There is a fundamental gap in understanding how platelet clearance is regulated. Until we gain a more
detailed understanding of this, we will lack effective therapies for some patients with low platelet counts
(thrombocytopenia) who are at risk for life-threatening bleeding. One important trigger for platelet clearance from
the body is desialylation, which refers to the removal of the sugar molecule sialic acid from glycoproteins on the
surface of the platelet. Platelet desialylation plays a role in accelerated clearance of platelets in immune
thrombocytopenia (ITP) and following transfusion of platelets that have been stored at cold temperatures.
Despite the clinical importance of desialylation, both the identity of the enzyme that cleaves platelet sialic
acid and its scope of glycoprotein substrates and products remain unknown, hampering efforts to develop
targeted therapies for ITP and other thrombocytopenic disorders. The central hypothesis is that human
neuraminidase 1 desialylates glycoprotein (GP) Iba O-glycans as well as other platelet surface glycoproteins,
thereby accelerating platelet clearance in ITP and after cold storage.
This hypothesis will be tested via the following specific aims: 1) Determine which GpIba glycans undergo
desialylation. Liquid chromatography/mass spectrometry (LC/MS) will be used to analyze GpIba purified from
platelets after desialylation is triggered in vitro either by incubation at 4°C or by incubation with sera from ITP
patients containing anti-GpIba autoantibodies. 2) Determine which human neuraminidase desialylates glycans
that are relevant for platelet clearance. The effect of a panel of potent and selective small molecule
neuraminidase isoenzyme inhibitors will be tested in in vitro platelet desialylation experiments. The in vivo half
life of platelets treated with these desialylation inhibitors will be measured. 3) Identify platelet neuraminidase
substrates. An established strategy for enrichment of membrane glycoproteins will be employed to allow LC/MS
proteomics analysis of desialylated vs control platelets. The outcome will be a delineation of the key platelet
neuraminidase enzyme which catalyzes desialylation and its glycoprotein substrates and products. This will
provide the first biochemical characterization of the process of platelet desialylation and will pave the way for
development of more effective therapies for thrombocytopenia.
The career development plan includes 1) training in chemical glycobiology as a member of the laboratory
of Prof. Carolyn Bertozzi at Stanford University and 2) training in platelet biology, mouse platelet transfusion
experiments, and immune thrombocytopenia under the mentorship of international experts at Stanford and
elsewhere, including Drs. Karin Hoffmeister, David Kuter, and Lawrence Leung. This will position Dr. Hollenhorst
to establish a unique niche as an independent physician-scientist investigator at the interface of chemical biology,
transfusion medicine, and non-malignant hematology.