High-affinity alpha-thrombin binding to platelet glycoprotein Ib alpha: identification of two binding domains.

Abstract

The platelet thrombin binding site(s) has been a controversial issue. Vu and coworkers (4-6) have cloned a platelet thrombin receptor which is a member of the 7-transmembrane loop-receptor family. This receptor is cleaved by thrombin between residues R41 and S42, exposing a new amino-terminal peptide, which serves as a tethered ligand whose binding site resides within the first six amino acids. This tethered ligand peptide binds to an undefined site that induces receptor activation (7-9).Prior to the discovery and cloning of the seven-transmembrane loop receptor, several studies had defined platelet glycoprotein lb (GPIb) as an a-thrombin receptor. These studies described the binding of thrombin to GPIb and the inhibition of a-thrombin binding to GPIb by glycocalicin (a proteolytic fragment of GPIba) and monoclonal antibodies directed against . The decrease or absence of high-affinity a-thrombin binding to Bernard-Soulier platelets, which lack GP~ba (17), provided further evidence that GPIb was a thrombin receptor. In experiments using crosslinking of a-thrombin to human platelets, the only identifiable site of binding was GPIb (18,19).The proposed roles of GPIba in thrombin binding include acting as a high-affinity receptor for localizing a-thrombin near the cell surface or serving as a site for excess thrombin binding (20). In this scheme, GPIba acts primarily as a neutralizing site for excess thrombin generation or as an independent modulator of platelet function through an undefined mechanism. We have studied the high-affinity binding of a-thrombin to human platelets and have examined the inhibitory effects of synthetic peptides derived from portions of the sequence of the GPIba chain and from the seventransmembrane loop thrombin receptor in order to assess the site(s) of high-and moderate-affinity thrombin binding to platelets and thrombin-induced platelet aggregation.MATERIALS AND METHODS Platelet Preparation. Blood was obtained by venipuncture using a two-syringe technique. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml of sodium citrate anticoagulant, final concentration 10.9 pM. Platelet-rich plasma was prepared from whole blood after centrifugation at 750 x g for 3 min at 250C. Then 6-10 ml of platelet-rich plasma was placed on a discontinuous Larcoll (Sigma) gradient, 20%o (3 ml) and 10% (5 ml), and the platelets were separated from the plasma proteins by centrifugation at 2000 x g for 30 min. The platelets were then resuspended in Hepes buffer at pH 7.35. All platelets were used within 2 hr of being obtained.Thrombin Lalin. Human a-thrombin was prepared and radiolabeled as described (21). Two hundred fifty microliters of a-thrombin solution (1 pg/2 units of activity) was treated with 5 Ad oflactoperoxidase beads, 5 td of0.5 mM KI, 0.5 mCi (18.5 MBq) of Na125I, and 1 A4 of 0.015% H202 for 5 min at room temperature. Free 125I was separated from bound 125I on a 10-ml column of Sephadex G-25 fine equilibrated with 0.05 M Tris/0.1 M NaCl at pH 7.35. Fractions were ...

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…6). At least one thrombin-binding site on GPIb␣ has been localized to residues 269 -287 (54) and is mediated by electrostatic interactions and perhaps direct contacts. FXI binding to GPIb␣ through its A3 domain is achieved when one of its homodimers is first complexed with HMWK or prothrombin (14,23,24).…”

Platelet Glycoprotein Ibα Binds to Thrombin Anion-binding Exosite II Inducing Allosteric Changes in the Activity of Thrombin

“…Glycocalicin can be released from the surface of platelets by proteolysis near the platelet membrane and consists of two subregions known as the macroglycopeptide and the amino-terminal domain (14). It is the amino-terminal domain, which consists of about 300 amino acids, that provides the sites within glycocalicin for ligand interaction (3), and within this the thrombin-binding site has been localized between residues 269 and 287 (17). The site on thrombin, however, where GPIb␣ binds is more controversial.…”

Thrombin Primes Responsiveness of Selective Chemoattractant Receptors at a Site Distal to G Protein Activation

“…In platelets, two glycoproteins (GPIb and GPV) interact with thrombin, but the biological significance of these interactions is not known (18, 19, 30 -32). GPIb is a high affinity thrombin receptor in human platelets (18,19). The demonstration that ␥-thrombin, which does not bind to GPIb in human platelets (33), causes priming in RBL-2H3 cells suggests that GPIb is unlikely to be involved in priming.…”

“…This tethered ligand binds to the thrombin receptor and induces its activation. Thrombin also binds to the platelet membrane glycoprotein Ib (GPIb) to activate Ca 2ϩ mobilization and platelet aggregation (18,19). However, thrombin-induced chemotaxis in monocytes and growth factor-like effect in fibroblasts appear to be mediated via the activation of as yet undefined thrombin receptors (14,20,21).…”