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.
Nurden
Paquita
P
UMR 5533 CNRS, Hôpital Cardiologique, Pessac and Académie des Sciences, and Institut de France, Paris, France.
Poujol
Christel
C
Winckler
Joelle
J
Combrié
Robert
R
Caen
Jacques P
JP
Nurden
Alan T
AT
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000368:Aged; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D016022:Case-Control Studies; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics* / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D009154:Mutation; D015539:Platelet Activation; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*; D011485:Protein Binding; D013268:Stimulation, Chemical; D013915:Thrombasthenia / Q000097:blood / Q000235:genetics*
D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D000244:Adenosine Diphosphate; D005340:Fibrinogen