Here we demonstrate a specific interaction between a platelet adhesion receptor, the GP Ib-IX-V complex, and P-selectin. This interaction represents a potential new paradigm for platelet adhesion, where the same receptor (GP Ib-IX-V) can support platelet adhesion to endothelial cells under the appropriate conditions of endothelial activation and to the subendothelial matrix when the endothelial cells have been removed by injury. Several interesting parallels exist between GP Ibα and PSGL-1 as cell adhesion receptors. Both are membrane mucins with elongated structures formed by the presence of a heavily O
-glycosylated region rich in threonine, serine, and proline residues 3839
. In both receptors, this mucin-like region separates the ligand-binding region from the plasma membrane 4041
, and in both it contains tandemly repeated sequences that account for polymorphism in the human population because of variable numbers of the tandem repeats 4243
. The major carbohydrate structures of the two receptors are very similar, with only a fucose in α(1,3) linkage in the major PSGL-1 carbohydrate chain differentiating it from the major carbohydrate species of GP Ibα. Furthermore, both receptors contain a highly acidic region with sulfated tyrosines that is important for ligand binding and lies immediately NH2
-terminal to the mucin-like region 4567141516
. The major ligands for both receptors (von Willebrand factor and P-selectin) are synthesized only in megakaryocytes and endothelial cells and are stored in the same subcellular compartments, the α-granules of platelets and megakaryocytes and the Weibel-Palade bodies of endothelial cells.
Despite all of these similarities, the interactions of the two mucins with P-selectin are quite different. Whereas PSGL-1 requires carbohydrate modification with core-2 branching and α(1,3)-fucosylation and the presence of calcium for its interaction with P-selectin, GP Ibα requires neither. This lack of a requirement for carbohydrate modification is consistent with the findings of Frenette et al. 8
that the platelet P-selectin counterreceptor does not require modification by FucTs for function.
The different carbohydrate requirements of the two P-selectin counterreceptors may not be absolute but rather may represent a difference in the relative importance of the two P-selectin binding determinants (carbohydrates and sulfated tyrosines). GP Ibα does not contain the fucosylated moiety that is so vital for PSGL-1; on the other hand, its anionic region has a much higher concentration of negative charge than does the analogous region of PSGL-1, as well as three tyrosines that are fully sulfated 1416
. This region of GP Ibα is known to bind tightly to thrombin and does so by interacting with the same region of thrombin that binds heparin 3132
. Thus, the anionic sulfated region of GP Ibα, with its high negative charge and sulfate groups, appears to resemble heparin. Several lines of evidence point to the importance of this heparin-like region of GP Ibα in the interaction with P-selectin. First, the calcium independence of the interaction resembles the interaction of P-selectin with heparin, as does the pattern of blockade with heparin and fucoidin, but not with chondroitin sulfate 17
. Second, the interaction is inhibited by SZ2, an mAb that binds within the region and also partially blocks botrocetin-induced von Willebrand factor binding, a phenomenon known to be sensitive to the removal of tyrosine sulfates 1516
. Finally, mocarhagin, the cobra venom metalloprotease that removes most of the heparin-like region, also inhibits binding. Thus, the GP Ibα–P-selectin interaction is almost completely independent of carbohydrate, although the failure of SZ2 or mocarhagin to fully inhibit the interaction suggests that carbohydrate may have a minor role.
The possible involvement of other regions of GP Ibα in the interaction is also suggested by the inhibition by the mAb AK2 of platelet rolling on stimulated endothelial cells. This antibody binds within the leucine-rich repeats of GP Ibα 16
. However, because the three-dimensional relationship of the leucine-rich repeats with the sulfated region in three dimensions is not known, we cannot definitively make a case for the involvement of the leucine-rich repeats in the interaction. The potential involvement of all of the regions of GP Ibα will have to await more definitive studies with mutant polypeptides.
The adhesion of platelets to regions of vessel wall injury mediated by von Willebrand factor can occur at very high shear stresses, approaching 100 dynes/cm2
. The interaction between platelets and endothelium, on the other hand, seems to occur at much lower shear stresses, although evidence exists that platelets can interact with endothelium at shear stresses above those able to support the interaction of leukocytes with the endothelium 44
. The studies reported here were carried out at venular shear stresses and do not address the possibility that the GP Ibα–P-selectin interaction may be able to support platelet rolling at shear stresses much higher than those that support leukocyte rolling.
The demonstration of an abundant receptor on the platelet surface for P-selectin brings into sharp focus the potential roles of this receptor–counterreceptor interaction in both physiological and pathological conditions. During hemostasis, the interface between the GP Ib-IX-V complex and P-selectin may be important in the interaction between activated and unactivated platelets. Activated platelets with surface-exposed P-selectin could associate with and activate previously unactivated platelets, thus providing a mechanism for propagation of platelet thrombi. Failure of this mechanism could explain the mild bleeding diathesis of P-selectin–deficient mice 45
and could contribute to the bleeding tendency in patients with Bernard-Soulier syndrome, the genetic disorder resulting from deficiency of the GP Ib-IX-V complex 46
. Nevertheless, this mechanism is likely to be only of secondary importance, as no defect in aggregation has been found in the platelets of patients with Bernard-Soulier syndrome (although a subtle defect might not be detected by conventional methods of performing aggregation).
Under certain circumstances, platelets may help neutrophils and other white cells to exit the vasculature by providing an additional ligand for recognizing activated endothelial cells. Such a role has been demonstrated for platelets in lymphocyte trafficking, where activated platelets bound to the lymphocytes can provide a ligand (P-selectin) for the endothelial addressins of lymph nodes 47
. Platelets interacting with activated endothelium may themselves become activated, releasing factors that may influence the behaviors of both the endothelial cells and underlying smooth muscle cells. When such a process is unchecked, such as during sepsis, the platelets may contribute to the deleterious effects of the underlying condition. In this regard, recent findings suggest that activated platelets also form stable attachments with endothelial cells mediated in part by other platelet GP receptors, such as the integrin GP IIb-IIIa 48
. At this point, however, the potential biological consequences of platelet interaction with endothelium are speculative. Nevertheless, with the demonstration of such a specific interaction involving endothelial selectins 9
and the current identification of a platelet receptor for these selectins, the endothelium can no longer be considered refractory to the overtures of the platelet.