Role of DNAJC10, An Hsp40 Type Chaperone, In αIIbβ3 Biogenesis In Human Megakaryocytes

Abstract 2111 INTRODUCTION: The integrin αIIbβ3 mediates platelet aggregation by binding to fibrinogen and other plasma proteins. Inherited defects of αIIbβ3 result in the mucocutaneous bleeding disorder, Glanzmann thrombasthenia (GT). However, pharmacological inhibition of αIIbβ3 can reduce mortality after myocardial infarction and percutaneous stent placement. Thus the degree to which αIIbβ3 functions is critical. One element influencing the degree of αIIbβ3 function is its level of surface expression on the platelet. The post-translational mechanisms controlling surface expression remain unclear, but their stringency is evident in certain GT patients whose full length αIIbβ3 complexes are completely retained and degraded intracellularly. A clearer understanding of the post-translational processes controlling αIIbβ3 surface expression can provide insight into a pathway that is a potential target for novel αIIbβ3-targeted therapies. Toward that end we previously reported the interaction of αIIb with DNAJC10, an hsp40 type chaperone protein, which was identified by a proteomic analysis of αIIb-interacting proteins. Continuing that investigation, we have explored the function of DNAJC10 in αIIbβ3 biogenesis pathway. METHODS: Megakaryocytes were derived from human umbilical cord blood (UCB) by enrichment of UCB leukocytes for CD34+ progenitor cells followed by culture in serum-free medium with thrombopoietin and stem cell factor. Under our conditions the UCB leukocytes differentiate into a population of large cells of which > 90% express αIIbβ3, > 80% express GPIb, and about 50% express α2β1. Interaction of αIIb and β3 with DNAJC10 was tested by co-immunoprecipitation with mAbs specific for αIIb (CA3, M148, B1B5), b3 (7H2), or the αIIbβ3 complex (10E5). Megakaryocytes were treated with a proteasome inhibitor (MG-132) to increase the capture of αIIb (and potential interacting proteins) that had been routed to the proteasome for degradation. DNAJC10, αIIb, and β3 localization within megakaryocytes and platelets was observed with immunofluorescence, and compared to localization of the endoplasmic reticulum (ER) and Golgi. The effect of siRNA-mediated knockdown of DNAJC10 on αIIbβ3 expression was assessed by flow cytometry and immunofluorescence. RNA depletion was confirmed by quantitative RT-PCR. RESULTS: DNAJC10 co-immunoprecipitated with αIIb-specific (CA3, M148, B1B5), β3-specific (7H2), and complex-specific (10E5) mAbs, indicating that it associated with αIIbβ3 in megakaryocytes. An antibody (B1B5) that recognized pro-αIIb and pro-αIIbβ3 co-immunoprecipitated much more DNAJC10 than antibodies preferentially recognizing mature αIIb and αIIbβ3. The amount of DNAJC10 immunoprecipitated was markedly increased after cells were treated with a proteasome inhibitor. siRNA against DNAJC10 resulted in significantly elevation of αIIbβ3 surface expression on megakaryocytes as judged by flow cytometry. Immunofluorescence with confocal microscopy surprisingly revealed that DNAJC10, nominally an endoplasmic reticulum protein, did not colocalize with ER markers, but was distributed in foci throughout the megakaryocytes, proplatelet processes, platelet buds, and mature peripheral blood platelets. CONCLUSIONS: DNAJC10 engages the precursor forms of αIIb and αIIbβ3 at a point upstream of degradation and may be an intermediate step in targeting these to the proteasome. Since siRNA mediated knockdown of DNAJC10 resulted in increased αIIbβ3 surface expression, this chaperone may be a negative regulator of αIIbβ3 surface expression. Disclosures: No relevant conflicts of interest to declare.