the supernatant, relative to intact GPVI in platelets (∼62 kDa) [9–11]. Shedding was blocked by treatment of platelets with EDTA, or with GM6001. In contrast, other membrane receptors, such as PECAM-1, were not shed from the platelet surface under the same conditions [10]. Nor were GPIbα and GPV shed under these conditions, suggesting a shedding mechanism specific to GPVI. In contrast, GPIbα and GPV are shed in response to platelet activation by low-dose thrombin, whereas GPVI is shed to a lesser extent by this agonist (unpubl. obs.). This and other evidence suggests that GPVI is shed by a different ADAM than ADAM17, possibly ADAM10 [15]. GPVI agonist-induced shedding was dependent at least on early GPVI dependent signaling and was blocked by inhibitors of Src family kinases (PP2), Syk (piceatannol) and PI 3-kinase (wortmannin) [10]. Calmodulin dissociation from GPVI on CRP-dependent platelet activation was also blocked by PP2 [65]. In contrast, W7- and NEM-induced shedding of GPVI is activation independent, as W7 causes calmodulin/receptor dissociation and NEM directly activates surface ADAM activity [22,23]. GPVI shedding can also be artificially induced in vivo using the antimouse GPVI monoclonal antibody, JAQ1, which selectively depletes GPVI expression on mouse platelets [68]. Alternately, human platelets injected into a NOD/SCID mouse can be depleted of GPVI in an activation-independent manner by antihuman GPVI antibodies [69].

Semaphorin 4D

The immune cell receptor, semaphorin 4D (Sema4D), and its binding partners, CD72 and plexin-B1, are expressed on human platelets, and Sema4D is also shed from the platelet surface [18]. Sema4D, CD72 (associated with the tyrosine phosphatase, SHP-1) and plexin-B1 contribute to the regulation of thrombus formation, and in particular, the soluble ectodomain fragment of Sema4D is functional as a competitive blocker of Sema4D-mediated aggregation. Sema4D-deficient mice exhibit decreased occlusive thrombi in arterial thrombosis models [18]. Surface expression of these proteins increases on activated platelets, and metalloproteinase-mediated shedding of Sema4D releases a soluble ectodomain fragment that may regulate angiogenesis or other vascular processes. Shedding of Sema4D is also inhibited in ADAM17-defective mice [18].


Platelet CD40 ligand (CD40L) regulates stability of αIIbβ3-dependent thrombi [19,70], and is shed from activated platelets by a mechanism that involves signaling through αIIbβ3 [70]. This is in contrast to GPVI shedding, which is independent of αIIbβ3 [10]. The physiological relationship between shedding of CD40L and other adhesion receptors, and the effects of shedding on thrombus formation in vivo, is not yet resolved. However, there is evidence that less than the full complement of these receptors impairs occlusive thrombus formation in animal models [71].

Intracellular proteolysis as a mechanism of receptor down-regulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References

A number of recent studies indicate that the intracellular proteinase, calpain, is involved in regulating platelet receptors, and that calpain may be activated under the same conditions that activate ectodomain sheddases in platelets, for example, following platelet activation or treatment with calmodulin inhibitors. Calpain is a ubiquitous intracellular cysteinyl proteinase, regulated, in part, by intracellular Ca2+ levels, and acting on >100 intracellular substrates [72–74]. Calpain, principally the μ isoform in platelets, plays a role in regulating cytoskeletal re-arrangements associated with cell motility and adhesion, and in other cell types, division. Like receptors subject to ectodomain shedding, the ability of a protein to bind calmodulin confers a strong likelihood that this protein is a substrate for calpain [75]. Therefore, mechanisms for activating extracellular shedding pathways of proteolysis also have the potential to activate intracellular calpain-dependent proteolytic pathways via changes in cellular calmodulin. EDTA inhibits both metalloproteinase-mediated receptor shedding and calpain activity [76], presumably by interfering with Ca2+ flux. In platelets, μ-calpain isoform is found at focal adhesions, where it regulates shape change, motility and adhesion, mainly through modulation of integrin clustering and function [77–81]. There is thus the possibility of dual extracellular (sheddase) and intracellular (calpain) proteolytic pathways operating in tandem in platelets.


Like GPVI, PECAM-1 is a member of the immunoglobulin superfamily, with six extracellular immunoglobulin domains, a transmembrane domain, and a cytoplasmic tail. In contrast to GPVI, the cytoplasmic domain of PECAM-1 contains an ITIM (immunoreceptor tyrosine-based inhibitory motif) sequence, involved in recruiting phosphatases and attenuating thrombus formation involving GPVI/FcRγ or other receptors [82–85]. Like GPVI, PECAM-1 contains a calmodulin-binding sequence in the juxtamembrane region of the cytoplasmic tail, and calmodulin inhibitors induce proteolysis of PECAM-1 [16]. However, proteolysis of PECAM-1 appears to involve cleavage of the cytoplasmic domain by calpain in activated platelets, at a site upstream of the ITIM, and consequently de-activating the receptor [17].


Proteolytic regulation of the platelet-specific integrin, αIIbβ3 (GPIIb-IIIa), involves intracellular and, potentially, extracellular pathways. Platelets express αIIbβ3, which binds VWF or fibrinogen to mediate platelet aggregation, in addition to the vitronectin receptor, αvβ3, and β1 integrins α1β1, α2β1, α5β1 and α6β1, which bind adhesive ligands including collagen, laminin and/or fibronectin [86]. The leukocyte integrin, αMβ2 (Mac-1), is involved in platelet-leukocyte adhesion, and is activated following initial contact of leukocyte P-selectin glycoprotein ligand-1 with P-selectin expressed on activated endothelial cells or activated mural platelets. This enables αMβ2-mediated adhesion to the endothelial receptor, intercellular adhesion molecule-1, or platelet GPIb–IX–V [37,40]. One mechanism for the proteolytic regulation of αIIbβ3 on platelets involves intracellular calpain-dependent cleavage of the β3 cytoplasmic tail, resulting in the removal of two NXXY motifs, and disrupting αIIbβ3-dependent signaling; four calpain-dependent cleavage sites flanking the NXXY motifs have been identified in platelets [87–89]. One of the critical functions of the cytoplasmic domain of αIIbβ3 is the regulation of clot contraction, involving association of the receptor with contractile actin filaments of the cytoskeleton in activated platelets, and αIIbβ3-dependent clot contraction is abolished by calpain-mediated proteolysis of the β3 cytoplasmic tail [87].

Recent studies involving another leukocyte β2 integrin, αLβ2, show that an unidentified sheddase(s) cleaves both a membrane-proximal region of β2 and a more upstream site of αL, releasing a soluble ectodomain fragment of αL (containing the ligand-binding ‘insert’ domain) in complex with the ectodomain of β2, from the surface of blister-fluid neutrophils [90,91]. Cell surface-expressed ADAM family receptors are known to interact via their distintegrin domains with integrins; for example, ADAM15, principally via its RGD-containing disintegrin domain, acts as a counter-receptor for αIIbβ3 [92]. Together, these results raise the possibility of a broader role for ADAM family metalloproteinases in platelet integrin shedding, as suggested for shedding of platelet GPIb–IX–V, GPVI and other receptors.

Mechanisms of thrombus formation and stability in vivo

  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References

In response to atherosclerotic plaque rupture or vascular injury, platelets initially translocate and then rapidly adhere to exposed VWF and collagen through the platelet mucin adhesion receptors, GPIb–IX–V and GPVI/FcRγ, respectively [1–8,32,33]. Contact adhesion through these receptor/matrix protein interactions generates signals leading to platelet activation, with resultant platelet spreading providing a cohesive platelet surface for further platelet accumulation by activation of the integrin αIIbβ3 [93,94]. Platelet accumulation on the developing thrombus also involves platelet translocation and subsequent firm platelet adhesion. Here, translocation is dependent on GPIb–IX–V in the interacting platelet and VWF on the surface of the developing thrombus bound through GPIb–IX–V and/or αIIbβ3, with firm adhesion mediated through αIIbβ3 binding of platelet associated VWF, fibronectin and/or fibrinogen [94–96]. Studies of thrombus formation in vivo using intravital microscopy indicate that the temporal development of the thrombus is a dynamic process, with large fragments of thrombus breaking off from the thrombus mass or from the thrombus/matrix interface as it develops and embolizing downstream, with subsequent replacement by fresh thrombus [95,97–99]. As activated platelets release ADP and other agonists, which facilitate platelet recruitment, and growth factors such as PDGF, which initiate vessel wound repair, it is probable that this process of thrombus formation and partial embolization acts to prolong the localized release of growth factors and chemokines that act in leukocyte recruitment and ultimately vessel repair. At a later stage, the thrombus can entirely occlude the vessel or the thrombus can cease to grow in size with no further platelet accumulation because of passification of the thrombus surface [99].

It is interesting to speculate that the ability of the platelet to shed GPIbα and GPVI postplatelet activation is fundamental in regulating and limiting thrombus formation. First, the capacity of platelets to form filopodia and lamellipodia and spread on a VWF and/or collagen matrix requires the dynamic breaking of existing receptor/matrix ligand bonds and formation of new receptor matrix/ligand interactions at the tips of filopodia or the spreading lamellipodial edge. One mechanism for how this could occur is through receptor ectodomain shedding. Secondly, shedding of GPIbα and associated VWF at the developing thrombus surface would regulate the number of translocating platelets and hence the rate of platelet accumulation. Ultimately, this would lead to the passification of the thrombus surface. Thirdly, as both GPIb- and GPVI-dependent signaling involve receptor cross-linking, shedding of GPIbα and GPVI could limit the time course of platelet signaling, and hence the degree of platelet activation and platelet secretion. Finally, shedding GPIbα and GPVI would destabilize thrombus strength, and thus facilitate embolization.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Ectodomain shedding as a mechanism of receptor down-regulation
  5. Intracellular proteolysis as a mechanism of receptor down-regulation
  6. Mechanisms of thrombus formation and stability in vivo
  7. Disclosure of Conflict of Interests
  8. References
  • 1
    Berndt MC, Shen Y, Dopheide SM, Gardiner EE, Andrews RK. The vascular biology of the glycoprotein Ib–IX–V complex. Thromb Haemost 2001; 86: 17888.
  • 2
    Andrews RK, Berndt MC, López JA. The glycoprotein Ib–IX–V complex. In: MichelsonA, eds. Platelets, 2nd edn. San Diego: Academic Press, 2006: 2.
  • 3
    Andrews RK, Gardiner EE, Shen Y, Whisstock JC, Berndt MC. Glycoprotein Ib-IX-V. Int J Biochem Cell Biol 2003; 35: 11704.
  • 4
    Kroll MH, Hellums JD, McIntire LV, Schafer AI, Moake JL. Platelets and shear stress. Blood 1996; 88: 152541.
  • 5
    Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the central receptor? Blood 2003; 102: 44961.
  • 6
    Moroi M, Jung SM. Platelet glycoprotein VI: its structure and function. Thromb Res 2004; 114: 22133.
  • 7
    Massberg S, Gawaz M, Gruner S, Schulte V, Konrad I, Zohlnhofer D, Heinzmann U, Nieswandt B. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J Exp Med 2003; 197: 419.
  • 8
    Bergmeier W, Piffath CL, Goerge T, Cifuni SM, Ruggeri ZM, Ware J, Wagner DD. The role of platelet adhesion receptor GPIbα far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis. Proc Natl Acad Sci USA 2006; 103: 169005.
  • 9
    Bergmeier W, Rabie T, Strehl A, Piffath C, Prostredna M, Wagner DD, Nieswandt B. GPVI down-regulation in murine platelets through metalloproteinase-dependent shedding. Thromb Haemost 2004a; 91: 9518.
  • 10
    Gardiner EE, Arthur JF, Kahn ML, Berndt MC, Andrews RK. Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase. Blood 2004; 104: 36117.
  • 11
    Stephens G, Yan Y, Jandrot-Perrus M, Villeval JL, Clemetson KJ, Phillips DR. Platelet activation induces metalloproteinase-dependent GPVI cleavage to down-regulate platelet reactivity to collagen. Blood 2005; 105: 18691.
  • 12
    Gardiner EE, Arthur JF, Berndt MC, Andrews RK. Role of calmodulin in platelet receptor function. Curr Med Chem Cardiovasc Hematol Agents 2005; 3: 2837.
  • 13
    Rabie T, Strehl A, Ludwig A, Nieswandt B. Evidence for a role of ADAM17 (TACE) in the regulation of platelet glycoprotein V. J Biol Chem 2005; 280: 144628.
  • 14
    Bergmeier W, Piffath CL, Cheng G, Dole VS, Zhang Y, Von Andrian UH, Wagner DD. Tumor necrosis factor-α-converting enzyme (ADAM17) mediates GPIbα shedding from platelets in vitro and in vivo. Circ Res 2004b; 95: 67783.
  • 15
    Aktas B, Pozgajova M, Bergmeier W, Sunnarborg S, Offermanns S, Lee D, Wagner DD, Nieswandt B. Aspirin induces platelet receptor shedding via ADAM17 (TACE). J Biol Chem 2005; 280: 3971622.
  • 16
    Wong MX, Harbour SN, Wee JL, Lau LM, Andrews RK, Jackson DE. Proteolytic cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is regulated by a calmodulin-binding motif. FEBS Lett 2004; 568: 708.
  • 17
    Naganuma Y, Satoh K, Yi Q, Asazuma N, Yatomi Y, Ozaki Y. Cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets exposed to high shear stress. J Thromb Haemost 2004; 2: 19982008.
  • 18
    Zhu L, Bergmeier W, Wu J, Jiang H, Stalker TJ, Cieslak M, Fan R, Boumsell L, Kumanogoh A, Kikutani H, Tamagnone L, Wagner DD, Milla ME, Brass LF. Regulated surface expression and shedding support a dual role for semaphoring 4D in platelet responses to vascular injury. Proc Natl Acad Sci USA 2007; 104: 16216.
  • 19
    Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derived CD40L: the switch-hitting player of cardiovascular disease. Circulation 2002; 106: 8969.
  • 20
    Blobel CP. ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005; 6: 3243.
  • 21
    Seals DF, Courtneidge SA. The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 2003; 17: 730.
  • 22
    Huovila APJ, Turner AJ, Pelto-Huikko M, Karkkainen I, Ortiz RM. Shedding light on ADAM metalloproteinases. Trends Biochem Sci 2005; 30: 41322.
  • 23
    Amit T, Amit T, Hochberg Z, Yogev-Falach M, Youdim MB, Youdim MB, Barkey RJ. Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase. J Endocrinol 2001; 169: 397407.
  • 24
    Srour N, Lebel A, McMahon S, Fournier I, Fugere M, Day R, Dubois CM. TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity. FEBS Lett 2003; 554: 27583.
  • 25
    Endres K, Anders A, Kojro E, Gilbert S, Fahrenholz F, Postina R. Tumor necrosis factor-α converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation. Eur J Biochem 2003; 270: 238693.
  • 26
    Leonard JD, Lin F, Milla ME. Chaperone-like properties of the prodomain of TNFα-converting enzyme (TACE) and the functional role of its cysteine switch. Biochem J 2005; 387: 797805.
  • 27
    Takeda S, Igarashi T, Mori H, Araki S. Crystal structures of VAP1 reveal ADAMs’ MDC domain architecture and its unique C-shaped scaffold. EMBO J 2006; 25: 238896.
  • 28
    Janes PW, Sah N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123: 291304.
  • 29
    Kahn J, Walcheck B, Migaki GI, Jutila MA, Kishimoto TK. Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism. Cell 1998; 92: 80918.
  • 30
    Zhao L-C, Shey M, Frnsworth M, Dailey MO. Regulation of membrane metalloproteolytic cleavage of L-selectin (CD62L) by the epidermal growth factor domain. J Biol Chem 2001; 276: 3063140.
  • 31
    Diaz-Rodriguez E, Esparis-Ogando A, Montero JC, Yuste L, Pandiella A. Stimulation of cleavage of membrane proteins by calmodulin inhibitors. Biochem J 2000; 346: 35967.
  • 32
    Goto S, Ikeda Y, Saldivar E, Ruggeri ZM. Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest 1998; 101: 47986.
  • 33
    Goto S, Tamura N, Handa S, Arai M, Kodama K, Takayama H. Involvement of glycoprotein VI in platelet thrombus formation on both collagen and von Willebrand factor surfaces under flow conditions. Circulation 2002; 106: 26672.
  • 34
    López JA, Andrews RK, Afshar-Kharghan V, Berndt MC. Bernard–Soulier syndrome. Blood 1998; 91: 4397418.
  • 35
    Jurk K, Clemetson KJ, De Groot PG, Brodde MF, Steiner M, Savion N, Varon D, Sixma JJ, Van Aken H, Kehrel BE. Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib(GPIb): an alternative/backup mechanism to von Willebrand factor. FASEB J 2003; 17: 14902.
  • 36
    Romo GM, Dong J-F, Schade AJ, Gardiner EE, Kansas GS, Li CQ, McIntire LV, Berndt MC, Lopez JA. The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin. J Exp Med 1999; 190: 80314.
  • 37
    Simon DI, Chen Z, Xu H, Li CQ, Dong J, McIntire LV, Ballantyne CM, Zhang L, Furman MI, Berndt MC, Lopez JA. Platelet glycoprotein Ibα is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J Exp Med 2000; 192: 193204.
  • 38
    Polgar J, Matuskova J, Wagner DD. The P-selectin, tissue factor, coagulation triad. J Thromb Haemost 2005; 3: 15906.
  • 39
    Katayama T, Ikeda Y, Handa M, Tamatani T, Sakamoto S, Ito M, Ishimura Y, Suematsu M. Immunoneutralization of glycoprotein Ibα attenuates endotoxin-induced interactions of platelets and leukocytes with rat venular endothelium in vivo. Circ Res 2000; 86: 10317.
  • 40
    Wang Y, Sakuma M, Chen Z, Ustinov V, Shi C, Croce K, Zago AC, Lopez J, Andre P, Plow E, Simon DI. Leukocyte engagement of platelet glycoprotein Ibαvia the integrin Mac-1 is critical for the biological response to vascular injury. Circulation 2005; 112: 29933000.
  • 41
    Baglia FA, Badellino KO, Li CQ, Lopez JA, Walsh PN. Factor XI binding to the platelet glycoprotein Ib–IX–V complex promotes factor XI activation by thrombin. J Biol Chem 2002; 277: 16628.
  • 42
    Yun TH, Baglia FA, Myles T, Navaneetham D, Lopez JA, Walsh PN, Leung LL. Thrombin activation of factor XI on activated platelets requires the interaction of factor XI and platelet glycoprotein Ibα with thrombin anion-binding exosites I and II, respectively. J Biol Chem 2003; 278: 481129.
  • 43
    Bradford HN, Pixley RA, Colman RW. Human factor XII binding to the glycoprotein Ib–IX–V complex inhibits thrombin-induced platelet aggregation. J Biol Chem 2000; 275: 2275663.
  • 44
    Joseph K, Nakazawa Y, Bahou WF, Ghebrehiwet B, Kaplan AP. Platelet glycoprotein Ib: a zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen. Mol Med 1999; 5: 55563.
  • 45
    Dumas JJ, Kumar R, Seehra J, Somers WS, Mosyak L. Crystal structure of the GPIbα-thrombin complex essential for platelet aggregation. Science 2003; 301: 2226.
  • 46
    Celikel R, McClintock RA, Roberts JR, Mendolicchio GL, Ware J, Varughese KI, Ruggeri ZM. Modulation of α-thrombin function by distinct interactions with platelet glycoprotein Ibα. Science 2003; 301: 21821.
  • 47
    Adam F, Bouton MC, Huisse MG, Jandrot-Perrus M. Thrombin interaction with platelet membrane glycoprotein Ibα. Trends Mol Med 2003; 9: 4614.
  • 48
    Vanhoorelbeke K, Ulrichts H, Romijn RA, Huizinga EG, Deckmyn H. The GPIbα-thrombin interaction: far from crystal clear. Trends Mol Med 2004; 10: 339.
  • 49
    De Cristofaro R, De Candia E. Thrombin domains: structure, function and interaction with platelet receptors. J Thromb Thrombolysis 2003; 15: 15163.
  • 50
    De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets. J Biol Chem 2001; 276: 46928.
  • 51
    Ramakrishnan V, DeGuzman F, Bao M, Hall SW, Leung LL, Phillips DR. A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V. Proc Natl Acad Sci USA 2001; 98: 18238.
  • 52
    Luo S-Z, Mo X, Afshar-Kharghan V, Srinivasan S, López JA, Li R. Glycoprotein Ibα forms disulfides with two glycoprotein Ibβ subunits in the resting platelet Blood 2006; 109: 6039.
  • 53
    Clemetson JM, Polgar J, Magnenat E, Wells TN, Clemetson KJ. The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcαR and the natural killer receptors. J Biol Chem 1999; 274: 2901924.
  • 54
    Jandrot-Perrus M, Busfield S, Lagrue AH, Xiong X, Debili N, Chickering T, Le Couedic JP, Goodearl A, Dussault B, Fraser C, Vainchenker W, Villeval JL. Cloning, characterization, and functional studies of human and mouse glycoprotein VI: a platelet-specific collagen receptor from the immunoglobulin superfamily. Blood 2000; 96: 1798807.
  • 55
    Bori-Sanz T, Suzuki-Inoue K, Berndt MC, Watson SP, Tulasne D. Delineation of the region in the glycoprotein VI tail required for association with the Fc receptor γ-chain. J Biol Chem 2003; 278: 3591422.
  • 56
    Locke D, Liu C, Peng X, Chen H, Kahn ML. Fc Rγ-independent signalling by the platelet collagen receptor glycoprotein VI. J Biol Chem 2003; 278: 154418.
  • 57
    Chen H, Locke D, Liu Y, Liu C, Kahn ML. The platelet receptor GPVI mediates both adhesion and signalling responses to collagen in a receptor density-dependent fashion. J Biol Chem 2002; 277: 30119.
  • 58
    Best D, Senis YA, Jarvis GE, Eagleton HJ, Roberts DJ, Saito T, Jung SM, Moroi M, Harrison P, Green FR, Watson SP. GPVI levels in platelets: relationship to platelet function at high shear. Blood 2003; 102: 28118.
  • 59
    Sarratt KL, Chen H, Zutter MM, Santoro SA, Hammer DA, Kahn ML. GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow. Blood 2005; 106: 126877.
  • 60
    Bigalke B, Lindemann S, Ehlers R, Seizer P, Daub K, Langer H, Schonberger T, Kremmer E, Siegel-Axel D, May AE, Gawaz M. Expression of platelet collagen receptor glycoprotein VI is associated with acute coronary syndrome. Eur Heart J 2006; 27: 21659.
  • 61
    Suzuki-Inoue K, Tulasne D, Bori-Sanz T, Inoue O, Jung SM, Moroi M, Shen Y, Andrews RK, Berndt MC, Watson SP. Association of Fyn and Lyn with the proline rich domain of GPVI regulates intracellular signalling. J Biol Chem 2002; 277: 215616.
  • 62
    Polgar J, Clemetson JM, Kehrel BE, Wiedemann M, Magnenat EM, Wells TN, Clemetson KJ. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem 1997; 272: 1357683.
  • 63
    Andrews RK, Gardiner EE, Asazuma N, Berlanga O, Tulasne D, Nieswandt B, Smith AI, Berndt MC, Watson SP. A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI. J Biol Chem 2001; 276: 280927.
  • 64
    Andrews RK, Munday AD, Mitchell CA, Berndt MC. Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib–IX–V complex. Blood 2001; 98: 6817.
  • 65
    Andrews RK, Suzuki-Inoue K, Shen Y, Tulasne D, Watson SP, Berndt MC. Interaction of calmodulin with the cytoplasmic domain of platelet glycoprotein VI. Blood 2002; 99: 421921.
  • 66
    Coller BS, Kalomiris E, Steinberg M, Scudder LE. Evidence that glycocalicin circulates in normal plasma. J Clin Invest 1984; 73: 7949.
  • 67
    Arthur JF, Matzaris M, Gardiner EE, Taylor SG, Wijeyewickrema LC, Ozaki Y, Kahn ML, Andrews RK, Berndt MC. Glycoprotein (GP)VI is associated with GPIb-IX-V on the membrane of resting and activated platelets. Thromb Haemostas 2005; 93: 71623.
  • 68
    Nieswandt B, Bergmeier W, Schulte V, Rackebrandt K, Gessner JE, Zirngibl H. Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRγ chain. J Biol Chem 2000; 275: 239984002.
  • 69
    Boylan B, Berndt MC, Kahn ML, Newman PJ. Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivo effectiveness of anti-human platelet agents. Blood 2006; 108: 90814.
  • 70
    Andre P, Prasad KS, Denis CV, He M, Papalia JM, Hynes RO, Phillips DR, Wagner DD. CD40L stabilizes arterial thrombi by a β3 integrin-dependent mechanism. Nat Med 2002; 8: 24752.
  • 71
    Brass LF, Jiang H, Wu J, Stalker TJ, Zhu L. Contact-dependent signaling events that promote thrombus formation. Blood Cells Mol Dis 2006; 36: 15761.
  • 72
    Goll DE, Thompson VF, Li H, Wei WEI, Cong J. The calpain system. Physiol Rev 2003; 83: 731801.
  • 73
    Franco SJ, Huttenlocher A. Regulating cell migration: calpains make the cut. J Cell Sci 2005; 118: 382938.
  • 74
    Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilagyi A, Banoczi Z, Hudecz F, Friedrich P. On the sequential determinants of calpain cleavage. J Biol Chem 2004; 279: 2077585.
  • 75
    Wang KK, Villalobo A, Roufogalis BD. Calmodulin-binding proteins as calpain substrates. Biochem J 1989; 262: 693706.
  • 76
    Satish L, Blair HC, Glading A, Wells A. Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of μ-calpain. Mol Cell Biol 2005; 25: 192241.
  • 77
    Taylor RG, Christiansen JA, Goll DE. Immunolocalization of the calpains and calpastatin in human and bovine platelets. Biomed Biochim Acta 1991; 50: 4918.
  • 78
    Croall DE, DeMartino GN. Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol Rev 1991; 71: 81347.
  • 79
    Schoenwaelder SM, Yuan Y, Cooray P, Salem HH, Jackson SP. Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin αIIbβ3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots. J Biol Chem 1997; 272: 1694702.
  • 80
    Fox JE. Cytoskeletal proteins and platelet signalling. Thromb Haemost 2001; 86: 198213.
  • 81
    Bialkowska K, Kulkarni S, Du X, Goll DE, Saido TC, Fox JE. Evidence that β3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active. J Cell Biol 2000; 151: 68596.
  • 82
    Wee JL, Jackson DE. The Ig-ITIM superfamily member PECAM-1 regulates the “outside-in” signalling properties of integrin αIIbβ3 in platelets. Blood 2005; 106: 381623.
  • 83
    Rathore V, Stapleton MA, Hillery CA, Montgomery RR, Nichols TC, Merricks EP, Newman DK, Newman PJ. PECAM-1 negatively regulates GPIb/V/IX signalling in murine platelets. Blood 2003; 102: 365864.
  • 84
    Falati S, Patil S, Gross PL, Stapleton M, Merrill-Skoloff G, Barrett NE, Pixton KL, Weiler H, Cooley B, Newman DK, Newman PJ, Furie BC, Furie B, Gibbins JM. Platelet PECAM-1 inhibits thrombus formation in vivo. Blood 2006; 107: 53541.
  • 85
    Thai le M, Ashman LK, Harbour SN, Hogarth PM, Jackson DE. Physical proximity and functional interplay of PECAM-1 with the Fc receptor FcγRIIa on the platelet plasma membrane. Blood 2003; 102: 363745.
  • 86
    Shattil SJ, Newman PJ. Integrins: dynamic scaffolds for adhesion and signalling in platelets. Blood 2004; 104: 160615.
  • 87
    Du X, Saido TC, Tsubuki S, Indig FE, Williams MJ, Ginsberg MH. Calpain cleavage of the cytoplasmic domain of the integrin β3 subunit. J Biol Chem 1995; 270: 2614651.
  • 88
    Xi X, Bodnar RJ, Li Z, Lam SC, Du X. Critical roles for the COOH-terminal NITY and RGT sequences of the integrin β3 cytoplasmic domain in inside-out and outside-in signalling. J Cell Biol 2003; 162: 32939.
  • 89
    Pfaff M, Du X, Ginsberg MH. Calpain cleavage of integrin β cytoplasmic domains. FEBS Lett 1999; 460: 1722.
  • 90
    Evans BJ, McDowall A, Taylor PC, Hogg N, Haskard DO, Landis RC. Shedding of lymphocyte function-associated antigen-1 (LFA-1) in a human inflammatory response. Blood 2006; 107: 35939.
  • 91
    Hemler ME. Shedding of heterodimeric leukocyte integrin. Blood 2006; 107: 34178.
  • 92
    Langer H, May AE, Bultmann A, Gawaz M. ADAM15 is an adhesion receptor for platelet GPIIb-IIIa and induces platelet activation. Thromb Haemost 2005; 94: 55561.
  • 93
    Maxwell MJ, Westein E, Nesbitt WS, Giuliano S, Dopheide SM, Jackson SP. Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation. Blood 2007; 109: 56676.
  • 94
    Kulkarni S, Dopheide SM, Yap CL, Ravanat C, Freund M, Mangin P, Heel KA, Street A, Harper IS, Lanza F, Jackson SP. A revised model of platelet aggregation. J Clin Invest 2000; 105: 78391.
  • 95
    Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000; 106: 38592.
  • 96
    Ni H, Yuen PS, Papalia JM, Trevithick JE, Sakai T, Fassler R, Hynes RO, Wagner DD. Plasma fibronectin promotes thrombus growth and stability in injured arterioles. Proc Natl Acad Sci USA 2003; 100: 24159.
  • 97
    Denis C, Methia N, Frenette PS, Rayburn H, Ullman-Cullere M, Hynes RO, Wagner DD. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci USA 1998; 95: 95249.
  • 98
    Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 2002; 8: 117581.
  • 99
    Celi A, Merrill-Skoloff G, Gross P, Falati S, Sim DS, Flaumenhaft R, Furie BC, Furie B. Thrombus formation: direct real-time observation and digital analysis of thrombus assembly in a living mouse by confocal and widefield intravital microscopy. J Thromb Haemost 2003; 1: 608.