Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo

Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers integrin-dependent adhesion and aggregation of platelets. The role of platelet beta1 integrins in these processes remains mostly undefined. Here, we demonstrate by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in alpha2-null mice and even in mice with a Cre/loxP-mediated loss of all beta1 integrins on their platelets. In contrast, inhibition of alphaIIbbeta3 integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, alphaIIbbeta3 inhibition had a comparable effect in alpha2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional alpha2beta1 and alphaIIbbeta3. These were identified to be alpha5beta1 and/or alpha6beta1 as alphaIIbbeta3 inhibition abrogated platelet adhesion in beta1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin-ligand interactions, none of which by itself is essential.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow

Resting platelet adhesion to inflammatory vascular endothelium is thought to play a causal role in secondary thrombus formation or microcirculatory disturbance after vessel occlusion. However, though adhesion receptors involved in platelet-matrix interactions have been extensively studied, the molecular mechanisms involved in platelet-endothelium interactions are incompletely characterized and have been mainly studied under static conditions. Using human platelets or platelets from wild-type and CD47-/- mice in whole blood, we demonstrated that at low shear rate, CD47 expressed on human and mouse platelets significantly contributes to platelet adhesion on tumor necrosis factor-alpha (TNF-alpha)-stimulated vascular endothelial cells. Using the CD47 agonist peptide 4N1K and blocking monoclonal antibodies (mAbs), we showed that CD47 binds the cell-binding domain (CBD) of endothelial thrombospondin-1 (TSP-1), inducing activation of the platelet alphaIIbbeta3 integrin that in turn becomes able to link the endothelial receptors intercellular adhesion molecule 1 (ICAM-1) and alphavbeta3. Platelet CD36 and GPIbalpha are also involved because platelet incubation with blocking mAbs directed against each of these 2 receptors significantly decreased platelet arrest. Given that anti-CD47 treatment of platelets did not further decrease the adhesion of anti-CD36-treated platelets and CD36 is a TSP-1 receptor, it appears that CD36/TSP-1 interaction could trigger the CD47-dependent pathway. Overall, CD47 antagonists may be potentially useful to inhibit platelet adhesion on inflamed endothelium.     

CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation

CD40 is a 48-kDa phosphorylated transmembrane glycoprotein belonging to the TNF receptor superfamily. CD40 has been demonstrated on a range of cell types, and it has an important role in adaptive immunity and inflammation. CD40 has recently been described on platelets but platelet activation by CD40 has not been described. In the present study, we use flow cytometry and immunoblotting to confirm that platelets constitutively express surface CD40. CD40 mRNA was undetectable, suggesting that the protein is synthesized early in platelet differentiation by megakaryocytes. Ligation of platelet CD40 with recombinant soluble CD40L trimer (sCD40LT) caused increased platelet CD62P expression, alpha-granule and dense granule release, and the classical morphological changes associated with platelet activation. CD40 ligation also caused beta3 integrin activation, although this was not accompanied by platelet aggregation. These actions were abrogated by the CD40L blocking antibody TRAP-1 and the CD40 blocking antibodies M2 and M3, showing that activation was mediated by CD40L binding to platelet CD40. beta3 integrin blockade with eptifibatide had no effect, indicating that outside-in signaling via alphaIIbbeta3 was not contributing to these CD40-mediated effects. CD40 ligation led to enhanced platelet-leukocyte adhesion, which is important in the recruitment of leukocytes to sites of thrombosis or inflammation. Our results support a role for CD40-mediated platelet activation in thrombosis, inflammation, and atherosclerosis.     

Activation of alphaIIbbeta3 is a sufficient but also an imperative prerequisite for activation of alpha2beta1 on platelets

Platelet integrins alpha2beta1 and alphaIIbbeta3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin alphaIIbbeta3 is not only sufficient, but also a prerequisite for alpha2beta1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of alpha2beta1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of alphaIIbbeta3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful alpha2beta1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of alphaIIbbeta3 for proper alpha2beta1 activation on platelets was demonstrated by using the alphaIIbbeta3 antagonist aggrastat, which was able to completely abolish alpha2beta1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking alphaIIbbeta3 confirmed the indispensability of alphaIIbbeta3 for alpha2beta1 activation.     

Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation.

Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of alphaIIbbeta3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin alpha2beta1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP-/- mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of alpha2beta1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and alpha2beta1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.     

UV-C irradiation disrupts platelet surface disulfide bonds and activates the platelet integrin alphaIIbbeta3

UV-C irradiation has been shown to be effective for pathogen reduction in platelet concentrates, but preliminary work indicated that UV-C irradiation of platelets can induce platelet aggregation. In this study, the mechanism underlying this phenomenon was investigated. Irradiation of platelets with UV-C light (1500 J/m(2)) caused platelet aggregation, which was dependent on integrin alphaIIbbeta3 activation (GPIIb/IIIa). This activation occurred despite treatment with several signal transduction inhibitors known to block platelet activation. UV-C also induced activation of recombinant alphaIIbbeta3 in Chinese hamster ovary (CHO) cells, an environment in which physiologic agonists fail to activate. Activation of alphaIIbbeta3 requires talin binding to the beta3 tail, yet alphaIIbbeta3-Delta724 (lacking the talin binding site) was activated by UV-C irradiation, excluding a requirement for talin binding. The UV-C effect appears to be general in that beta(1) and beta(2) integrins are also activated by UV-C. To explain these findings, we investigated the possibility of UV-C-induced photolysis of disulfide bonds, in analogy with the activating effect of reducing agents on integrins. Indeed, UV-C induced a marked increase in free thiol groups in platelet surface proteins including alphaIIbbeta3. Thus, UV-C appears to activate alphaIIbbeta3 not by affecting intracellular signal transduction, but by reduction of disulfide bonds regulating integrin conformation.     

Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state

Platelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.     

Calreticulin-independent regulation of the platelet integrin alphaIIbbeta3 by the KVGFFKR alphaIIb-cytoplasmic motif

The platelet integrin alphaIIbbeta3 alters conformation in response to platelet activation and ligand binding, although the molecular mechanisms involved are not known. We previously showed that a lipid modified peptide, corresponding to the membrane proximal 989KVGFFKR995 portion of the alphaIIb cytoplasmic tail, independently activates platelet alphaIIbbeta3. Calreticulin (CRT) is a potential integrin regulatory protein based on its interaction with the highly conserved alpha-integrin sequence KxGFFKR. We therefore examined the possible interaction of calreticulin and alphaIIbbeta3 in human platelets. We demonstrate that calreticulin in platelets is localised to the granulomere. In contrast, the known integrin-binding protein talin accumulates at the periphery of spreading platelets and colocalises with alphaIIbbeta3 during the process of adhesion. An interaction between calreticulin and alphaIIbbeta3 could not be demonstrated using co-immunoprecipitation techniques under various platelet activation states, even in the presence of covalent chemical crosslinkers. Thus, calreticulin does not functionally interact with the major integrin in human platelets. In order to identify proteins that interact with the integrin KVGFFKR motif we then used a peptide 'pull-down' assay from platelet lysates with biotinylated peptides and demonstrate that only the alphaIIb and beta3 subunits selectively and individually interact with this sequence. This interaction is divalent cation-dependent, has high-affinity, and occurs both with purified alphaIIbbeta3 complex and with electroeluted alpha and beta subunits. Thus, our data show that the conserved integrin KVGFFKR domain interacts primarily with the alpha and beta cytoplasmic tails and not with CRT in human platelets.     

Soluble CD40 ligand induces beta3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling

We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of alphaIIbbeta3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the alphaIIbbeta3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a alphaIIbbeta3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40-/- platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of beta3. Platelets from the diYF mouse strain, expressing beta3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to alphaIIbbeta3, triggering outside-in signaling by tyrosine phosphorylation of beta3.     

The tetraspanin superfamily member CD151 regulates outside-in integrin alphaIIbbeta3 signaling and platelet function

The tetraspanin family member CD151 forms complexes with integrins and regulates cell adhesion and migration. While CD151 is highly expressed in megakaryocytes and to a lesser extent in platelets, its physiologic role in platelets is unclear. In this study, we investigate the physical and functional importance of CD151 in murine platelets. Immunoprecipitation/Western blot studies reveal a constitutive physical association of CD151 with integrin alpha(IIb)beta(3) complex under strong detergent conditions. Using CD151-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta(3) signaling with defective platelet aggregation responses to protease-activated receptor 4 (PAR-4) agonist peptide, collagen, and adenosine diphosphate (ADP); impaired platelet spreading on fibrinogen; and delayed kinetics of clot retraction in vitro. This functional integrin alpha(IIb)beta(3) defect could not be attributed to altered expression of integrin alpha(IIb)beta(3). CD151(-/-) platelets displayed normal platelet alpha granule secretion, dense granule secretion, and static platelet adhesion. In addition, CD151(-/-) platelets displayed normal "inside-out" integrin alpha(IIb)beta(3) signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol 1,4,5 triphosphate (IP(3)) levels. This study provides the first direct evidence that CD151 is essential for normal platelet function and that disruption of CD151 induced a moderate outside-in integrin alpha(IIb)beta(3) signaling defect.     

ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib-IX-V and other agonists

Interaction between von Willebrand factor (VWF) and platelet GP Ib-IX-V is required for hemostasis, in part because intracellular signals from VWF/GP Ib-IX-V activate the ligand-binding function of integrin alphaIIbbeta3. Because they also induce tyrosine phosphorylation of the ADAP adapter, we investigated ADAP's role in GP Ib-IX-V signal transduction. Fibrinogen or ligand-mimetic POW-2 Fab binding to alphaIIbbeta3 was stimulated by adhesion of ADAP+/+ murine platelets to dimeric VWF A1A2 but was significantly reduced in ADAP-/- platelets (P<.01). alphaIIbbeta3 activation by ADP or a Par4 thrombin receptor agonist was also decreased in ADAP-/- platelets. ADAP stabilized the expression of another adapter, SKAP-HOM, via interaction with the latter's SH3 domain. However, no abnormalities in alphaIIbbeta3 activation were observed in SKAP-HOM-/- platelets, which express normal ADAP levels, further implicating ADAP as a modulator of alphaIIbbeta3 function. Under shear flow conditions over a combined surface of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and alphaIIbbeta3, respectively, ADAP-/- platelets displayed reduced alphaIIbbeta3-dependent stable adhesion. Furthermore, ADAP-/- mice demonstrated increased rebleeding from tail wounds. These studies establish ADAP as a component of inside-out signaling pathways that couple GP Ib-IX-V and other platelet agonist receptors to alphaIIbbeta3 activation.     

New insights on disintegrin-receptor interactions: eristostatin and melanoma cells

Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors alphavbeta3, alpha5beta1 or alphaIIbbeta3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to alphaIIbbeta3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified.     

Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin alpha2beta1

Integrin alpha2beta1 is the principal adhesive receptor for collagen but platelets also adhere through glycoprotein VI (GPVI). Integrin alphaIIbbeta3 may augment platelet adhesion. We have shown that disulfide exchange is necessary for platelet adhesion to fibrinogen, fibronectin, and collagen. However 2 questions remained: (1) Can activated alphaIIbbeta3 explain the observed role of disulfide exchange in adhesion to collagen, or is this role common to other integrins? (2) Is disulfide dependence specific to the integrin receptors or shared with GPVI? To discriminate adhesive functions of alpha2beta1 from those of alphaIIbbeta3 we used Glanzmann platelets and alphaIIbbeta3-specific antibodies applied to normal platelets. To resolve adhesive events mediated by alpha2beta1 from those of GPVI we used synthetic peptides specific to each receptor. We addressed direct integrin ligation using purified alpha2beta1 and recombinant I domain. We observed the following: adhesion to the alpha2beta1-specific peptide was disulfide-exchange dependent and protein disulfide isomerase (PDI) mediated; membrane-impermeant thiol blockers inhibited alpha2beta1, but not GPVI mediated, adhesion; direct blockade of PDI revealed that it is involved in adhesion through alpha2beta1 but not GPVI; and purified alpha2beta1, but not recombinant I domain, depended on free thiols for ligation. These data suggest that the enzymatically catalyzed adhesion-associated reorganization of disulfide bonds is common to members of the integrin family and specific to this family.     

Characterization of a genetic difference in the platelet aggregation response of two inbred mouse strains, C57BL/6 and C3H/He

An in vitro platelet aggregation assay has been used to characterize the platelet response of two inbred mouse strains, C57BL/6J and C3H/J. Arachidonic acid, ADP, ristocetin, and collagen, but not epinephrine, were effective inducers of platelet aggregation in C57BL/6 mice. In C3H mice, however, platelets responded differently to some, but not all, inducers of platelet aggregation. The difference between the two strains was particularly striking for arachidonic acid; at 0.375 microM arachidonic acid, platelets from C57BL/6 mice aggregated well, but platelets from C3H mice failed to aggregate. The two strains also differed in serotonin release from platelets. When the pool of platelet serotonin was labeled by incubating platelets with [14C]serotonin, the amount of label subsequently released from platelets in response to 0.375 microM arachidonic acid was 20.5% +/- 2.5 SE for C57BL/6 mice and 1.5% +/- 1.5 SE for C3H mice. The platelets from F1 progeny of a cross between C57BL/6 and C3H mice were indistinguishable in aggregation response from the C57BL/6 parent. The defect in aggregation response found in C3H mice appears to reside in the platelets rather than in the plasma. Experiments which involved adding the plasma of one strain to the washed platelets of the other strain indicated that C57BL/6 platelets aggregated well whether they were resuspended in plasma from C57BL/6 or C3H mice, while C3H platelets failed to aggregate regardless of the origin of the plasma. Strain C57BL/6 is susceptible to diet-induced formation of atherosclerotic lesions, but strain C3H is resistant to lesion formation. This genetic difference in atherosclerosis susceptibility is not related to the genetic difference in platelet aggregation since a recombinant inbred strain, BXH-9, is susceptible to atherosclerosis like the C57BL/6 parent but has the reduced platelet aggregation response of the C3H parent.     

Platelet and osteoclast beta3 integrins are critical for bone metastasis

Mice with a targeted deletion of beta3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of beta3+/+ mice by 14 days. In contrast, only 4% of beta3-/- mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in beta3-/- mice, but no associated trabecular bone resorption as seen inbeta3+/+ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrow-derived cell. To dissect the roles of osteoclast and platelet beta3 integrins in this model of bone metastasis, osteoclast-defective src-/- mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated alphaIIbbeta3 prevented B16 metastases. These data demonstrate a critical role for platelet alphaIIbbeta3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors.     

Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.     

Differential roles of integrins alpha2beta1 and alphaIIbbeta3 in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha2beta1 and possibly to other receptors. In this study, we have compared the role of integrins alpha2beta1 and alphaIIbbeta3 in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A2 (TxA2) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alphaIIbbeta3 activation. This adhesion was substantially mediated by alpha2beta1. The alphaIIbbeta3 antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alphaIIbbeta3 outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha2beta1 and alphaIIbbeta3 in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA2 greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha2beta1.     

Role of alphavbeta6 integrin in acute biliary fibrosis

Acute biliary obstruction leads to periductal myofibroblasts and fibrosis, the origin of which is uncertain. Our study provides new information on this question in mice and humans. We show that bile duct obstruction induces a striking increase in cholangiocyte alphavbeta6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF-beta). Administration of blocking antibody to alphavbeta6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in beta6-null mice subjected to the injury, fibrosis is reduced by 50% relative to that seen in wild-type mice, whereas inflammation occurs to the same extent. The data indicate that alphavbeta6, rather than inflammation, is linked to fibrogenesis. It is known that alphavbeta6 binds latent TGF-beta and that binding results in release of active TGFbeta. Consistent with this, intracellular signaling from the TGFbeta receptor is increased after bile duct ligation in wild-type mice but not in beta6(-/-) mice, and a competitive inhibitor of the TGFbeta receptor type II blocks fibrosis to the same extent as antibody to alphavbeta6. In a survey of human liver disease, expression of alphavbeta6 is increased in acute, but not chronic, biliary injury and is localized to cholangiocyte-like cells. CONCLUSION: Cholangiocytes respond to acute bile duct obstruction with markedly increased expression of alphavbeta6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of alphavbeta6 integrin or TGFbeta for down-regulating fibrosis in the setting of acute or ongoing biliary injury.