Loss of the WASP-Interacting Protein, CIP4, Results In Murine Thrombocytopenia: Insights Into the Pathophysiology of WASP-Related Thrombocytopenia

Abstract 382

Wiskott-Aldrich Syndrome (WAS) is an X-linked disorder associated with thrombocytopenia and is due to loss of function mutations in the Wiskott-Aldrich Syndrome Protein (WASP) gene. WASP serves to trigger actin nucleation upon its binding to activated form of Cdc42, phosphorylation by Src kinases, and membrane phospholipid. Loss of function mutations in WASP results in thrombocytopenia and small platelets, but the precise mechanism remains elusive. Using the Src kinase Lyn as bait in yeast two-hybrid screen, we isolated Cdc42 interaction protein 4 (CIP4). CIP4 is a member of the F-BAR family of proteins, which generate membrane curvature and promote cortical actin polymerization. CIP4 possesses an SH3 domain that almost exclusively interacts with WASP and Dynamin, a GTPase involved in membrane scission. To better understand the physiologic role of CIP4, we made the CIP4 knockout mouse strain. We have reported that these mice display defects in clathrin-dependent and clathrin-independent endocytosis (Feng et al J Biol Chem 285:4348, 2010). Because of CIP4's interaction with WASP, we hypothesized that there may be defects in platelet production. Complete blood counts performed on C57BL/6 male mice between the ages of 3 and 6 months showed a decrease in platelets in CIP4^-/- mice comparable to that observed in WAS^−/0 mice (Table 1). No other defects were observed in hemoglobin and total leukocyte, absolute neutrophil, and absolute lymphocyte counts. Mean platelet volume was normal in CIP4^-/- as in WAS^−/0 mice. Bone marrow examination showed no dysmorphology. There was no difference in numbers of CFU-MK, CFU-GEMM, or CFU-GM between CIP4^-/- and wild-type mice. Since there are other cytoskeletal proteins that interact with WASP and may contribute to thrombocytopenia, we are currently generating CIP4, WASP double knockouts. Available data will be presented. Should the double knockout strain have the same degree of thrombocytopenia, this would strongly suggest that CIP4-WASP interaction constitutes the critical pathway for WASP-dependent platelet production. Propidium iodine staining of CD41+ bone marrow cells showed similar greater ploidy in wild-type than in CIP4^-/-. Also, CIP4^-/- megakaryocytes displayed fewer proplatet protrusions. Proplatelet protrusions are due to membrane remodeling and elongation due to actin and microtubule polymerization. This effect is similar to our observation of fewer invadopodia/MDA-MB-231 breast cancer cells. These findings suggest that CIP4 contributes to WASP-mediated thrombocytopenia by affecting membrane remodeling and protrusion stability. Since CIP4's ancestral protein Cdc15p is involved in the contractile actomyosin ring during cytokinesis in fission yeast, there may also be defects in endomitosis of the CIP4-deficient megakaryocyte.