Page 142 - Haematologica Vol. 107 - September 2022

ARTICLE - A mouse model of humanized type 2B VWD
S. Kanaji et al.
Our type 2B VWD mouse model exhibited exaggerated pla- telet phenotype compared to human patients. In fact, pla- telet count in VWF2Bhet hGPIba mice was about 5% of the control mice with normal VWF A1 sequence (VWFhA1 hGPIba), which was more severe than human patients having the same mutation.5 In addition, hGPIba was cleaved from platelet surface, and VWF2Bhomo hGPIba mice were more severely affected than VWF2Bhet hGPIba mice. Shed- ding of mouse GPIba has also been reported in a study ex- pressing mutant VWF by hydrodynamic gene transfer.24 In their study, mouse GPIba cleavage was not reduced when mutant VWF was expressed in mice deficient in ADAM17, indicating ADAM17-independent cleavage.24 Mechanical cleavage by shear stress caused by engagement of hGPIba binding to VWF A1 may contribute to the shedding of hGPIba. Shedding of hGPIba from the platelet surface has not been previously documented in human type 2B VWD patients.22 Thus, cleavage of hGPIba observed in type 2B VWD model might be caused by mouse-specific factors. Of note, both VWF2Bhet hGPIba and VWF2Bhomo hGPIba mice presented marked splenomegaly (see Figure 4). In the de- veloping embryo, hematopoiesis occurs in the liver and spleen. In mouse but not in hu-man, the spleen remains as hematopoietic organ throughout the life.29 In our study, histological analysis revealed abundant presence of MK in the spleen of VWF2Bhet hGPIba and VWF2Bhomo hGPIba mice, suggesting extramedullary megakaryopoiesis and potential exposure of MK and platelets to plasma VWF in the open circulatory system of spleen. In support of this idea, trans- plantation of VWF-/- hGPIba mouse BM into VWF2Bhomo hGPIba (2B MKnull) showed splenic MK bound to and inter- nalized plasma-derived mutant VWF, and presence of VWF- containing platelets in the circulation of 2B MKnull mice. Thus, we speculate that the extramedullary megakaryo- poiesis in the spleen contributes to exaggerated platelet phenotype observed with our type 2B VWD model mice. Another interesting finding is that the expression of gain- of-function mutant VWF protein leads to MK ploidy shift to mature population (see Figure 4). Plasma TPO level was not significantly changed in VWF2Bhet hGPIba compared to VWFhA1 hGPIba, indicating that the ploidy shift was not caused by elevated TPO levels but by direct influence of aberrant VWF binding to MK. In support of this idea, MK ploidy shift observed in VWF2Bhet hGPIba mouse was re- versed by administration of NMC-4 which blocks VWF A1- GPIba interaction. Taken together, these results demonstrate that aberrant VWF-hGPIba occurs not only in platelets but also in MK, the latter leads to altered mega- karyopoiesis and possibly platelet generation. We have pre- viously reported signaling effects of GPIba cytoplasmic tail on MK proliferation and maturation.30 Crosstalk of GPIba signaling with TPO-Mpl pathway, mediated by 14-3-3ξ and phosphoinositol-3-kinase/Akt (PI3K/Akt) pathways, might be involved in the effect of gain-of-function mutant VWF
on MK proliferation and maturation.30 Further study is required to elucidate the mechanism of gain-of-function VWF A1 on GPIba signaling.
As to the functional defect of platelets, impaired activation of the small GTPase Rap1 has been previously reported as a cause of thrombocytopathy and bleeding tendency in type 2B VWD model mice.22 Subsequent study determined preactivation and exhaustion of the PKC pathway as the cause of impaired Rap1 signaling and thrombopathy in type 2B VWD.24 In addition, the same group identified the effect of dysregulated RhoA/ROCK/LIMK/cofilin pathway on Mk and macrothrombocytopenia.23 In the current study, we found enhanced degradation of intracellular FlnA, a major substrate for m-calpain in VWF2Bhet hGPIba and VWF2Bhomo hGPIba mouse platelets (Online Supplementary Figure S1).21,31 FlnA is a scaffold protein which links cytoskeletal proteins to GPIb-IX in platelets and plays an important role in the regulation of contractile force and production of nor- mal size platelets.32,33 We have previously reported in a mouse model of sitosterolemia that activation of m-calpain and enhanced degradation of FlnA led to cytosolic redis- tribution of FlnA, thereby contributing to generation of large platelets.21 In the current study, there was a signifi- cant population of VWF2Bhet hGPIba and VWF2Bhomo hGPIba mouse platelets refractory to agonist stimulation and failed to bind fibrinogen, which was similar to what was pre- viously observed with sitosterolemia mice. Thus, m-calpain activation and FlnA degradation may partly explain the mechanism for platelet abnormalities observed in type 2B VWD.21 Further studies are warranted to elucidate the in- volvement of FlnA in generation of large platelets in type 2B VWD.
Currently, type 2B VWD is treated mostly with VWF replace- ment therapy with adjunct therapies used for other types of VWD.34 1-Desamino-8-D-arginine vasopressin (DDAVP) needs to be carefully considered because of the concern for the exacerbation of thrombocytopenia. VWF A1-GPIba has been focused as a target for the development of thera- peutics against type 2B VWD.35-37 Interestingly, MK ploidy shift,thrombocytopenia,andGPIbacleavagewerefoundto be corrected by administration of an antibody against VWF A1 which blocks binding to GPIba (NMC-4). However, in agreement with our previous report on VWFhA1 hGPIba mice,13 tail bleeding time of our type 2B VWD mice was not corrected after normalization of platelet counts by high- dose NMC-4 administration. Due to the primary role in hemostasis, targeting VWF A1-GPIba needs to be carefully studied and our animal models serve as useful tools for in vivo evaluation. Dysregulated VWF-GPIba interaction can be seen not only in type 2B VWD but also in platelet-type VWD (PT-VWD), which is caused by mutations in GP1BA.38 PT-VWD mouse models have been previously generated and platelet phenotypes similar to type 2B VWD have been reported.25,39-42 Thus, results obtained from this study will
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