In Vivo Relevance for Platelet Glycoprotein Ibα Residue Tyr²⁷⁶ in Thrombus Formation.

Background: GPIbα is a critically important platelet receptor best recognized for its ability to bind vWF and mediate platelet adhesion. In addition, a GP Ibα binding site for thrombin has been described and characterized for several decades. However, the physiologic relevance of GPIbα/thrombin binding has remained elusive. Site-directed mutagenesis has shown that a sulfated GP Ibα residue, Tyr²⁷⁶, is essential for thrombin binding to GP Ibα but not critical for vWF binding. The importance of Tyr²⁷⁶ was further substantiated in the crystal structure of a GP Ibα/thrombin complex confirming the sulfated Tyr²⁷⁶ GP Ibα residue directly participates with other neighboring negatively charged residues in establishing a first contact with the anion-binding exosite II of an α-thrombin molecule. To determine the physiologic relevance, if any, of the GP Ibα Tyr²⁷⁶ residue we generated mice with platelets containing a mutant human GP Ibα subunit containing a Tyr²⁷⁶ to Phe²⁷⁶ substitution (Y276F) and did comparative studies with mice expressing a normal human (WT) GP Ibα subunit.

Methods: Mouse colonies expressing the normal or variant GP Ibα subunits were established by transgenic technology and bred into a mouse colony devoid of murine GP Ibα. Surface expression of WT or Y276F GP Ibα subunits was measured by flow cytometry. Relevant assays included platelet counts, FeCl₃-induced thrombus formation, tail bleeding times, hemoglobin content following tail resection (measured as 575 nm absorbance of lysed erythrocytes), and washed platelet aggregation induced by different agonists (fibrillar type I collagen, thrombin, ristocetin, arachidonic acid, and PAR activating peptides).

Results: Founder mice were selected with nearly identical levels of surface-expressed WT and Y276F GPIbα subunits. Circulating platelet counts were similar between the 2 colonies. Stirred platelet aggregation assays induced by 0.05, 0.1, 0.5 units/ml thrombin, 10 and 20 mg/ml fibrillar collagen, 1.25 mg/ml ristocetin, 0.25 mM arachidonic acid, 100 mM PAR3 activating peptide and 100 mM PAR4 activating peptide revealed no significant differences between WT and Y276F samples. No statistically significant values were obtained in tail bleeding time assays or hemoglobin loss comparing WT and Y276F animals. However, the time required for a reduction in carotid artery blood flow following FeCl₃ injury was significantly prolonged in Y276F animals (10.6±1.6 min vs. 5.8±1.4 min). All WT animals displayed complete occlusion following the initial reduction in blood flow. In contrast, 3 out of 6 Y276F animals did not show complete occlusion and had evidence of fluctuating blood flow suggestive of embolization.

Discussion: FeCl₃ injury of the mouse carotid artery revealed an essential role for GP Ibα residue Tyr²⁷⁶. In contrast, no differences were seen in tail bleeding time assays or in any platelet aggregation assay. These results are suggestive that thrombin binding to human GPIbα may be relevant for the pathological development of a thrombus. This conclusion is based on both the failure of some carotid arteries from Y276F animals to fully occlude and our observation that all Y276F animals showed an increased length of time for the reduction of blood flow. The results suggest thrombin binding to GPIbα could be significant in terms of enhancing fibrinogen cleavage and therefore stabilizing the development of a platelet-rich thrombus.