A deficiency of glycoprotein (GP) VI in platelets of a patient with gray platelet syndrome

Abstract number: OC068

Nurden^* P., Kunicki† T., Combrie^* R., Winckler^* J., Paponneau^* A., Pasquet^* J. M., Jandrot-Perrus‡ M., Nurden^* A.

‡INSERM E9907, France †Scripps Research Institute IInstitute, USA; ^*UMR 5533 CNRS, France;

We report a novel case of gray platelet syndrome associated with the absence of the platelet glycoprotein (GP) VI collagen receptor. As classically described, the patient, a middle aged woman, with marrow myelofibrosis and a low platelet count has platelets lacking distinguishable alpha-granules in electron microscopy. P-selectin as observed on platelet sections by immunogold staining was largely confined to the membranes of the open canalicular system. Estimation of alpha-granule proteins such as fibrinogen, VWF, PDGF, showed platelet contents of about 5% of normal values. Dense granules appeared to be normal with a usual ADP/ATP content and mepacrine labeling. Platelet aggregation to ADP, arachidonic acid, ristocetin was normal, but slightly decreased with TRAP. In contrast, platelet aggregation with collagen was severely decreased as was the response to convulxin (Cvx). The alpha2beta1 collagen receptor was normally present on her platelets as assessed by flow cytometry as were other major GPs including GPIb and alphaIIbbeta3. However, analysis of GPVI using FITC-labeled Cvx and flow cytometry showed severely decreased binding. A severe GPVI deficiency was confirmed by Western blotting performed with two monoclonal antibodies which failed to detect either the intact protein or degradation products. The FcRgamma chain constitutively associated with GPVI in normal platelets was found both in permeabilized platelets of the patient by flow cytometry and by Western blotting, albeit in subnormal amounts. When the patient's platelets were stimulated by cross-linking the FcgammaRII receptor with the monoclonal antibody IV3 in the presence of antimouse IgG, they aggregated normally, showing that the phospholipase Cgamma-dependent activation pathway, used by GPVI, was functional. Because auto-antibodies to GPVI have been shown to induce the loss of the receptor in mice, four MoAbs to GPVI have been investigated with the patient's serum in a modified MAIPA test. No human antibodies have been found. Sequencing of coding regions of the GPVI gene has failed to show genetic abnormalities. In conclusion, a new case of gray platelet syndrome is described with an additional defect in GPVI receptor leading to a more severely deficient collagen-induced platelet aggregation than has been usually reported for this syndrome in the literature. A genetic defect causing both an abnormal alpha-granule maturation and GPVI synthesis is speculated in this case.