The Gray platelet syndrome is a rare inherited disorder of platelets linked to undefined molecular abnormalities that prevent the normal formation and maturation of alpha-granules. This leads to the spontaneous release of endogenously synthesized alpha-granule proteins from megakaryocytes into the marrow, a factor favouring the development of fibrosis. The platelet functional response varies greatly in patients in the GPS. Here we examine patients from two unrelated families, patient 1 has platelets that have a much reduced aggregation response to collagen, and patient 2 has a normal response. Both patients possess platelets severely deficient in alpha-granules. Features of their internal stucture include numerous vacuoles, thin channels and a reduced surface-connected canalicular system (SCCS) and small vesicular structures (SVs) some of which are open to the surface and rapidly accessed by abciximab used as a probe for ligand trafficking in immunoelectron microscopy (I-EM). Residual amounts of fibrinogen, and VWF are often associated with the SVs, P-selectin delimits some elements of the SCCS and vacuoles while most internal and surface membranes are labelled for seven transmembrane receptors such as PAR-1 and TPalpha, Immunoblotting showed that unlike platelets of patient 1 where GPVI was severely decreased (
Nurden P et al, Blood 2004; 104:107–114
), those of patient 2 normally expressed this collagen receptor. TREM-like transcript-1 (TLT-1), a membrane glycoprotein, has recently been predominantly localized to the alpha-granules of human platelets on the basis of its codistribution with P-selectin. Using a rabbit antibody to a cytoplasmic domain sequence we have confirmed this distribution in normal platelets. Significantly, we then further showed by Western blotting (WB) that TLT-1, like GPVI, is severely reduced in the platelets of patient 1 but normally expressed in those of patient 2. Flow cytometry using permeabilized platelets showed that this decrease was selective compared to normal levels of JAM-3 and claudin-5, also members of the immunoglobulin family. I-EM showed residual TLT-1 associated with both internal and external membranes. One possibility is that a pathology-related metalloprotease activity (MMP) leads to spontaneous cleavage of the extracellular domains of GPVI and TLT-1 in the platelets of some patients. We therefore examined the platelets of patient 1 to see if MMP-2 and MMP-9, two proteases expressed in normal platelets, were present. I-EM first showed that while for normal platelets both proteases were in their majority localized to alpha-granules, other pools were present in the cytoplasmic and surface-linked membrane systems. The latter were retained in the platelets of patient 1 but flow cytometry with permeabilized platelets failed to show over-expression. Our results identify TLT-1 as a new glycoprotein potentially cleaved in GPS platelets and further show that so-far undefined protease activity contributes to phenotypic heterogeneity in this rare disease.
Disclosures: NIH (USA), INSERM/CNRS (France).
