Generation and rescue of a murine model of platelet dysfunction: The Bernard-Soulier syndrome

Abstract

The human Bernard-Soulier syndrome is an autosomal recessive disorder of platelet dysfunction presenting with mild thrombocytopenia, circulating ''giant'' platelets and a bleeding phenotype. The bleeding in patients with the Bernard-Soulier syndrome is disproportionately more severe than suggested by the reduced platelet count and is explained by a defect in primary hemostasis owing to the absence of the platelet glycoprotein (GP) Ib-IX-V membrane receptor. However, the molecular basis for the giant platelet phenotype and thrombocytopenia have remained unresolved but assumed to be linked to an absent receptor complex. We have disrupted the gene encoding the ␣-subunit of mouse GP Ib-IX-V (GP Ib␣) and describe a murine model recapitulating the hallmark characteristics of the human Bernard-Soulier syndrome. The results demonstrate a direct link between expression of a GP Ib-IX-V complex and normal megakaryocytopoiesis and platelet morphogenesis. Moreover, using transgenic technology the murine Bernard-Soulier phenotype was rescued by expression of a human GP Ib␣ subunit on the surface of circulating mouse platelets. Thus, an in vivo model is defined for analysis of the human GP Ib-IX-V receptor and its role in the processes performed exclusively by megakaryocytes and platelets.

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

“…A second mouse colony expressing the normal human GP Ib␣ subunit in platelets (hTg WT ) has been previously described and serves as the control for the G233V phenotype. 15 Phenotypic comparison of the two colonies confirms that the salient features of human Pt-vWD, such as impaired hemostasis, are present in the model. Histology and skeletal phenotyping of the transgenic animals revealed a significant increase in splenic megakaryocytes and the unexpected identification of a high bone mass phenotype, the result of a decrease in osteoclast number that results in an overall increase in the biomechanical strength of the femurs of hTg G233V mutants.…”

“…Multiple founders expressing the G233V transgene were initially identified, but most of the subsequent work was performed from one founder because by flow cytometry the expression of GP Ib␣ antigen was similar to the previously described hTg WT control. 15 Expansion of the hTg WT and hTg G233V colonies was accomplished by breeding to a mouse GP Ib␣ Null colony, a colony that has been backcrossed into the C57BL/6J strain since its inception. Briefly, hTg G233V animals were generated by breeding mice expressing the mutant human transgene into a knockout colony to produce offspring with heterozygous mouse GP Ib␣ alleles (mGPIb␣ ϩ/Ϫ ) and a portion of the offspring expressing the transgene.…”

“…15 A variant GP Ib␣ transgene cassette was generated by site-directed mutagenesis replacing the Gly 233 codon with a Val 233 codon. Expression of the transgene used a megakaryocyte-specific promoter in a identical manner as described for hTg WT15,19,20 and was confirmed by flow cytometry using fluorescein isothiocyanate-labeled anti-human monoclonal antibodies.…”

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

“…For this, we replaced the Asp277 codon with Asn (h-GPIba D277N ) in the same EcoRI DNA fragment. Replacement of the endogenous homologue with huGPIba in mouse platelets was achieved as previously described 68 by crossing C57BL/6J GPIba À / À and h-GPIba WT or h-GPIba D277N transgene-expressing mice. Expression of the human transgene in the absence of murine GPIba was determined using flow cytometry, and mice with the desired phenotype were bred to establish colonies for experimental use.…”

Genetic variability of platelet glycoprotein Ibα gene

“…Loss of function mutations in the human GPIba gene is the most common molecular defect associated with the bleeding disorders known as Bernard-Soulier syndrome [2]. Disruption of the murine GPIba gene recapitulates closely the human disease [3]. Platelets also participate in atherogenesis and subsequent formation of occlusive thrombi is dependent on platelet adhesion to the atherosclerotic plaque rupture [4].…”