Thrombin adhesive properties: induction by plasmin and heparan sulfate

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild- type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.     

Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand.

Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD-dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein-protein association.     

Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.     

The small GTPase Rac3 interacts with the integrin-binding protein CIB and promotes integrin alpha(IIb)beta(3)-mediated adhesion and spreading.

There are only three human isoforms of the small GTPase Rac, which together regulate a variety of cellular processes, including those related to actin cytoskeletal reorganization. A role for Rac3 in integrin-mediated adhesion and spreading has not been defined. We here report that CIB, a protein that binds to the alpha(IIb)beta(3) fibrinogen receptor, interacts exclusively with activated (V12) Rac3 but not Rac1 or Rac2. Binding of V12Rac3 to CIB was mediated by the C-terminal end of Rac3 and by Rac3 membrane localization. Adhesion of cells on fibrinogen was accompanied by a specific increase in the levels of Rac3 but not Rac1 or Rac2 in the Triton-insoluble fraction of the cell. Also, CIB co-localized with active Rac3 to the periphery of cells adhering to fibrinogen. Expression of V12Rac3 and CIB stimulated alpha(IIb)beta(3)-mediated adhesion and spreading on fibrinogen. Moreover, adhesion through alpha(IIb)beta(3) caused a marked increase in the levels of endogenous GTP-bound Rac3 but not Rac1. These combined results strongly implicate Rac3 and CIB in integrin-associated cytoskeletal reorganization during alpha(IIb)beta(3)-mediated adhesion.     

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.     

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.     

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.     

Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, and alpha(2)-macroglobulin. The chemically inactivated alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only anti-integrin (alpha(v)beta(3) and alpha(5)beta(1)) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.     

The activation state of alphavbeta 3 regulates platelet and lymphocyte adhesion to intact and thrombin-cleaved osteopontin

Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the alpha(v)beta(3)-mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin- and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of alpha(v)beta(3) to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the alpha(v)beta(3) binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross-linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mm Mn(2+). Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for alpha(v)beta(3) on osteopontin, but this enhancement is also a function of the activation state of alpha(v)beta(3). Moreover, they indicate that the sequence RGDSVVYGLR contains sufficient information to specify activation-dependent alpha(v)beta(3)-mediated platelet and lymphocyte adhesion.     

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.