José A. LópEz

Glycoprotein (GP) Ibα, a member of the leucine-rich repeat (LRR) protein family, mediates platelet adhesion to immobilized von Willebrand factor (VWF). We investigated the role in VWF binding of charged residues in the LRR region of GP Ibα that are conserved in human, canine, and murine proteins. Substitution of His86 with either Ala or Glu resulted in a gain of VWF-binding function as judged by increased VWF binding in the presence of the modulators ristocetin and botrocetin and by enhanced adhesion of Chinese hamster ovary (CHO) cells expressing the mutant GP Ibα to immobilized VWF under conditions of flow. This is the first report of a gain-of-function phenotype resulting from mutations in the LRR region of GP Ibα. Because His86 is 2 nm away from the region of GP Ibα with the largest surface of contact with VWF, the data suggest that the LRRs regulate GP Ibα affinity for VWF allosterically.

In this review, the authors summarize how platelets interact with subendothelium when the vessel wall is damaged or with intact endothelium in the inflammatory state. When subendothelium is exposed to rapidly flowing blood upon vessel damage, platelets adhere rapidly to the exposed surface, decelerate, and aggregate to arrest bleeding. Under high shear stress, such as is found in the microcirculation, the interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor, glycoprotein (GP) Ib‐IX‐V, is required to slow down platelets and allow the platelet collagen receptors α₂β₁ and GP VI to bind to collagen. GP VI and α₂β₁ play important roles to activate platelets in the early stage and work with GP Ib‐IX‐V to fully activate platelets to form thrombi. GP Ib‐IX‐V and GP VI employ similar signaling pathways for platelet activation and the signals from both receptors are down‐modulated by PECAM‐1 (platelet–endothelial–cell adhesion molecule 1) to prevent unnecessary platelet activation under high shear. During inflammatory states, intact endothelial cells release VWF and P‐selectin from their Weibel‐Palade bodies. Both molecules are ligands for GP Ib‐IX‐V. The newly released VWF is larger and stickier than the form normally found in plasma and binds platelets spontaneously. Normally, VWF is processed by proteolysis by the plasma metalloprotease ADAMTS‐13. Failure of this processing results in the microvascular thrombotic disorder thrombotic thrombocytopenic purpura. In this review, the authors also use available crystal structures of platelet receptors and ligands to explain the details of their interactions.

The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ibα. Of the many GP Ibα monoclonal antibodies described, AP1 is of particular interest because it blocks static VWF binding induced by 2 modulators, ristocetin and botrocetin, and platelet adhesion to VWF surfaces under flow. We mapped the AP1 binding site to a region encompassing Arg218 to Tyr228, comprising the α1 helix and β13 strand defined by the GP Ibα crystal structure. AP1 binding absolutely required Arg218, Asp222, and Glu225. We evaluated the ability of cells expressing mutants of this region to bind VWF under static conditions in the presence of modulators, and to attach to and roll on a VWF matrix under flow. These data indicate that 2 regions within the sequence Arg218 to Tyr228 have important roles in VWF binding: the α1 helix has a regulatory role and the β turn and β13 strand bind VWF directly. Despite this, the only effect of a synthetic peptide corresponding to Leu214 to Val229 was to slightly increase the rolling velocity of GP Ibα-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ibα, thereby facilitating the interaction with VWF. (Blood. 2004;104:3971-3978)

The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V–associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcγRIIA. The importance of this association was demonstrated by the ability of the FcγRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V–dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.

A dominant feature of the structure of platelet glycoprotein (GP) Ibα, the von Willebrand factor (VWF)-binding subunit to the GP IbIX-V complex, is the presence of an elongated, heavily glycosylated mucin-like stalk between the plasma membrane and the N-terminal 45-kDa ligand-binding domain. Here, we investigated the function of that region by expressing a mutant lacking residues 318-452 as part of a recombinant GP Ib-IX complex. We studied the VWF-binding function of this mutant under both static conditions and flow. The mutant GP Ibα was expressed normally on the surface of CHO bIX cells (stably expressing GP Ibβ and GP IX) and the proper conformation of the ligand-binding region was verified by the normal binding of 5 conformation-sensitive monoclonal antibodies. Under static conditions, cells expressing mutant GP Ibα bound VWF (binding induced by either botrocetin or ristocetin) in a manner indistinguishable from cells expressing wild-type GP Ibα. We also evaluated the ability of the mutant to mediate cell adhesion to immobilized VWF in the presence of fluid shear stress (at 2 and 10 dyn/cm²). When the mutant-expressing cells were incubated with immobilized VWF for 1 min before being exposed to shear, they rolled on the VWF surface in a manner similar to wild-type cells. However, if the cells were not first allowed to settle on the surface before the application of shear stress, the mutant GP Ibα was unable to capture the cells onto the VWF surface from the fluid stream, an indication that steric hindrance from other cell surface molecules may prevent access of the GP Ibα ligand-binding site to the surfaceimmobilized VWF. ^† This work was supported by a Specialized Centers of Research Grant from the National Institutes of Health (1 P50 HL65967)

Platelet GP Ibα and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) are membrane mucins with a number of structural and functional similarities. It was investigated whether, like GP Ibα, PSGL-1 is affected by a variable number of tandem repeat polymorphism in its mucin-like region. By polymerase chain reaction amplification of the genomic region encoding the PSGL-1 repeats, 3 allelic variants were identified in the human population. The 3 alleles—A, B, and C—from largest to smallest, contained 16, 15, and 14 decameric repeats, respectively, with the B variant lacking repeat 2 and the C variant retaining repeat 2 but lacking repeats 9 and 10. Allele frequencies were highest for the A variant and lowest for the C variant in the 2 populations studied (frequencies of 0.81, 0.17, and 0.02 in white persons and 0.65, 0.35, and 0.00 in Japanese). Thus, PSGL-1 is highly polymorphic and contains a structural polymorphism that potentially indicates functional variation in the human population.

We have identified platelet glycoprotein (GP) Ibα as a counterreceptor for P-selectin. GP Ibα is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibα, and GP Ibα–expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin–dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibα. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibα required neither calcium nor carbohydrate core-2 branching or α(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibα, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.