Binding of Thrombin to Glycoprotein Ib Accelerates the Hydrolysis of Par-1 on Intact Platelets

Abstract

The activation of human platelets by ␣-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ib␣ also has a high affinity binding site for ␣-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIb␣ may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by ␣-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k cat /K m value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 ؎ 0.1 ؋ Platelet activation by the coagulation protease ␣-thrombin plays a crucial role in physiologic hemostatic processes and in thrombotic diseases. The activation of human platelets by thrombin is mediated by at least two receptors belonging to the family of protease-activated receptors (PARs), 1 i.e. PAR-1 and PAR-4 (1-2). These receptors are activated upon cleavage by thrombin and mediate transmembrane signaling by coupling to G-proteins (1-2). ␣-Thrombin also binds with high affinity to the platelet glycoprotein Ib (GpIb) that belongs to the leucinerich repeat family of proteins (3). Thrombin binding to GpIb contributes to platelet activation by the enzyme, as demonstrated by the finding that Bernard-Soulier platelets, which lack the GpIb-IX-V complex, have a delayed response to thrombin stimulation (4). In addition, several in vitro studies have demonstrated that the inhibition of thrombin binding to GpIb by different strategies causes a reduction in thrombin-induced platelet activation (5-9).The mechanism by which the binding of ␣-thrombin to GpIb contributes to platelet activation is not clear. Numerous studies have shown that a proteolytic-active enzyme is required to activate platelets. PPACK-thrombin, which retains its ability to bind to GpIb, does not induce platelet aggregation (10). Moreover, GpIb does not undergo cleavage by thrombin. On the other hand, the cleavage of G-protein-coupled PARs seems to be essential in platelet activation and transmembrane signaling. The finding that thrombin binding to GpIb involves a distinct thrombin domain, the HBS, which is far from the thrombin catalytic site and the fibrinogen recognition site (7-9), would suggest that a ternary complex thrombin⅐GpIb⅐PAR-1 may form on the platelet membrane that could be responsible for optimal hydrolysis and signal transduction.In the present study, the hypothesis that thrombin binding to GpIb may affect the hydrolysis of PAR-1 by the enzyme on intact platelets was investigated. The hydrolysis of PAR-1 was evaluated as a paradigm to construct a model where GpIb acts as a cofactor for PAR(s) cleavage. EXPERIMENTAL PROCEDURESMaterials-Human ␣-thrombin w...

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

“…This PAR1 sequence extends from the active site to ABE I. Hydrolysis of full-length PAR1 on intact platelets is also hindered by increasing concentrations of Fbg ␥Ј peptide. By contrast, GpIb␣ binding can promote hydrolysis of PAR1 on platelets (44). Similar effects were not observed with a comparable PAR4 peptide substrate sequence (11).…”

“…Prior studies demonstrated that the presence of GpIb␣ at thrombin ABE II leads to enhanced cleavage of PAR 1 (44,45). PAR1 hydrolysis within the thrombin active site region benefits from anchoring a distal portion of PAR1 to ABE I.…”

Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4

“…Human platelets, however, express PAR1 and PAR4, which signal independently but with different thresholds (higher in PAR4) for thrombin activation. In addition, GPIba acts as a cofactor to enhance thrombin cleavage of PAR1 on intact platelets 57 . Therefore, predicting what the net effects of disrupting thrombin binding to GPIba might be on leukocyte responses in humans is not straightforward.…”

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

“…Platelets from healthy volunteers were gel-filtered on Sepharose 2B columns (GE Healthcare) as reported previously (22). Born's aggregation of gel-filtered platelets, performed on a 4-channel PACKS-4 aggregometer (Helena Laboratories, Sunderland, UK) as detailed previously (22), was induced by 1 nM thrombin in the absence or presence of different concentrations of ␥Ј peptide and fibrinogen fragment D*.…”

“…Cleavage of these peptides by 0.1-1 nM thrombin was monitored by RP-HPLC as detailed previously (22). The Michaelis-Menten parameters k cat and K m were calculated in the absence and presence of fixed concentrations of the ␥Ј peptide ranging from about 2.5 to 320 M. The k cat /K m values of PAR1 peptide hydrolysis in the presence of fragment D* (from 0.2 to 6.4 M) was calculated at a peptide concentration of 1 M, which is a concentration lower than the K m value of the thrombin-PAR interaction.…”