Perturbing Hemostasis by Altering the Kinetics of the Interactions between GPIb Apha and the A1 Domain of VWF: A Mouse Tale.

Effective hemostasis relies on ability of circulating platelets to be rapidly deposited at sites of vascular injury. Critical to this process is the formation of adhesive bonds between the platelet receptor GPIb alpha and the A1 domain of VWF, an interaction that is rapid and reversible due to the relatively high rate of bond formation and dissociation. The importance of maintaining the kinetic properties of this interaction within a critical range is suggested by the bleeding phenotype that results from gain- or loss-of function points mutations associated with VWD. To demonstrate the an alteration in bond kinetics can have a dramatic effect of hemostasis, we generated a mouse in which a single point mutation was introduced into the vWF-A1 domain that results in a shortening of the time it can maintain contact with GPIb alpha (aka - increase in off-rate). The mutation was chosen based on our crystal structure data of the recombinant mouse A1 domain protein and its effect on mouse platelet adhesion to the immobilized mutant protein under flow conditions. Homozygous animals were viable and exhibited normal Mendelian ratios. Both VWF antigen levels as determined by ELISA and multimer banding pattern were identical to WT littermate controls. Moreover, transcription of the A1-A2-A3 region of VWF was comparable to controls as determined by RT-PCR. Despite the similarities in biochemical and molecular parameters to WT littermates, mutant A1 domain mice had a significant prolongation in bleeding time as measured by severing a small portion of their tail. Moreover, perfusion of whole blood from these animals over surface-immobilized collagen resulted in >50% reduction in platelet accumulation at 1600 s-1. This abnormality in platelet adhesion was also observed in vivo. Although mutant A1 domain mice were able to form thrombi upon laser-induced injury to arterioles located within the cremaster muscle, these thrombi were unstable and rapidly dissolved. In conclusion, we are the first to demonstrate that an alteration in the kinetic properties of a receptor-ligand bond can have a significant effect on biological processes such as hemostasis. Our results also provide insight into mechanisms by which point mutations associated with certain types of VWD may result in bleeding in afflicted individuals.