Previous studies have shown that fibrinogen can associate with endothelial cells via an Arg-Gly-Asp (RGD) recognition specificity. In the present study, we have characterized the specificity of fibrinogen binding to endothelial cells under different cation conditions. Fibrinogen binding to suspended endothelial cells was selectively supported by Mn 2؉ and was suppressed by Ca 2؉ . The Mn 2؉ -supported interaction was completely inhibited by RGD peptides but not by ␣ v ␤ 3 blocking monoclonal antibodies. In contrast, the interaction was completely blocked by two ␣ 5 ␤ 1 monoclonal antibodies. This interaction was not mediated by fibronectin bound to the integrin; could be demonstrated with purified ␣ 5 ␤ 1 ; and also was observed with a second ␣ 5 ␤ 1 -bearing cell type, platelets. The binding of fibrinogen to ␣ 5 ␤ 1 on endothelial cells in the presence of Mn 2؉ was time-dependent, specific, saturable, and of high affinity (K d ‫؍‬ 65 nM). By employing anti-peptide monoclonal antibodies, the carboxyl-terminal RGD sequence at A␣ 572-574 was implicated in fibrinogen recognition by ␣ 5 ␤ 1 . Two circumstances were identified in which ␣ 5 ␤ 1 interacted with fibrinogen in the presence of Ca 2؉ : when the receptor was activated with monoclonal antibody (8A2) or when the fibrinogen was presented as an immobilized substratum. These results identify fibrinogen as a ligand for ␣ 5 ␤ 1 on endothelial and other cells, an interaction which may have broad biological implications.The luminal surface of endothelial cells (EC) 1 is continuously exposed to a high concentration of plasma fibrinogen (Fg). Disruption of the endothelium results in local thrombus formation, and Fg/fibrin accumulates at such sites of vascular injury. Based upon these proximal relationships, the molecular mechanisms and functional consequences of the interaction of Fg with EC have been topics of considerable interest and investigation (1-6). Indeed, it has been shown that Fg can induce EC attachment, spreading, and migration (1, 7) and can support an angiogenic response (8). Several distinct receptors have been implicated in mediating Fg binding to EC. These include ␣ v ␤ 3 (9, 10), a member of the integrin family of cell adhesion receptors, as well as several non-integrin binding sites (6, 11). Transglutaminase-mediated Fg cross-linking to EC also has been demonstrated (12).␣ v ␤ 3 interacts with Fg via an Arg-Gly-Asp (RGD) recognition specificity; i.e. RGD-containing peptides block Fg binding to this receptor (13). This tripeptide sequence is recognized not only by ␣ v ␤ 3 but also by several other integrins (14 -16), including ␣ 5 ␤ 1 , which serves as a fibronectin (Fn) receptor on EC and many other cell types (17)(18)(19)(20). Fg contains two RGD sequences within its A␣-chain: RGDF at A␣ 95-98 and RGDS at A␣ 572-575. Previous studies have shown that EC adhesion to immobilized Fg is blocked by a monoclonal antibody (mAb) to the carboxyl-terminal peptide containing RGD sequence at A␣ 572-574 (3). Moreover, this adhesion was blocked by mAbs against ␣...

AlphaIIbbeta3 (platelet membrane glycoprotein IIb-IIIa) and alphavbeta3 are members of the beta3 subfamily of integrin adhesion receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD), originally described by Scarborough et al. (Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol. Chem. 268, 1066-1073) has been employed as a high affinity ligand for alphaIIbbeta3 to examine the specificity of the beta3 integrins. cHarGD interacted with high affinity with purified alphaIIbbeta3 (Kd = 10 nM) or with platelets (Kd = 120 nM). While cHarGD was specific for alphaIIbbeta3 in the presence of Ca2+, it bound to both beta3 integrins in the presence of Mn2+. Barbourin, a snake venom disintegrin containing a reactive KGD sequence, remained alphaIIbbeta3-specific, even in the presence of Mn2+. cHarGD became cross-linked to a site in beta3 of alphaIIb beta3, which is distinct from that of RGD peptides. These results allow identification of at least four classes of beta3 ligands: Class I, represented by RGD peptides and vitronectin, react similarly with alphaIIbbeta3 and alphavbeta3; Class II, represented by cHarGD, gamma-chain peptides and fibrinogen, react with both receptors in the presence of Mn2+ but only with alphaIIbbeta3 in the presence of Ca2+; Class III, represented by barbourin, are alphaIIbbeta3-specific under all cation conditions; Class IV, represented by osteopontin, bind primarily to alphavbeta3.

Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).

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Integrins are alpha/bets heterodimeric adhesion receptors. aubƒÀ3 (GPIIb-IIIa) and avƒÀ3 (the vitronectin receptor) share the same beta subunit, ƒÀ3, but have distinct alpha subunits. These sister integrins not only recognize many of the same ligands but also have certain unique ligands. Based upon current information in the literature, we propose that four classes of ƒÀ3 ligands can be distinguished. Since the ƒÀ3 integrins have multiple functions in vivo and are targets for therapy, this classification system may be useful in the design and characterization of therapeutic agents.

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Coagulation factor VIII inhibitor arising in a patient with autoimmune disease was immunologically analyzed. A 63-year-old man who had been diagnosed as suffering from polyarteritis nodosa was treated with prednisolone for 10 years. Severe bleeding tendency developed and coagulation studies demonstrated a high titer of inhibitor to factor VIII:C. As a result of immunological analysis, the inhibitor was found to be IgG type autoantibody having both k and λ light chains. The subclasses were IgG₁ and IgG₄. The inhibitor recognized the COOH-terminal light chain (72-kDa thrombin fragment) on the factor VIII molecule as an epitope.