A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen.

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.

Immunolocalization of P2Y1 and TPalpha receptors in platelets showed a major pool associated with the membranes of alpha -granules and the open canalicular system.

P2Y(1) and thromboxane-prostanoid-alpha (TPalpha) receptors on platelets belong to the G-protein-coupled 7-transmembrane domain family. They transmit signals for shape change, mobilization of calcium, and platelet aggregation. Immunogold labeling with a monoclonal antibody (MoAb) to the amino-terminal domain of P2Y(1) and a polyclonal antibody to the C-terminal domain of TPalpha revealed that while present at the platelet surface, both receptors were abundantly represented inside the platelet. Specifically, receptors were found in membranes of alpha-granules and elements of the open-canalicular system. A similar organization was found in mature megakaryocytes. Activation of platelets by adenosine diphosphate (ADP) and the thromboxane A(2) (TXA(2)) analog, I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha)-7-(3-(3- hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid], increased the labeling of both P2Y(1) and TPalpha at the surface and in intracellular pools, suggesting that activation resulted in greater antibody accessibility to the receptor. A return to a platelet discoid shape and to basal values of labeling accompanied receptor desensitization. Platelets lacking the P2Y(12) ADP receptor normally expressed P2Y(1) and TPalpha, both before and after activation. Studies with the anti-ligand-induced binding site (anti-LIBS) MoAb, AP-6, confirmed that stored fibrinogen associated with internal pools of alpha(IIb)beta(3) at the start of secretion in a microenvironment containing agonist receptors. Pharmacologic antagonism of ADP or TXA(2) receptors in antithrombotic therapy may need to take into account blockade of internal receptor pools.

Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome.

We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking alpha-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor gamma-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C gamma 2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the literature.