Hyperfunctional Platelets in a Murine Model of Platelet-Type von Willebrand Disease.
Authors: Ware Jerry , Russell Susan , Dent Judith , Kanaji Taisuke 
Published: April 2020

Abstract
Insights into the regulation of platelet adhesion have historically been aided by congenital bleeding disorders. Two clinically similar disorders, distinct at the molecular level, are platelet-type von Willebrand disease (Pt-vWD) and type 2B von Willebrand disease (vWD). In each case, the receptor (Pt-vWD) or ligand (type 2B vWD) has undergone a structural change supporting the binding of soluble von Willebrand factor (vWF) to platelets. The net result is an increased affinity between circulating vWF and the platelet glycoprotein (GP) Ib-IX receptor leading to thrombocytopenia and the risk of a bleeding event. In the case of Pt-vWD several missense mutations within the glycoprotein (GP) Ibα subunit of the GP Ib-IX complex have been described as the cause of Pt-vWD. The first described genetic mutation produces a Gly233 to Val233 (G233V) substitution within the GP Ibα amino-terminus. Crystallographic studies of isolated recombinant domains of GP Ibα have documented structural perturbations within the vWF binding site as a consequence of the G233V substitution. The hypothesis exists that these structural changes lead to an increased affinity between GP Ib-IX and vWF. We report the generation of mice expressing a human GP Ibα subunit with a G233V substitution. The animals were generated by traditional transgenic techniques using the human GP Ibα promoter sequence. Human Pt-vWD is autosomally dominant where both wild-type GP Ibα and Pt-vWD variant alleles are present. Mice expressing the G233V transgene were bred to mice deficient in mouse GPIbα producing a number of different genotypes for characterization. Mouse platelets were characterized from animals with (i) wild-type mouse GP Ibα alleles (mGp1b+/+); (ii) devoid of murine GP Ibα but expressing a wild-type human GPIbα allele (mGp1b−/−;hGp1b+); (iii) a single mouse GPIbα wild-type allele and the human G233V variant (mG1b+/−;hGp1bG233V); and (iv) devoid of murine GP Ibα and expressing the human G233V variant (mGp1b−/−;hGp1bG233V). In vivo, the effect of the Pt-vWD G233V human variant produces a mouse thrombocytopenia with increased bleeding as determined in tail bleeding time assays. The most severe phenotype was observed in mice with the mGp1b−/−;hGp1bG233V genotype. One of the diagnostic characteristics of human Pt-vWD is the agglutination of platelets with subthreshold levels of the agonist, ristocetin. Platelet-rich plasma from mice expressing the G233V variant (mGp1b-/-;hGp1bG233V) aggregate with ristocetin (0.25 mg/ml) unlike their counterparts expressing a wild-type human GP Ibα sequence (mGp1b−/−;hGp1b+) that require at least 0.8 mg/ml ristocetin. To observe the ristocetin effect required supplementing mouse platelet-rich plasma with purified human vWF illustrating the species-specificity of ristocetin for human vWF. The characterization of this model documents an animal with hyperfunctional platelets owing to an increased vWF/GP Ib-IX interaction. The generation of a Pt-vWD murine model provides an in vivo setting to examine the role of platelets with increased adhesive potential in diseases where the platelet is gaining recognition as important mediators of inflammatory responses, tumor metastases and angiogenesis.