A Unique Recognition Site Mediates the Interaction of Fibrinogen with the Leukocyte Integrin Mac-l (CDllb/CDl@*

Mac-1 (CD11b/CD18), a leukocyte-restricted integrin receptor, mediates neutrophil/monocyte adhesion to vascular endothelium and phagocytosis of complement-opsonized particles. Recent studies have shown that Mac-1 also functions as a receptor for fibrinogen in a reaction linked to fibrin deposition on the monocyte surface. In this study, we have used extended proteolytic digestion of fibrinogen to identify the region of this molecule that interacts with Mac-1. We found that an Mr approximately 30,000 plasmic fragment D of fibrinogen (D30) produced dose-dependent inhibition (IC50 = 1.6 microM) of the interaction of intact 125I-fibrinogen with stimulated neutrophils and monocytes. 125I-D30 bound saturably to these cells with specific association of 136,200 +/- 15,000 molecules/cell in a reaction inhibited by OKM1 and M1/70, monoclonal antibodies specific for the alpha subunit of Mac-1. Direct microsequence analysis and an epitope-mapped monoclonal antibody showed that D30 lacks the COOH-terminal dodecapeptide of the gamma chain as well as the Arg-Gly-Asp sequences in the A alpha chain. We conclude that fibrinogen interacts with the leukocyte integrin Mac-1 through a novel recognition site that is not shared with other known integrins that function as fibrinogen receptors.

Recent studies have shown that Mac-l also functions as a receptor for fibrinogen in a reaction linked to fibrin deposition on the monocyte surface.
In this study, we have used extended proteolytic digestion of fibrinogen to identify the region of this molecule that interacts with Mac-l. We found that an M, -30,000 plasmic fragment D of fibrinogen (D& produced dose-dependent inhibition UC60 = 1.6 PM) of the interaction of intact '261-fibrinogen with stimulated neutrophils and monocytes. "'I-Dso bound saturably to these cells with specific association of 136,200 f 15,000 molecules/cell in a reaction inhibited by OKMl and M1/70, monoclonal antibodies specific for the a subunit of Mac-l. Direct microsequence analysis and an epitope-mapped monoclonal antibody showed that DSO lacks the COOH-terminal dodecapeptide of the y chain as well as the Arg-Gly-Asp sequences in the Aa chain. We conclude that fibrinogen interacts with the leukocyte integrin Mac-l through a novel recognition site that is not shared with other known integrins that function as fibrinogen receptors.
The deposition of fibrinogen on the leukocyte surface is a hallmark of a variety of inflammatory responses (1). Fibrinogen association with monocytes or neutrophils has in fact been recognized as a causal component of delayed type hypersensitivity (2, 3), incompatible transplant rejection (4), and in the physiopathology of vascular obstruction and atherogenesis (5-B).
In this study we sought to determine the molecular requirements supporting the interaction of fibrinogen with the leukocyte integrin [22][23][24][25][26]

AND DISCUSSION
Previous studies have shown that the leukocyte integrin Mac-l (CDllb/CD18) (14, 22) functions as a receptor for fibrinogen on monocytes, PMN, and various transformed cell lines of myelomonocytic lineage (23-26).
In preliminary experiments, various proteolytic fragments derived from sequential plasmic digestion of fibrinogen (29, 30) were tested for their ability to inhibit the binding of intact "'I-fibrinogen to stimulated PMN or monocytic THP-1 cells. As previously reported, THP-1 cells provide a suitable in vitro model of the mono&e phenotype with respect to surface expression of Mac-l (CDllb/CD18) and for the inducible high affinity ligand recognition of this receptor (23). The results of these experiments are summarized in Fig. 1 ogen fragments X, D:E, and D inhibited the binding of lZ51fibrinogen to stimulated PMN in a concentration-dependent fashion with ICsO ranging between 25 and 50 pg/ml added competitor (Fig. 1). Under the same experimental conditions, 1 mM concentrations of the synthetic peptides RGDS and HI2 alone or in combination did not inhibit '251-fibrinogen binding to PMN, in agreement with previous observations (23) (Fig. 1).
These inhibitory fibrinogen fragments were further analyzed for their reactivity with the epitope-mapped monoclonal antibody 4A5, specific for the COOH-terminal dodecapeptide of the fibrinogen y chain (31). This region plays a crucial role in mediating the interaction of fibrinogen with the platelet integrin air& (28,34,35) and was also previously implicated in Mac-l recognition of fibrinogen (24). As shown in Fig. 2, monoclonal antibody 4A5 bound specifically to fibrinogen, fragment X, and D:E while it failed to react with the M, -80,000 fragment D (Fig. 2B). In contrast, a rabbit polyclonal antibody directed to the region 95-264 of the y chain (29) strongly reacted with fragment D under comparable experimental conditions (not shown), thus verifying the authenticity of this fragment. Proteolytic cleavage to delete the COOH terminus of the y chain is a well established structural characteristic of the plasmic-derived M, -80,000 fragment D of fibrinogen (36-38). On the other hand, these experiments raised the possibility that fibrinogen interaction with the leukocyte integrin Mac-l occurred through a novel recognition site, structurally distinct from the previously described regions that mediate fibrinogen binding to platelet or endothelial cell integrins (15,18,27,28,34,35,39,40). To further investigate this possibility, the inhibitory M, -80,000 fragment D was subjected to 24-h plasmic proteolysis to originate an M, -30,000 advanced fragment D (DaO) (30) that was isolated by ion exchange chromatography. Increasing concen- conditions, HPLCpurified fibrinogen fragment E was ineffective (Fig. 3). Similar concentrations of Dso also inhibited '""I-fibrinogen binding to fMLP-stimulated THP-1 cells in a dose-dependent manner (I&, = 50 pg/ml). Analysis of the binding data by the Lineweaver-Burk double-reciprocal plot indicated that DsO behaves as a competitive inhibitor of '""I-fibrinogen binding to PMN (y' = 1.98 X lOs, fibrinogen without competitor; y' = 2.57 x lo*, fibrinogen in the presence of D:rc). The molecular prerequisite for the inhibition of Mac-lfibrinogen interaction mediated by Dso was further established in direct '""I-labeled binding experiments. As shown in Fig. 4, suspensions of PMN bound Y-DsO in a specific and saturable reaction, competitively inhibited by a molar excess of unlabeled fibrinogen or Dso and approaching steady state at 15-20 pg/ml added protein, with association of -136,200 + 15,000 molecules of DZO/cell (Fig. 4). Binding of 12hI-D30 occurred with quantitatively identical characteristics to THP-1 cells (not shown), and as previously described for the binding of the intact fibrinogen molecule (12, 23), it required cell stimulation (10 pM fMLP) and divalent cations (2.5 mM CaClJ. Finally, the '""I-labeled cell-bound material showed a molecular size and structural organization indistinguishable from the D:," fragment when analyzed by SDS-gel electrophoresis and autoradiography under reducing or nonreducing conditions ( Fig. 4, inset).
The possible role of the leukocyte fibrinogen receptor Mac-1 (23-26) in mediating the inducible recognition of Da0 was investigated further using monoclonal antibody strategy. Saturating concentrations of monoclonal antibodies OKMl or M1/70, both directed against the (Y subunit of Mac-l, CDllb (23), completely abolished the binding of ""I-D:,o to fMLPstimulated suspensions of PMN (Table I). In contrast, monoclonal antibodies reacting with a different epitope on the cy subunit (60.1) or against the p subunit, CD18 (60.3, IB4), did not interfere with '""I-DZro binding to Mac-l (Table I).
To conclusively establish the structural organization of DZro, direct microsequence analysis was carried out. We found that the new NH? termini in the Aa chain remnant of this plasmic fragment originated with residues Leu'"G, Gin'"', Lys"", and Asn"'". This (Y chain origin of Dso thus excludes the NH,- Suspensions of PMN were incubated as described in Fig. 1 in the presence of increasing concentrations of ""I-D:, for 20 min at room temperature. Specific binding was calculated in the presence of loo-fold molar excess unlabeled fibrinogen or Dm, and was subtracted from the total to calculate specific binding (0). Each point is the mean + S.E. of three independent experiments. Inset, analysis of l'"I-D:l,, bound to the cell surface by SDS electrophoresis on a 15% polyacrylamide gel under nonreducing (lane 1) or reducing conditions (lane 2). terminal RGD sequence at residues 95-97 (41), while for its M, -ll,~O-13,000 DsO must terminate to omit the COOHterminal RGD at position 572-574 (41). The origin of DSO was also assigned at residues AspX3*, Asn'35, GIu'~~, and Asn'37 in the BP chain and at residues Met", Leu"', Glu'i, and G1us2 in the y chain. Furthermore, as demonstrated above, and in agreement with previous observations (36-38), plasmic digestion of fibrinogen originating the M, -80,000 fragment D is already associated with proteolytic cleavage and removal of the COOH terminus of the y chain. These findings are difficult to reconcile with a previously reported inhibition of fibrinogen interaction with PMN mediated by synthetic analogues of the COOH terminus of the y chain unless an entirely distinct recognition is involved in Mac-l binding to immobilized fibrinogen (24). In addition to the inhibition of lz51-DS0 binding to PMN mediated by intact fibrinogen, we have further substantiated the specificity of this interaction using a panel of monoclonal antibodies. Complete inhibition of lz51-DS0 binding to stimulated PMN was achieved with the same monoclonal antibodies that in previous studies abolished the binding of Y-fibrinogen to leukocytes (23) and directed it to the cy, Mac-l-specific, subunit of this receptor, Interestingly, monoclonal antibodies against the fl subunit of Mac-l did not reduce binding of '251-D30, leading to the speculation that the complementary fibrinogen interacting site on Mac-l might reside on its (Y subunit, in analogy with the preferential platelet Lunb-mediated recognition of fibrinogen (39,40,42). In summary, we have identified a novel interacting region that mediates the assembly of fibrinogen on the leukocyte surface. Integrin receptors homologous to Mac-l display fibrinogen binding capacity on a variety of ceils in a reaction crucially regulated by recognition of the RGD sequence and/ or the COOH-terminal dodecapeptide of the y chain (15,18,27,28,34,35,39). The platelet integrin aI& well exemplifies the complexity of this simultaneous recognition of two noncontiguous regions in the fibrinogen molecule (40), coordinating a process of central importance in the maintenance of normal hemostasis and vessel wall integrity. On leukocytes, ~brinogen deposition participates in broad mechanisms of inflammation associated with fibrin formation (9-12). The relevance of this process is underscored by its biologic aberrations when leukoc~es play a major role in the pathophysiology of vascular obst~ction and in atherothrombotic disease (5-8). These important functional differences are perhaps reflected in the unique structural recognition of Mac-l for fibrinogen that does not involve the RGD sequence nor the COOH-terminal dodecapeptide of the y chain. The remarkable affinity of this interaction combined with its rigorous specificity suggest its therapeutic potential as an inhibitor of leukocyte adhesion without interfering with hemostatic functions dependent upon 39).