ROLE OF GLYCOPROTEIN-V IN THE FORMATION OF THE PLATELET HIGH-AFFINITYTHROMBIN-BINDING SITE

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ibn, GP Ib beta, GP IX, and GP V, The site within the com plex that binds thrombin has been localized to a 45-kD region at the a mino terminus of GP Ib alpha, which also contains the site through whi ch the complex interacts with von Willebrand factor. A GP Ib-IX comple x that lacks GP V can be efficiently expressed on the surface of trans fected cells, We examined the ability of L cells expressing the GP Ib- IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells, Becau se it is one of the few substrates for thrombin on the platelet surfac e, GP V has also been implicated as possibly participating in thrombin 's actions on the platelet, To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of throm bin to L2H cells versus cells that express the entire GP Ib-IX-V compl ex (L2H/V cells). Surface expression of GP Ib alpha was equivalent in these two stable cell lines, Thrombin binding to L2H/V cells was detec table at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L, No bi nding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently e xpressing GP V was compared with its binding to sham-transfected L2H c ells. Again, only cells transiently expressing GP V bound thrombin spe cifically. As with the platelet polypeptide, thrombin cleaved GP V fro m the surface of L2H/V cells. To test whether GP V cleavage was requir ed for enhancing thrombin binding to the complex, we tested the bindin g of enzymatically inactive D-phenylalanyl-Lprolyl-L-arginine chlorome thylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thromb in, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating t hat GP V cleavage is not a prerequisite for the formation of the high- affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the hi gh-affinity thrombin receptor on the platelet surface has complex allo steric requirements. (C) 1997 by The American Society of Hematology.