Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin ␣v␤3 at a high affinity (K D ‫؍‬ 2 ؋ 10 ؊7 M). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to ␣4␤1. We showed that sPLA2-IIA competed with VCAM-1 for binding to ␣4␤1, and bound to a site close to those for VCAM-1 and CS-1 in the ␣4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins ␣v␤3 and ␣4␤1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrinsPLA2-IIA interaction is a novel therapeutic target. The phospholipase A2 (PLA2)2 family is a group of intracellular and secreted enzymes that hydrolyzes the sn-2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes to form nonesterified fatty acids and lysophospholipids. These products act as intracellular second messengers or are further metabolized into potent mediators of a broad range of cellular processes, including inflammation, apoptosis, and atherogenesis (1). The mammalian secretory PLA2 isoforms are comprised of the groups named IB, IIA, IIC, IID, IIE, IIF, V, X, and XII (2, 3). All secretory PLA2 isoforms have in common a Ca 2ϩ -dependent catalytic mechanism, a low molecular mass (13-16 kDa), several disulfide bridges, and a wellconserved overall three-dimensional structure (2, 4, 5). Secretory PLA2 type IIA (sPLA2-IIA) was first isolated and purified from rheumatoid synovial fluid. sPLA2-IIA is an acute phase reactant and is found in markedly increased plasma concentrations in diseases that involve systemic inflammation such as sepsis, rheumatoid arthritis, and cardiovascular disease (up to 1000-fold and Ͼ1 g/ml). Inflammatory cytokines such as IL-6, TNF-␣, and IL-1␤ induce synthesis and release of sPLA2-IIA in arterial smooth muscle cells and hepatocytes, which are the major sources of the plasma sPLA2-IIA in these systemic inflammatory conditions (6, 7). In addition to being a pro-inflammatory ...

Fibrinogen is a major plasma protein (350 kDa) that induces proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing, angiogenesis, and tumor growth. Fibrin(ogen) degradation products generated during fibrinolysis are implicated in tissue injury. The fibrinogen ; chain has a COOH-terminal globular domain (;C, residues 151-411 of the ; chain, 30 kDa) to which several integrin cell adhesion receptors (e.g., platelet A IIb B 3 , endothelial A v B 3 , and leukocyte A M B 2 ) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that ;C and its truncation mutant (designated ;C399tr), with a deletion of the COOHterminal 12 residues, induced apoptosis of endothelial cells and blocked tube formation of endothelial cells. DLD-1 human colon cancer cells that secrete ;C or ;C399tr grew at similar levels in vitro but grew much slower in vivo than mocktransfected cells. The recombinant purified ;C399tr fragment markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis in vivo in the highly metastatic Met-1 breast cancer model. The determinant responsible for binding to endothelial cells is cryptic in native fibrinogen but is exposed in ;C and ;C399tr. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential. (Cancer Res 2006; 66(19): 9691-7)

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Fibrinogen is a major plasma protein (350 Kd) that induces proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing, angiogenesis, and tumor growth. Plasma concentrations of fibrin(ogen) degradation products (FDPs) generated during fibrinolysis are markedly elevated in the adult respiratory distress syndrome, disseminated intravascular coagulation, and septic shock, and FDPs are implicated in tissue injury associated with these disorders. However, the basis of how FDPs induce the vascular injury is unclear. The fibrinogen gamma chain has a C-terminal globular domain (gammaC, residues 151-411 of gamma chain, 30 Kd) to which several integrin cell adhesion receptors (e.g., platelet alphaIIbbeta3, endothelial alphavbeta3, and leukocyte alphaMbeta2) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that gammaC and its truncation mutant (designated gammaC399tr), with a deletion of the C-terminal 12 residues, effectively blocked proliferation of endothelial cells in tissue culture at concentrations of less than 10 micro g/ml, while native fibrinogen and fragment D did not have a noticeable effect. We demonstrated that the effect of gammaC is due to induction of apoptosis, as evidenced by the detection of apoptotic cells by using fluorescence-labeled annexin V and of activated caspase 3/7 in gammaC-treated cells. gammaC also blocked tube formation from endothelial cells in matrigel. GammaC did not induce such effects in several other cell types tested (e.g., CHO cells, keratinocytes, and tumor cells). We demostrated that tumor cells that secrete gammaC or gammaC399tr grew much slower than nonsecreting control cells in vivo. Also recombinant gammaC399tr protein markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis when injected intraperitoneally in vivo. We showed that the structural determinant responsible for binding to endothelial cells is cryptic in native fibrinogen, but is exposed in gammaC. We showed that the alphavbeta3-binding site is also crypric in fibrinogen and Fragment D, but is exposed in gammaC. However, we were unable to determine whether integrins are directly involved in gammaC-induced apoptosis of endothelial cells, since antagonists and antibodies to integrins induce apoptosis of endothelial cells. In conclusion, these results suggest that fibrinogen has a novel cryptic determinant that can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential.