GPIbα As a Selective Bidirectional Modulator Of α-Thrombin Prothrombotic Functions Influencing Fibrin Deposition and PAR-Dependent Platelet Activation

Generation of α-thrombin (FIIa) in response to vascular injury is a key host defense mechanism influencing thrombus formation and inflammation. Blood platelets express glycoprotein (GP) Ibα as the most abundant FIIa membrane binding site, as well as different protease activated receptors (PARs) with an effector role in platelet activation after proteolytic cleavage. The functional role of GPIbα, which is not a substrate for FIIa, relative to that of different PARs remains unclear.

Goal of these studies was to define with mechanistic understanding whether and how binding to GPIbα can modulate FIIa prothrombotic functions in vivo and ex vivo.

Endogenous mouse platelet GPIbα was replaced by the human (hu) counterpart with wild type (WT) sequence; or containing the single substitution of Asp277 (mutated to Asn), which interacts selectively with a site involving FIIa exosite 2; or with the combined substitution of post-translationally sulfated Tyr276, Tyr278 and Tyr279 (each mutated to Phe), which interact with FIIa residues in proximity of exosite 1 as well as exosite 2. These mice were evaluated in intravital models of arterial thrombosis. Moreover, their platelets were tested ex vivo for the response to FIIa-induced activation measuring changes in intracytoplasmic Ca²⁺ levels; and for effects on fibrinogen clotting and fibrin formation. Comparative ex vivo experiments were conducted with human and huGPIbα-WT mouse platelets in which FIIa binding was similarly blocked by the anti-human GPIbα monoclonal antibody, LJ-Ib10. Ex vivo FIIa effects on platelet activation/aggregation and fibrin clot formation were also evaluated concurrently in a model of thrombus formation in blood perfused over a thrombogenic surface under controlled flow conditions.

Genetically modified mouse platelets expressed ≈9000 WT or mutant huGPIbα molecules; platelets with huGPIbα-WT bound ≈10,000 FIIa molecules with 1:1 stoichiometry and K_D of ≈3 nM. FIIa binding to mutant huGPIbα was essentially abolished. Mice with defective FIIa binding to GPIbα exhibited a pronounced prothrombotic phenotype, with a shorter time to carotid artery occlusion following ferric chloride injury (median 550.5 seconds in 18 mutant huGPIbα, vs. 1980 seconds in 19 huGPIbα-WT mice; P<0.01). Accordingly, the platelet-rich plasma (PRP) of mutant huGPIbα mice exhibited a significantly shorter clotting time in the presence of 4 nM FIIa and significantly enhanced intracytoplasmic Ca²⁺ transients and platelet aggregation following stimulation by 0.5 nM FIIa. Human platelets, similar to mouse platelets, bound FIIa with a 1:1 stoichiometry relative to GPIbα and K_D of ≈3 nM. Remarkably, blocking FIIa binding to GPIbα with antibody LJ-Ib10 essentially abolished activation by 1 nM FIIa in human platelets, in which FIIa effects are mediated predominantly by PAR1; this was in contrast to the enhanced activation seen under the same conditions in hu GPIbα-WT mouse platelets, in which FIIa acts through PAR3 and PAR4. Accordingly, the volume of platelet aggregates and fibrin formed in huGPIbα-WT mouse blood perfused over a thrombogenic surface was enhanced by blocking FIIa binding to platelets; in contrast, the volume of platelet aggregates, but not that of fibrin clots, was decreased under the same conditions in human blood. Antibody LJ-Ib10 shortened the clotting time of both huGPIbα-WT mouse and human PRP; however, in the absence of GPIbα-bound FIIa, fibrin associated with platelet aggregates had a less ordered fibrillar structure.

Our findings identify GPIbα as a relevant FIIa activity modulator. Through distinct mechanisms influenced by the expression of specific PAR subtypes, GPIbα can modulate FIIa function in hemostasis and thrombosis both enhancing and controlling prothrombotic responses and, thus, size and structure of platelet/fibrin thrombi. The effect of GPIbα on PAR4-mediated platelet activation, as well as fibrinogen clotting, can be explained by competition for FIIa exosites required for substrate binding, but the mechanism supporting the distinct GPIbα-PAR1 functional association remains to be elucidated.

No relevant conflicts of interest to declare.