Direct interaction between GpIbα and thrombin is essential for initiation of platelet procoagulant activity, and exposure to very small concentrations (<1 nM) of thrombin is sufficient to induce aggregation and activation at sites of vascular injury. The portion of the GpIbα receptor studied is the extracellular domain, which resides outside the platelet membrane. The crystal structure of the complex, obtained to a resolution of 2.6 Å by the Wyeth group working at ALS Beamline 5.0.1 operated by the Berkeley Center for Structural Biology, provides important details of GpIbα–thrombin interactions, thereby opening promising avenues both for further investigation and for development of novel antithrombotic drugs to treat coronary artery and other diseases that involve reduced blood flow.

The charge distribution on the surface of thrombin (red is negative, blue is positive). The GpIbα receptor is shown in green with the surface rendered transparent.

Through Thick or Thin

Most of us are fortunate indeed that our blood clots. Otherwise, like those suffering from bleeding disorders such as hemophilia, we would find injuries as trivial as paper cuts to be life-threatening. Sometimes clotting is not so beneficial, as those surviving heart attacks and strokes resulting from narrowed arteries blocked by clots know full well. Clotting itself is a quite complex process. The passage of blood plasma, a largely watery fluid, over the rough surface associated with an injury, results in adhesion of platelets in the blood to damaged tissue, the first step in clotting. The subsequent chain of biochemical reactions eventually causes the generation of the enzyme thrombin, which then catalyzes the production of fibrin molecules that clump together into an insoluble mesh and capture red blood cells and platelets, thereby forming a clot.

One of the many key steps is that by which the platelets become sticky (activated) and more easily enmeshed in the fibrin. It turns out that thrombin plays a role here as well, when it binds to a structure called a receptor on the surface of platelets. Understanding the precise molecular mechanism by which the binding occurs offers opportunities for developing drugs to treat coronary artery and related diseases by controlling overzealous clotting. Dumas et al. have contributed an important insight into the binding mechanism by determining the molecular structure of the part of the receptor involved in the binding while the binding is underway.

The structure reveals that both the GpIbα and thrombin molecules are bivalent. Two highly electronegative regions on the surface of GpIbα simultaneously interact with two positively charged surface patches or "exosites" located on opposite poles of thrombin (exosites I and II). In the crystals, pairs of GpIbα and thrombin molecules associate as crystallographically independent complexes. Two types of GpIbα–thrombin interfaces that are related by crystal symmetry are evident within adjacent complexes of oligomeric arrays in which the two distinct binding sites continuously alternate throughout the crystal.

The first interface involves interactions between thrombin exosite I and a site on GpIbα that spans a surface area of about 1400 Ų. The binding interactions between GpIbα residues and residues of exosite I are predominantly electrostatic. A second binding interface lies between exosite II of a second thrombin molecule and two adjacent regions of GpIbα (the carboxy-terminal end of the extracellular domain and the convex surface). This extensive interface covers a surface area of about 2000 Ų. About 60% of this interface consists of a network of hydrogen bonds between the sulfated anionic region of GpIbα and highly basic residues in thrombin exosite II. Three tyrosine (Tyr) residues in this anionic region, including attached sulfate groups (necessary for optimal binding in vivo), participate in ionic and hydrophobic interactions with exosite II. In the remaining part of the GpIbα-exosite II interface, residues on the convex face of GpIbα make mostly hydrophobic contacts with thrombin.

Ribbon representation of two GpIbα–thrombin complexes related by crystal symmetry. The asymmetric unit contains one molecule of GpIbα (green) and one molecule of thrombin (blue), which associate as a crystallographically independent complex. The anionic region of GpIbα is colored red, and a region corresponding to a peptide that inhibits thrombin-mediated aggregation is gold. Thrombin exosite I and exosite II are colored dark blue.

In the context of cell–cell recognition and GpIbα-dependent platelet adhesion, the high affinity of thrombin for platelets and the associated thrombotic activity at physiologically relevant, low thrombin concentrations are likely accounted for by additive energetic contributions from individual GpIbα–exosite interfaces. In this regard, fortuitous crystal packing of the GpIbα receptor relative to thrombin provides a scaffold that could support tight multivalent adhesive interactions between platelets in vivo.

Schematic diagram of thrombin and GpIb-IX complexes in the region between platelets. GpIbα and thrombin molecules are arranged as an adhesive ribbon structure, as observed in the crystals. The region between the last residue observed in GpIbα and the stalk region is depicted as green dotted lines.

Given the mode of assembly depicted, a GpIbα receptor projecting from the cell membrane would interact with another GpIbα receptor indirectly through an intervening thrombin molecule. In this arrangement, GpIbα receptors from different membranes are aligned in alternating fashion, and the active site of thrombin remains accessible for other physiological substrates. This linear zipper configuration resembles the "cell-adhesion zipper" seen in other proteins involved in cell adhesion.

Research conducted by J.J. Dumas, R. Kumar, J. Seehra, W.S. Somers, and L. Mosyak (Wyeth Research).

Research funding: Wyeth Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: J.J. Dumas, R. Kumar, J. Seehra, W.S. Somers, and L. Mosyak, "Crystal structure of the GpIbα-thrombin complex essential for platelet aggregation,"Science 301, 222 (2003).