Altered microtubule equilibrium and impaired thrombus stability in mice lacking RanBP10

Abstract

The crucial function of blood platelets in hemostasis is to prevent blood loss by stable thrombus formation. This process is driven by orchestrated mechanisms including several signal transduction cascades and morphologic transformations. The cytoplasmic microtubule modulator RanBP10 is a Ran and ␤1-tubulin binding protein that is essential for platelet granule release and mice lacking RanBP10 harbor a severe bleeding phenotype. In this study, we demonstrate that RanBP10-nullizygous platelets show normal adhesion on collagen and von Willebrand factor under flow conditions. However, using a ferric chloride-induced arterial thrombosis model, the formation of stable thrombi was significantly impaired, preventing vessel occlusion or leading to recanalization and thromboembolization. Deltagranule secretion was normal in mutant mice, whereas platelet shape change in aggregometry was attenuated. Lack of RanBP10 leads to increased ␤1-tubulin protein, which drives ␣-monomers into polymerized microtubules. In mutant platelets agonists failed to contract the peripheral marginal band or centralize granules. Pretreatment of wild-type platelets with taxol caused microtubule stabilization and phenocopied the attenuated shape change in response to collagen, suggesting that RanBP10 inhibits premature microtubule polymerization of ␤1-tubulin and plays a pivotal role in thrombus stabilization. (Blood. 2012;120(17): 3594-3602) IntroductionMammals share a unique mechanism to ensure that injured blood vessels are sealed and loss of blood is minimized. Although specialized cells for this purpose are even found in invertebrates, the generation of anuclear cell fragments, designated platelets, are only present in mammals where they bud off cytoplasmic protrusions emanating from the megakaryocyte periphery. 1 These precursor cells form proplatelets and release nascent platelets across the endothelial barrier into the blood stream. 2 Endothelial cell lesions trigger platelets to make direct cellular contact with the subcellular matrix, which leads to platelet adhesion at the site of injury. Circulating platelets bind to an adhered layer of platelets, resulting in aggregation and thrombus formation, which is restricted to the site of injury and may ultimately occlude the vessel. 3 This process is tightly regulated as false activation leads to thrombosis and infarction, one of the major causes of death in the Western countries. Platelet activation is a multistep process. A hallmark of activation is the release of bioactive small molecules and proteins stored in 3 defined sets of granules present within the platelet cytoplasm: release of ␣-granules leads to surface expression of CD62P; CD63 is a marker for lysosomal granules; and dense granules harbor nucleotides such as ATP, ADP, amines, and bivalent cations. Although the granule markers are wellcharacterized, the molecular and cell biologic mechanisms underlying the process of granule release are less well understood. [4][5][6] Platelets harbor a cortical ring of bundled microtubules. In r...

Aggresome formation is regulated by RanBPM through an interaction with HDAC6

“…RanBPM and RanBP10 display 67% amino acid sequence identity and, while having divergent N-terminal domains, share a SPRY, LisH, and CTLH domains (Deshmukh and Johnson, 1998; Wang et al, 2004). Thus, microtubule association and regulation may be a common feature of both proteins; however, their functions at microtubules appear to be distinct since the RanBP10 knockout mouse platelet microtubule defects are not compensated for by RanBPM (Kunert et al, 2009; Meyer et al, 2012). Interestingly, HDAC6 was recently shown to regulate tubulin deacetylation during platelet activation, raising the intriguing possibility of a potential interplay between HDAC6 and RanBP10 in platelet activation (Sadoul et al, 2012).…”

miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease

“…Microtubule depolymerization and repolymerization were determined according to the method developed in a previous study 13. N2a cells were transfected with Ds-Red, miR-196a-DsRed or RANBP10-dsRed , cultured at 37℃ for 48 hours, and some cells were fixed using 4% paraformaldehyde as the control stage.…”

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss

“…Incubation of platelets from controls at 4°C caused disassembly of microtubules, which then reassembled to the marginal band after rewarming to 37°C, as previously reported. [22][23][24] In contrast, cold incubation or rewarming did not grossly affect the microtubules in platelets from the DIAPH1 R1213* cases ( Figure 6A-B), suggesting that the increased microtubule content resulted from increased microtubule stability.…”

“…Reassembly was allowed by subsequent rewarming at 37°C as previously reported. [22][23][24] Detailed methods and uncropped images of western blots are provided in supplemental Methods and supplemental Figures, respectively, available on the Blood Web site.…”