αvβ3 and αvβ5 integrin antagonists inhibit angiogenesis in vitro

Although angiogenesis is believed to require cell-extracellular matrix interactions which are mediated in part via integrins alphav beta 3 and alphav beta 5, a formal demonstration that alphav beta 3 and alphav beta 5 are involved in endothelial-cell invasion and capillary-like tube formation is still required. This has arisen from the cellular complexities which occur in vivo and the difficulty in finding appropriate in vitro model systems. Here we have used a three-dimensional assay which employs bovine aortic and microvascular endothelial cells, to show that alphav beta 3 and alphav beta 5 regulate angiogenesis in vitro. We cloned and characterized 350-450 bp regions of the bovine homologues of alphav, beta 3 and beta 5, covering much of the beta -propeller and A-domain regions, and show that they are >95% identical to their human orthologues. We used cyclic peptides EMD 121974, 85189 and 66203, which selectively inhibit alphav beta 3 and alphav beta 5, but not gpIIbIIIa or alpha5 beta 1, to probe in vitro angiogenesis induced by angiogenic cytokines in three-dimensional fibrin or collagen gels. We found that these peptides are potent inhibitors of endothelial cell invasion and differentiation induced by vascular endothelial growth factor-A or fibroblast growth factor-2 but do not affect the unstimulated cells in 3D culture. Inhibition was greatest when cells were grown on fibrin, but also occurred on collagen I which is not a recognized ligand for alphav beta 3. These findings demonstrate the requirement for endothelial cell alphav beta 3 and alphav beta 5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of alphav beta 3 and alphav beta 5 antagonists.     

Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

Herpes simplex virus (HSV) is an important human pathogen. It enters cells through an orchestrated process that requires four essential glycoproteins, gD, gH/gL, and gB, activated in cascade fashion by receptor-binding and signaling. gH/gL heterodimer is conserved across the Herpesviridae family. HSV entry is enabled by gH/gL interaction with αvβ6- or αvβ8-integrin receptors. We report that the interaction of virion gH/gL with integrins resulted in gL dissociation and its release in the medium. gL dissociation occurred if all components of the entry apparatus—receptor-bound gD and gB—were present and was prevented if entry was blocked by a neutralizing monoclonal antibody to gH or by a mutation in gH. We propose that (i) gL dissociation from gH/gL is part of the activation of HSV glycoproteins, critical for HSV entry; and (ii) gL is a functional inhibitor of gH and maintains gH in an inhibited form until receptor-bound gD and integrins signal to gH/gL.Entry of herpesviruses into the cell is an orchestrated process that necessitates a multipartite glycoprotein system, rather than one-two glycoproteins, as is the case for the vast majority of viruses (1–4). For herpes simplex virus (HSV), the entry-fusion apparatus consists of four essential glycoproteins —gD, the heterodimer gH/gL, and gB—plus cognate cellular receptors. gD is the major determinant of HSV tropism. Structurally, it exhibits an Ig-folded core with N- and C-terminal extensions (5, 6). The structure of the gH/gL heterodimer does not resemble that of any known protein (7–9). The binding site of gL in gH maps to the N-terminal domain I. Whether gH/gL has a profusion activity in itself or is only an intermediate between gD and gB in the chain of activation of the glycoproteins is an open question (10–13). gB is considered the fusogenic glycoprotein, based on structural features, including a trimeric fold and a bipartite fusion loop (14, 15). gH, gL, and gB constitute the conserved-entry glycoproteins across the Herpesviridae family. The ability of gH to heterodimerize with gL is also conserved across the family, highlighting that the heterodimeric structure is critical to the function of two glycoproteins.The prevailing model of HSV entry envisions that following a first virion attachment to cells mediated by heparan sulfate glycosaminoglycans, the interaction of gD with one of its receptors, nectin1 and HVEM (herpesvirus entry mediator), results in conformational changes to gD, in particular to the ectodomain C terminus, which harbors the profusion domain (5, 6, 16, 17). The activated gD recruits gH/gL, which, in turn, recruits gB. gB executes the virus–cell fusion (2–4, 18, 19). We observed that the glycoproteins are already in complex in resting virions (17, 18). In contrast with the view that glycoproteins are stepwise-recruited to a complex, we favor the view that the process of activation of the viral glycoproteins results from the interaction of preassembled glycoproteins’ complexes with cellular receptors and from a signaling cascade, which is likely triggered by receptor-induced conformational changes (18).The more speculative part of the HSV-entry model concerns the roles of gH and of gL and why gH has evolved to be a heterodimer with gL. Recently, we discovered that αvβ6- and αvβ8-integrins serve as interchangeable receptors for HSV gH/gL. They play two distinct roles in infection (20). They enable virus entry, as inferred by inhibition of infection following integrin depletion by siRNAs or exposure of cells to anti-integrin antibodies. Second, they promote HSV endocytosis into acidic endosomes (20); the latter function is nonessential because the virus may enter some cells also by fusion with plasma membranes or with neutral endosomes (21–23). Remarkably, the use of integrins as receptors is a common feature among herpesviruses. Integrins serve as receptors also for gH/gL of EBV (Epstein Barr virus), of human cytomegalovirus and equine herpesvirus, and for gB of Kaposi’s sarcoma-associated herpesvirus (24–28). Most likely, they play a common role.Here, we asked whether αvβ6- or αvβ8-integrin induce conformational changes to HSV gH/gL, as part of the process of glycoprotein activation in virus entry. We report that αvβ6- and αvβ8-integrin promote the dissociation of gL from gH/gL. Conditions for the dissociation were the presence of gD, its receptor nectin1, and gB, i.e., conditions that lead to activation of the entry machinery, including the virion glycoproteins.     

Lipopolysaccharide stimulates adrenal steroidogenesis in rodent cells by a NFκB-dependent mechanism involving COX-2 activation

Hydroxysteroid (17β) dehydrogenase 10 (HSD10), the HSD17B10 gene product, is a mitochondrial NAD(+)-dependent dehydrogenase. There are two outstanding features of this vital enzyme: (a) the versatility of its catalytic endowment is attributed to the flexibility of its active site to accommodate diverse substrates such as steroids, fatty acids, bile acid, and xenobiotics; (b) its capacity to bind other proteins and peptides. For example, it tightly binds with three identical subunits to compose a homotetramer. The homotetramer then binds with two other proteins, namely, RNA (guanine-9-)methyl-transferase domain containing-1 and KIAA0391, to form mitochondrial RNase P. Furthermore, various HSD10 functions are inhibited when the enzyme is bound by amyloid-β peptide or estrogen receptor alpha. Missense mutations of HSD10 may cause neurodegeneration related to HSD10 deficiency, whereas a silent mutation of HSD10 results in mental retardation, choreoathetosis and abnormal behavior (MRXS10). The clinical condition of some HSD10 patients mimics mitochondrial disorders. Since normal HSD10 function is essential for brain cognitive activity, elevated levels of HSD10 found in brains of Alzheimer disease (AD) patients and mouse AD model might counterbalance the inhibition of HSD10 by amyloid-β peptide. The investigation of HSD10 may lead to a better understanding of AD pathogenesis.     

Molecular basis for complement recognition by integrin αXβ2

Integrin α(X)β(2) functions as complement receptor for iC3b and mediates recognition and phagocytosis of pathogens. We used negative-stain EM to examine the α(X)β(2) interaction with iC3b. EM class averages of α(X)β(2) in complex with iC3b define the binding sites on both the integrin and iC3b. iC3b contains C3c and thioester domain moieties linked by a long flexible linker. The binding site is on the key ring of the C3c moiety, at the interface between the MG3 and MG4 domains. Similar complexes are seen between α(X)β(2) and the C3c fragment. α(X)β(2) binds through the α(X) αI domain, on the face known to bear the metal ion-dependent adhesion site, at the opposite end of the αI domain from its site of insertion in the β-propeller domain.     

Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbβ3

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet–bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbβ3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5’-diphosphate (ADP) and thromboxane A₂ (TxA₂) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.     

Differential roles of integrins α2β1 and αIIbβ3 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 α₂β₁ and possibly to other receptors. In this study, we have compared the role of integrins α₂β₁ and α_IIbβ₃ 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 α_IIbβ₃ activation. This adhesion was substantially mediated by α₂β₁. The α_IIbβ₃ antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that α_IIbβ₃ outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of α₂β₁ and α_IIbβ₃ 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 α₂β₁.     

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

Negative stain electron microscopy (EM) and adhesion assays show that α_Xβ₂ integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the β₂ leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that α_Xβ₂ in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.Integrins are a major family of αβ heterodimeric, cell surface adhesion receptors that transmit both chemical signals and mechanical forces bidirectionally across the plasma membrane (1). The integrin α and β subunits are each type I transmembrane proteins with large N-terminal extracellular domains, single-spanning transmembrane domains, and usually short C-terminal cytoplasmic domains (Fig. 1 A–C). The β₂ integrin subfamily, α_Dβ₂, α_Mβ₂ (CR3, Mac-1), α_Lβ₂ (LFA-1), and α_Xβ₂ (CR4, p150,95), is exclusively expressed on leukocytes. All β₂ integrins contain an inserted I domain in the α subunit, which is the ligand-binding domain. The head comprises the α-subunit β-propeller and αI domains, and the β-subunit βI domain (Fig. 1D). The upper legs comprise the α-subunit thigh domain, and the β-subunit PSI, hybrid, and I-EGF1 domains. The head and upper legs together are called the headpiece. The lower legs comprise the α-subunit calf-1 and calf-2 domains, and the β-subunit I-EGF domains 2–4 and β-tail domain (Fig. 1D).Open in a separate windowFig. 1.Integrin conformational states and preparation of α_xβ₂–Fab complexes. (A–C) Schematic of three conformational states of integrin. (D) Schematic of the α_xβ₂ ectodomain construct used in this study. (E and F) Ribbon diagrams of the α_xβ₂ ectodomain in extended-closed headpiece (E) and extended-open headpiece (F) conformations. Spheres show Cα atoms of residues to which epitopes are mapped. Epitopes are labeled in the headpiece conformation for which mAb are found to be specific in this study. Models are based on the bent α_xβ₂ crystal structure (4) and open α_IIbβ₃ headpiece (13). (G and H) Superdex 200 size exclusion chromatography profiles of α_xβ₂ in the absence and presence of the indicated Fabs.Crystal structures of the ectodomain of both αI-less (2, 3) and αI-containing integrins (4) revealed a bent conformation, in which the lower α and β legs fold back against the head and upper legs (Fig. 1A). It is believed that the bent conformation represents the physiological low affinity state, whereas during activation, integrins extend with a switchblade-like motion (5–9). EM images of the αI-less integrin, α_Vβ₃, showed the extended conformation upon activation by Mn²⁺ or ligand binding (6).Over the years, an array of mAbs has been developed that can inhibit, induce, or report β₂ integrin activation on cell surfaces. Therefore, correlating the functional effects of mAbs on intact cells with their effects on integrin conformational changes revealed by EM provides a powerful method for studying the mechanism of integrin activation. mAbs that bind to epitopes that are buried in the bent conformation can induce extension, as found with CBR LFA-1/2 Fab to I-EGF3; or report extension, as found with KIM127 Fab to I-EGF2 (5, 8, 10). Upon extension, two headpiece conformations were observed: closed, as when bent (Fig. 1 B and E), and open, with the hybrid domain swung out (Fig. 1 C and F) (6, 8).The open headpiece conformation has been widely speculated to be the activated state with high affinity for ligand, because ligand binding induces and stabilizes the open headpiece conformation (6, 11–13). Moreover, mutations that stabilize the open headpiece conformation activate ligand binding (14, 15). However, direct evidence for distinct functional roles for the extended conformation with the closed headpiece (Fig. 1B) and the extended conformation with the open headpiece (Fig. 1C) is yet to be acquired for any integrin. Furthermore, there has been much speculation about an intermediate affinity state between the bent, low affinity and the activated, high affinity states. At the leading edge of migrating T lymphocytes, a distinct population of LFA-1 was defined as in an intermediate affinity state by the exposure of different monoclonal antibody epitopes (16). Lateral mobility measurements of LFA-1 on T cell surfaces suggested an intermediate conformational state with distinct diffusion profile (17). In vitro flow chamber experiments showed that an intermediate affinity of LFA-1 on lymphocytes could be induced by immobilized chemokine (18). Moreover, different affinity and conformational states of LFA-1 have been suggested on the basis of differential effects of antibodies and metal ions on binding to the higher affinity ligand ICAM-1 and the lower affinity ligand ICAM-3 (19–22). Nevertheless, it remains unclear which conformational state corresponds to the observed intermediate affinity and what affinity state the extended-closed headpiece conformation represents. Finally, the adhesiveness of the bent conformation, the extended-closed headpiece conformation, and the extended-open headpiece conformation has never been compared for any integrin.We herein examine the effects of representative activating and inhibitory Fabs on the headpiece conformation and function of α_Xβ₂ integrin. The results show that extension in the absence of headpiece opening is not sufficient for integrin activation and that the open headpiece conformation is required for α_Xβ₂ integrin activation.     

Metal ion and ligand binding of integrin α5β1

Integrin α₅β₁ binds to an Arg–Gly–Asp (RGD) motif in its ligand fibronectin. We report high-resolution crystal structures of a four-domain α₅β₁ headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α₅β₁ complexed with a Fab that allosterically inhibits ligand binding by stabilizing the closed conformation. Soaking experiments show that binding of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β₁-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast, binding of a linear RGD peptide induces no shape shifting. However, linear peptide binding induces shape shifting when Ca²⁺ is depleted during soaking. Ca²⁺ bound to the adjacent to metal ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca²⁺ is depleted. The results directly demonstrate that Ca²⁺ binding to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible integrin leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin–semaphorin–integrin (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens.Integrins are adhesion receptors that transmit bidirectional signals across the plasma membrane. The α₅β₁ integrin and its primary extracellular matrix ligand fibronectin (Fn) are of great biological importance (1). Studies of this prototypic integrin–ligand pair led to the remarkable observation that much of the specific recognition by α₅β₁ of the ∼2,000-residue fibronectin molecule was encoded by a 3-residue sequence, Arg–Gly–Asp (RGD), in Fn3 domain 10 (2). Eight of 24 integrin αβ heterodimers are now known to recognize RGD motifs within their ligands. Higher-affinity and more α₅β₁-selective cyclic RGD peptide antagonists have been discovered (3) and mutationally characterized (4) but remain uncharacterized structurally.Integrin αI and βI domains coordinate acidic residues in their ligands, such as the Asp side chain of RGD, through an Mg²⁺ ion held in a metal ion-dependent adhesion site (MIDAS) (5–8). βI domains also contain flanking Ca²⁺ ions coordinated by residues in the adjacent to MIDAS (ADMIDAS) and synergistic metal ion binding site (SyMBS). The ligand-binding headpiece of integrins has two states: a high-affinity, open conformation with the β-subunit hybrid domain swung out at its interface with the βI domain, and a low-affinity, closed conformation with the hybrid domain swung in (8). The altered conformation at the βI–hybrid domain interface is transmitted to an ∼3-Å rearrangement in the MIDAS- and ADMIDAS-coordinating βI domain β1-α1 loop, which alters affinity by ∼1,000-fold (9). The SyMBS and ADMIDAS have important roles in regulating ligand binding affinity (10, 11). The ADMIDAS is a negative regulatory site responsible for integrin inhibition by high concentration of Ca²⁺ and for activation by Mn²⁺, which competes with Ca²⁺ for binding to the ADMIDAS (10, 11). However, the mechanism by which metals at the ADMIDAS regulate integrin affinity remains mysterious.A recent structural study (12) revisited the role of Ca²⁺ at the ADMIDAS. The α₅β₁ headpiece was characterized at 2.9-Å resolution bound to the SG/19 Fab that stabilizes the closed conformation of β₁ integrins by binding to the βI–hybrid domain interface. Soaking with RGD peptide induced movement of the βI domain β1-α1 loop and α1-helix, resulting in a conformational state intermediate between closed and open. A decrease in electron density for the ADMIDAS Ca²⁺ ion was also observed (12).Here we report high-resolution crystal structures of the α₅β₁ headpiece, alone or with soaked-in linear or cyclic peptides. Our results include important advances in understanding the role of Ca²⁺ binding to the ADMIDAS in ligand binding by β₁ integrins, how integrin α₅β₁ binds cyclic peptides with high affinity, and the observation that Ca²⁺ at the ADMIDAS inhibits RGD-induced β1-α1 loop and α1-helix movement.     

Interleukin-1β promotes proliferation and inhibits differentiation of chondrocytes through a mechanism involving down-regulation of FGFR-3 and p21

The proinflammatory cytokine IL-1β is elevated in many childhood chronic inflammatory diseases as well as obesity and can be associated with growth retardation. Here we show that IL-1β affects bone growth by directly disturbing the normal sequence of events in the growth plate, resulting in increased proliferation and widening of the proliferative zone, whereas the hypertrophic zone becomes disorganized, with impaired matrix structure and increased apoptosis and osteoclast activity. This was also evident in vitro: IL-1β increased proliferation and caused a G1-to-S phase shift in the cell cycle in ATDC5 chondrocytes, accompanied by a reduction in fibroblast growth factor receptor-3 (FGFR-3) and its downstream gene, the cell-cycle inhibitor p21 and its family member p57, whereas the cell-cycle promoter E2F-2 was increased. The reduction in FGFR-3, p21, and p57 was followed by delayed cell differentiation, manifested by decreases in proteoglycan synthesis, mineralization, alkaline phosphatase activity, and the expression of Sox9, RunX2, collagen type II, collagen type X, and other matrix proteins. Taken together, we suggest that IL-1β alters normal chondrogenesis and bone growth through a mechanism involving down-regulation of FGFR-3 and p21.     

Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression

AIM: To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices.METHODS: An immunohistochemical study of integrin αvβ6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors.RESULTS: High immunoreactivity for αvβ6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvβ6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvβ6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvβ6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvβ6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvβ6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvβ6 expression and promoted MMP-9 secretion compared with low density.CONCLUSION: Integrin αvβ6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανβ6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.     

A novel mechanism of sustained platelet αIIbβ3 activation via PEAR1

Because single nucleotide polymorphisms (SNPs) in platelet endothelial aggregation receptor 1 (PEAR1) are associated with differential functional platelet responses in healthy subjects, we studied the function of PEAR1 in human platelets. During platelet aggregation by various agonists, the membrane expression of PEAR1 and its tyrosine phosphorylation increased. The recombinant PEAR1 EMI domain (GST-EMI) competitively reduced platelet adhesion to surface-coated PEAR1, diminished platelet aggregation, and eliminated PEAR1 phosphorylation. Polyclonal antibodies against the extracellular PEAR1 domain triggered PEAR1 phosphorylation in a src family kinase (SFK)-dependent manner. Such resulted in downstream signaling, culminating in extensive platelet degranulation and irreversible aggregation reactions interrupted by excess monovalent anti-GST-EMI F(ab) fragments. In resting platelets, the cytoplasmic tail of PEAR1 was found complexed to c-Src and Fyn, but on its phosphorylation, phospho-PEAR1 recruited p85 PI3K, resulting in persistent activation of PI3K and Akt. Thus, αIIbβ3 activation was amplified, hence stabilizing platelet aggregates, a signaling cascade fully interrupted by the SFK inhibitor PP1 and the PI3K inhibitor LY294002. This study is the first demonstration of a functional role for PEAR1 in platelet activation, underpinning the observed association between PEAR1 and platelet function in genome-wide association studies.     

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Integrin α2β1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3Kβ(KD)), but occurred normally in PI3Kγ(KD) platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)-knockout mice, and we found that intracellular Ca(2+) linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca(2+). Whereas activation of Pyk2 occurred normally in PI3Kβ(KD) platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.     

Single and combined effects of αvβ3- and α5β1-integrins on capillary tube formation in a human fibrinous matrix

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-α and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the αvβ3-integrin inhibiting mAb LM609 or the α5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when αvβ3- and α5β1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. αvβ5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. αv-specific cRGD only inhibited angiogenesis when α5β1-integrin was simultaneously blocked. Endostatin mimicked the effect of α5β1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of αvβ3- and α5β1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.