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PubMed1998

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen.

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2003

Modulation of matrix metalloproteinase-9 in hepatic stellate cells by three-dimensional type I collagen: its activation and signaling pathway.

Background/AIMS: Hepatic stellate cells (HSCs) play a key role in the production and degradation of extracellular matrix (ECM) in the liver. In the present study, we investigated the interaction between ECM and HSCs in vitro with emphasis on the modulation of matrix metalloproteinases (MMPs) by ECM. METHODS: Freshly isolated rat HSCs were cultured in several conditions on type I collagen- or matrigel-coated dishes, on thick matrigel or in three-dimensional type I collagen (3D-gel), and MMPs expression in HSCs was examined. In addition, activation and signaling pathway of MMP-9 expression modulated by 3D-gel in HSCs were examined. RESULTS: Increased expression of MMP-3, -9, -13 and -14 was markedly detected only in the 3D-gel-treated HSCs. Zymography demonstrated that only 3D-gel-treated cells showed active gelatinase activity of MMP-9 at 82 kDa. MMP-9 expression was inhibited by neutralizing antibody against integrin alpha2beta1, tyrosine kinase inhibitors, or MEK1,2 inhibitor PD 98059, but not by p38 inhibitor SB 203580. Western blotting also showed phosphorylated p38, ERK1,2, and JUN/SAPK was quickly induced in HSCs by 3D-gel. CONCLUSIONS: MMP-9 expression and activation is induced in HSCs by 3D-gel and this observed collagen-dependent induction of MMP-9 requires ERK1,2 activity.

Journal Article↗
PubMed2003

Structural characterization of EMS16, an antagonist of collagen receptor (GPIa/IIa) from the venom of Echis multisquamatus.

Snake venoms contain a number of hemostatically active C-type lectin-like proteins (CLPs), which affect the blood coagulation system, endothelial cells, and platelets. CLPs have broad similarities in structure and possess distinct biological functions. EMS16, a CLP from Echis multisquamatus venom, which is a potent and selective inhibitor of the collagen receptor, glycoprotein Ia/IIa (integrin alpha2beta1), has been used in the present study to examine structure-function relationships in venom CLPs by X-ray crystallography. The structure of EMS16, determined at a resolution of 1.9 A, revealed a heterodimer involved with domain swapping of the central loop as observed in the structures of other CLPs. A part of the glycan was observed and identified as N-acetyl-D-glucosamine (GlcNAc) in the electron density map at Asn21 of subunit B, an expected glycosylation site. EMS16 had a unique, positively charged electrostatic potential patch on the concave surface that may qualify as a site for interaction with the I-domain of the glycoprotein Ia/IIa.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of peptide-amphiphiles possessing cellular activation sequences.

Numerous approaches have been described for modifying biomaterials to incorporate extracellular matrix components. "Peptide-amphiphiles", whereby monoalkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to (a) form specific molecular architecture with enhanced stability and (b) promote cell adhesion, spreading, and signaling. The present study has examined the use of chimeric peptide-amphiphiles for inducing protein-like structures and peptide-amphiphile mixtures for enhancing surface bioactivity. The alpha-helical propensity of a 21 residue peptide, incorporating the SPARC(119-122) angiogenesis-inducing sequence and either unmodified or acylated with a C(6), C(10), C(14), C(16), C(18), C(18:1), or C(18:1-OH) monoalkyl hydrocarbon chain, has been examined. Peptide and peptide-amphiphile structures were characterized by circular dichroism and one- and two-dimensional NMR spectroscopic techniques. The 21 residue peptide alone does not form a distinct structure in solution, whereas N-terminal acylation by monoalkyl hydrocarbon chains results in the 21 residue peptide-amphiphile adopting a predominantly alpha-helical structure in solution. The thermal stability of the alpha-helix increases with increasing hydrocarbon chain length. The SPARC(119-122) peptide-amphiphiles were then screened for promotion of endothelial cell adhesion and spreading. The greatest activity was achieved by using a mixture of the alpha-helical SPARC(119-122) peptide-amphiphile, a triple-helical peptide-amphiphile incorporating the alpha2beta1 integrin binding site from type I collagen, and a pseudolipid. The pseudolipid is most likely required for a spatial distribution of the peptide-amphiphiles that allows for optimal cellular interactions. Overall, we have found that incorporation of bioactive sequences within peptide-amphiphiles results in the induction of an ordered structure of the bioactive sequence and that mixtures of peptide-amphiphiles can be used to promote endothelial cell behaviors comparable to extracellular matrix components.

Amino Acid Sequence↗
PubMed2002

The regulation of prostate cancer cell adhesion to human bone marrow endothelial cell monolayers by androgen dihydrotestosterone and cytokines.

A previous study from our laboratory suggested that prostate cancer metastasis to bone may be mediated, in part, by preferential adhesion to human bone marrow endothelial (HBME) cells. Tumor cell adhesion to endothelial cells may be modulated by the effect of cytokines on cell adhesion molecules (CAMs). Tumor necrosis factor-alpha (TNF-alpha) regulates VCAM expression on the endothelium and this effect is enhanced by dihydrotestosterone (DHT). Transforming growth factor-beta (TGF-beta) stimulates the expression of alpha2beta1 integrin on PC-3 cells. The current study investigated the effects of the above cytokines and DHT (singularly and in various combinations) upon HBME and prostate cancer cell expression of VCAM, alpha2 integrin subunit, and beta1 integrin subunit by flow cytometry. We also monitored the effects of the above treatments on PC-3 cell adhesion to HBME monolayers. The data demonstrate that none of the treatments significantly altered the expression of selected CAMs on HBME cell and neoplastic prostate cell lines. The treatment of HBME monolayers with various combinations of cytokines and DHT prior to performing adhesion assays with PC-3 demonstrates that treatments containing TGF-beta reduced PC-3 cell adhesion to HBME monolayers by 32% or greater (P < 0.05). The reduction in PC-3 cell adhesion to TGF-beta-treated HBME monolayers was dose dependent. Interestingly, LNCaP cells but not PC-3 cells treated with TGF-beta had a reduced ability to adhere to untreated HBME monolayers. These results suggest that TGF-beta may reduce tumor cell adhesion to bone marrow microvascular endothelium, in vivo. The biological significance of this observation is discussed.

Adenocarcinoma↗
PubMed2002

Differential effects of reduced glycoprotein VI levels on activation of murine platelets by glycoprotein VI ligands.

We have investigated the effects of decreased levels of the complex between glycoprotein VI (GPVI) and the Fc receptor gamma-chain (FcRgamma) on responses to collagen and GPVI-specific ligands in murine platelets. We show that levels of GPVI-FcRgamma of the order of 50% and 20% of wild-type levels caused 2- and 5-fold shifts to the right respectively in the dose-response curve for aggregation in response to collagen, the snake toxin convulxin and the monoclonal antibody JAQ1. In addition, there is a delay in the onset of aggregation in response to collagen. In contrast, the stimulation of protein tyrosine phosphorylation by collagen (as measured after 150 s) and adhesion to a collagen-coated surface under static conditions were unaffected in platelets with 50% and 20% of wild-type levels of GPVI. In contrast, responses to a collagen-related peptide (CRP), made up of repeat glycine-proline-hydroxyproline motifs, were markedly inhibited and abolished in platelets expressing 50% and 20% of wild-type levels of GPVI respectively. We suggest that the marked effect of a reduction in GPVI levels on the CRP-induced activation of platelets is due to the multivalent nature of CRP and the fact that GPVI is its sole receptor on platelets. Thus it appears that the interaction of CRP with GPVI is determined by a combination of affinity and avidity. The observation that collagen does not behave like CRP in platelets expressing reduced levels of GPVI, even in the combined presence of blocking antibodies against integrin alpha2beta1 and GPV, suggests that collagen has a greater affinity than CRP for GPVI, and/or that other receptors are involved in its binding to platelets. The clinical significance of these results is discussed.

Animals↗
PubMed2005

Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor.

The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin alpha2beta1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.

Amino Acid Sequence↗
PubMed1997

Identification of the epitope for a monoclonal antibody that blocks platelet aggregation induced by type III collagen.

A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with alpha2beta1-integrin of platelets.

Amino Acid Sequence↗
PubMed1998

Antigenic phenotype of early intra-embryonic lymphoid progenitors in the chicken.

The stem cells for the definitive haematopoiesis are derived from intra-embryonic sources originally described in an avian model and later also in mammals. However, the molecular make-up of the early embryonic haematopoietic progenitors is not yet clearly defined. We have recently characterized the phenotype of prethymic intra-embryonic progenitors capable of thymic colonization. Here we studied the ontogeny of cell-surface antigens HEMCAM, alpha2beta1 integrin, thrombomucin, chL12 and c-kit and their co-expression on prethymic T-cell progenitors. The early intra-embryonic expression of avian B-cell antigen chB6 was also demonstrated on cells derived from the intra-embryonic areas. We suggest that in the chicken, embryonic B-cell progenitors segregate earlier than T-cell progenitors in the differentiation of multipotent haematopoietic stem cells to committed progenitors.

Animals↗
PubMed2002

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

Antigenic Modulation↗
PubMed2003

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

Animals↗
PubMed2006

Genetics of diabetic retinopathy.

Familial aggregation as well as racial and ethnic differences in incidence suggest that genetic components play a role in the development of diabetic retinopathy (DR), several approaches have been used to identify genes contributing to the development of retinopathy. We searched the literature database using the keywords [diabetes], [gene], for publications dealing with retinopathy. 88 original publications reporting data on genetics of retinopathy were found. For the purpose of this review, a simple scoring system was applied, that results in a score for each considered gene to indicate its potential relevance in genetic control of retinopathy. Based on published studies, the most intriguing genes for further genetic studies are aldose receptor, advanced glycation end products receptor, vascular endothelial growth factor, intercellular adhesion molecule 1, beta3-adrenergic receptor gene, hemochromatosis, and alpha2beta1 integrin. Pathways involving these gene products may represent a fruitful area for further studies aimed at investigating the genetics and pathophysiology of DR. Meta-analyses of candidate gene studies may provide further useful insights into their role. In addition, our paper addresses several issues challenging genetic studies of retinopathy such as replication of associations, patient ascertainment schemes, or accurately defined phenotypes.

Aldehyde Reductase↗
PubMed2004

Intersubunit signal transmission in integrins by a receptor-like interaction with a pull spring.

The function of some multidomain proteins is regulated by interdomain communication. We use second-site suppressor cysteine mutations to test a hypothesis on how the inserted (I)-like domain in the integrin beta-subunit regulates ligand binding by the neighboring I domain in the integrin alpha-subunit [Huth, J. R., Olejniczak, E. T., Mendoza, R., Liang, H., Harris, E. A., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5231-5236; and Alonso, J. L., Essafi, M., Xiong, J. P., Stehle, T. & Arnaout, M. A. (2002) Curr. Biol. 12, R340-R342]. The hypothesis is that an interaction between the beta I-like metal ion-dependent adhesion site (MIDAS) and an intrinsic ligand in the linker following the alpha I domain, Glu-310, exerts a pull that activates the alpha I domain. Individual mutation of alpha(L) linker residue Glu-310 or beta(2) MIDAS residues Ala-210 or Tyr-115 to cysteine abolishes I domain activation, whereas the double mutation of alpha(L)-E310C with either beta(2)-A210C or beta(2)-Y115C forms a disulfide bond that constitutively activates ligand binding. The disulfide-bonded mutant is resistant to small molecule antagonists that bind to the beta I-like domain near its interface with the alpha I domain and inhibit communication between these domains but remains susceptible to small molecule antagonists that bind underneath the I domain alpha 7-helix and certain allosteric antagonistic antibodies. Thus, the alpha 7-helix and its linker are better modeled as a pull spring than a bell rope. The results suggest that alpha(L) residue Glu-310, which is universally conserved in all I domain-containing integrins, functions as an intrinsic ligand for the beta I-like domain, and that when integrins are activated, the beta I-like MIDAS binds to Glu-310, pulls the spring, and thereby activates the alpha I domain.

Animals↗
PubMed1997

Identification of cell binding sequences in mouse laminin gamma1 chain by systematic peptide screening.

Laminin-1, a major component of basement membranes, consists of three different chains designated alpha1, beta1, and gamma1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin gamma1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the gamma1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-alpha2beta1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin gamma2 chain. These results suggest that the specific sequences on the laminin gamma1 chain are biologically active and interact with distinct cell surface receptors.

Amino Acid Sequence↗
PubMed2002

Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1.

Polymerization of the ECM proteins fibronectin and laminin has been shown to take place in close vicinity to the cell surface and be facilitated by beta(1) integrins (Lohikangas, L., Gullberg, D., and Johansson, S. (2001) Exp. Cell Res. 265, 135-144 and Wennerberg, K., Lohikangas, L., Gullberg, D., Pfaff, M., Johansson, S., and Fassler, R. (1996) J. Cell Biol. 132, 227-238). We have studied the role of collagen receptors, integrins alpha(11)beta(1) and alpha(2)beta(1), and fibronectin in collagen polymerization using fibronectin-deficient mouse embryonic fibroblast cell lines. In contrast to the earlier belief that collagen polymerization occurs via self-assembly of collagen molecules we show that a preformed fibronectin matrix is essential for collagen network formation and that collagen-binding integrins strongly enhance this process. Thus, collagen deposition is regulated by the cells, both indirectly through integrin alpha(5)beta(1)-dependent polymerization of fibronectin and directly through collagen-binding integrins.

Animals↗
PubMed2004

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration.

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.

Acetylglucosamine↗
PubMed2004

Integrin-mediated cell adhesion to type I collagen fibrils.

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.

Animals↗
PubMed2004

Impaired platelet activation in familial high density lipoprotein deficiency (Tangier disease).

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.

ATP Binding Cassette Transporter 1↗