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Role of glycoprotein V in the formation of the platelet high-affinity
thrombin-binding site
JF Dong, G Sae-Tung and JA Lopez
Department of Internal Medicine, Baylor College of Medicine and Veterans
Affairs Medical Center, Houston, TX 77030, USA.
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 Ib
alpha, GP Ib beta, GP IX, and GP V. The site within the complex that binds
thrombin has been localized to a 45-kD region at the amino terminus of GP
Ib alpha, which also contains the site through which the complex interacts
with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be
efficiently expressed on the surface of transfected 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. Because it is one of the few substrates for
thrombin on the platelet surface, 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 thrombin to L2H cells versus cells that express the entire GP
Ib-IX-V complex (L2H/V cells). Surface expression of GP Ib alpha was
equivalent in these two stable cell lines. Thrombin binding to L2H/V cells
was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L.
No binding to L2H cells was detectable at these concentrations. Comparable
results were obtained when thrombin binding to L2H cells transiently
expressing GP V was compared with its binding to sham- transfected L2H
cells. Again, only cells transiently expressing GP V bound thrombin
specifically. As with the platelet polypeptide, thrombin cleaved GP V from
the surface of L2H/V cells. To test whether GP V cleavage was required for
enhancing thrombin binding to the complex, we tested the binding of
enzymatically inactive D-phenylalanyl-L-prolyl-L- arginine
chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native
thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating
that 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
high-affinity thrombin receptor on the platelet surface has complex
allosteric requirements.
Volume 89,
Issue 12,
pp. 4355-4363,
06/15/1997
Copyright © 1997 by The American Society of Hematology
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