Platelet Cellular Prion Protein (PRPC) Is Associated with alpha Granules.
Authors: Holada Karel , Brouckova Adela , Simak Jan , Gahl William A. , Vostal Jaroslav G. 
Published: October 2019

The cellular prion protein (PrPc) is a membrane glycoprotein expressed on many human cells including blood platelets. We have previously shown that human platelets rapidly up-regulate PrPc on their plasma membranes after activation (Holada et al., Br J Haematol.1998;103(1):276–82.). Our preliminary results also showed that platelet PrPc is resistant to cleavage by phosphatidylinositol specific phospholipase C (PIPLC). In this study we investigated intracellular localization of platelet PrPc and its resistance to PIPLC of different origin and under different experimental conditions. To determine the intracellular localization of platelet PrPc, we used flow cytometry and PrPc directed monoclonal antibodies (MAb) 1562 and 6H4, which recognize residues 109–112, and 144–152, in human PrPc sequence, respectively. The specificity of MAb binding was confirmed by its inhibition with competing peptides. We compared the increase in PrPc expression after platelet activation with increase in expression of P-selectin (CD62) - an α-granular protein, and LIMP (CD63) - a lysosomal and δ-granular protein. Gel filtered platelets were activated by ADP (1–100 μM) or TRAP (0.5–50 μM). Increasing concentrations of agonists induced coexpression of P-selectin and PrPc on the platelet surface at lower concentrations than were required for expression of LIMP (e.g. 5 μM ADP vs. 50 μM ADP to reach 40% of maximal expression), suggesting that PrPc did not associate with lysosomes. To further address the question of the origin of intraplatelet PrPc, we evaluated the expression of platelet PrPc in two patients with Hermansky-Pudlak (H/P) syndrome and two patients with Grey platelet syndrome (GPS). H/P platelets have a low number of δ-granules, but normal numbers of α-granules and lysosomes. When compared to controls, the H/P platelets had decreased mepacrine staining, demonstrating a defect of δ-granules. The expression of LIMP was equivalent on resting control (15.2 geometric mean of FL1 (GMF)) and H/P platelets (14.4 GMF), but was substantially decreased on H/P platelets after full platelet activation (38.2 vs. 168.8 GMF). In comparison, similar levels of PrPc and α-granular P-selectin were expressed on normal (35.7 and 726 GMF) and H/P patient (33.9 and 689 GMF) activated platelets. Platelets of GPS patients are deficient in α-granules. Resting GPS platelets demonstrated higher expression of P-selectin (9.9 GMF) and PrPc (25.8 GMF) than normal platelets (4.9 and 14.3 GMF, respectively). In contrast to normal platelets, GPS platelets failed to up-regulate P-selectin (76 vs. 653 GMF) and PrPc (30.1 vs. 59.9 GMF) after activation. In order to confirm a resistance of PrPc to PIPLC in normal platelets, the presence of glycosyl-phosphatidylinositol (GPI) anchor was verified by the treatment of platelet PrPc with hydrofluoric acid, which resulted in a 2–4 kDa decrease of its molecular weight corresponding to the removal of GPI anchor. Interestingly, PIPLC enzymes of three different origins (B. thuringiensis, B. cereus, B. subtilis) did not cleave GPI anchor even after the solubilization of platelet membranes by Triton X-100, which should make anchor more accessible to the enzyme. In conclusion, our results suggest that platelet intracellular PrPc is associated with α-granules, but not with lysosomes, and δ-granules. Platelet membrane PrPc is resistant to PIPLC, likely due to GPI-modification rather than GPI accessibility.