In this study, we have identified a direct interaction between the leukocyte integrin Mac-1 and platelet GP Ibα. The following evidence was obtained for this interaction: (a) mAbs to both Mac-1 and GP Ibα inhibited THP-1 cell adhesion to purified GP Ibα; (b) 293 cells that express Mac-1, but not LFA-1, bound strongly to GP Ibα, and this adhesion was inhibited specifically by mAbs; (c) wild-type, but not Mac-1–deficient, neutrophils adhered to platelets and to purified GP Ibα; (d) neutrophil adhesion to platelets was inhibited by mAbs to Mac-1 and GP Ibα and by pretreatment of the platelets with the snake venom metalloprotease, mocarhagin, whose major platelet substrate is GP Ibα 35
; and (e) basal and agonist-stimulated leukocyte–platelet aggregates were decreased in whole blood of a patient with BSS compared with a normal control.
By virtue of binding diverse ligands including, among others, fibrin(ogen) 5556
, ICAM-1 57
, factor X 58
, C3bi 55
, high molecular weight kininogen 59
, and heparin 4
, Mac-1 regulates important leukocyte functions including adhesion, migration, coagulation, proteolysis, phagocytosis, oxidative burst, and signaling 49606162
. However, these ligands do not account for all of Mac-1's adhesive interactions. Although previous studies have shown that Mac-1, the primary fibrin(ogen) receptor on leukocytes, directly facilitates the recruitment of leukocytes at sites of platelet and fibrin deposition 121314
, the precise platelet counterreceptor was unidentified.
I or A domains are regions of ~200 amino acids that are present in 1 or more copies in many proteins involved in cell–cell, cell–matrix, and matrix–matrix interactions 2531
. This superfamily motif is present in integrin α subunits, including CD11a, CD11b, CD11c, CD11d, CD49a, CD49b, and αE 25
, the complement proteins Factors B and C2 6364
, collagens 6566
, and vWf 29
. For CD11b, experimental evidence supports the notion that the I domain is responsible for the binding of all Mac-1 ligands except for factor X 2467
. vWf has three similar domains, in this case termed A domains. The first and third of these, A1 and A3, mediate binding to GP Ibα and collagen, respectively 68
. High-resolution crystal structures of the CD11b I domain and the vWf A1 domain show that both of these domains adopt a classic α/β “Rossman” fold 2569
. The Mac-1 I domain also contains a metal ion–dependent adhesion site (MIDAS) for binding protein ligands, a motif, however, not present in the vWf A1 domain due to the presence of an arginine and an alanine instead of a serine and an aspartate, respectively, at two of the critical amino acids forming the MIDAS motif. The observation that mutations of the αM I domain that correspond to gain-of-function mutations of the vWf A1 domain also alter the binding activity of Mac-1 48
supports the notion that Mac-1 and vWf may be functionally similar with respect to GP Ib-IX-V binding.
The binding of GP Ibα to vWf and Mac-1 has several similarities and some interesting differences. First, as expected, the binding involves the homologous I or A domains. In addition, in both cases binding requires a conformational change of the A or I domain, in the case of vWf requiring ristocetin, botrocetin, or shear stress, and in the case of Mac-1 requiring activation of the integrin to its ligand-competent form. Distinguishing the two interactions is the distinct pattern of inhibition by GP Ibα antibodies. AK2, for example, is a potent inhibitor of vWf binding to GP Ibα, whether induced by ristocetin, botrocetin, or shear stress. This antibody failed to inhibit the interaction of Mac-1 with GP Ibα. VM16d, on the other hand, does not inhibit vWf binding to GP Ibα, except as induced by botrocetin, but is a potent inhibitor of Mac-1 binding. However, the sites are not completely distinct, as indicated by the observation that the isolated vWf A1 domain blocks the interaction of Mac-1–expressing cells with GP Ibα and by the ability of the mAb AP1, which also blocks ristocetin- and botrocetin-induced vWf binding, to inhibit the interaction.
The identification of the interaction between GP Ibα and Mac-1 provides a tantalizing lead into the nature of leukocyte–platelet adhesion, helping to clarify the sequential adhesion model of neutrophil attachment to surface-adherent platelets proposed by Diacovo et al. 12
. Nevertheless, our data do not rule out the possibility of additional platelet surface receptors for Mac-1. Other potential Mac-1 ligands present on the platelet membrane include fibrinogen (bound to GP IIb-IIIa) 5556
, ICAM-2 70
, high molecular weight kininogen 59
, and glycosaminoglycans 4
. A leukocyte–platelet interaction mediated by fibrinogen bridging between Mac-1 and GP IIb-IIIa has been discounted by Ostrovsky et al. 22
, who found that neither RGDS peptides nor the replacement of normal platelets with thrombasthenic platelets (i.e., lacking GP IIb-IIIa) affected the accumulation of the leukocytes on platelets, and recently by Furman et al. 71
, who found that GP IIb-IIIa antagonists and RGDS peptides did not reduce leukocyte–platelet aggregate formation in whole blood.
Other interactions contributing to Mac-1–independent leukocyte–platelet complex formation include thrombospondin bridging between GP IV receptors on platelets and monocytes 72
, and P-selectin on activated platelets binding with leukocyte PSGL-1 7374
. Nevertheless, under the experimental conditions employed in the present study, which assayed the adhesion of activated neutrophils (i.e., thioglycollate-elicited peritoneal neutrophils) to surface-adherent platelets after vigorous washing, the predominant interaction between neutrophils and platelets appeared to be between Mac-1 and GP Ibα.
These present observations also suggest a possible target for therapeutic intervention. In particular, the distinct difference in the inhibitory patterns of GP Ibα antibodies suggests that it might be possible to prevent leukocyte attachment to platelets by targeting GP Ibα without inhibiting platelet adhesion to the vessel wall. Our recent observations have identified Mac-1 as a molecular determinant of neointimal thickening after experimental arterial injury that produces endothelial denudation and platelet and/or fibrin deposition. We found that antibody-mediated blockade 7
or selective absence 75
of Mac-1 impaired transplatelet leukocyte migration into the vessel wall, diminishing medial leukocyte accumulation and neointimal thickening after experimental angioplasty or endovascular stent implantation. Therefore, future studies aimed at identifying the precise binding site(s) responsible for Mac-1–GP Ibα binding might provide a molecular strategy for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.