title: Quantitation of platelet fibrinogen and thrombospondin in Glanzmann's thrombasthenia by electroimmunoassay.
abstract: Fibrinogen and thrombospondin are major constituents of human platelet alpha-granules and contribute to cell-cell interactions following their release. Glanzmann's thrombasthenia is characterized by the absence of platelet aggregation and reduced levels of GP IIb-IIIa complexes and platelet fibrinogen. The level of thrombospondin is thought to be normal but has not so far been quantified. Using an electroimmunoassay method adapted from Laurell, we have measured fibrinogen and thrombospondin in platelet extracts of four patients with classical Glanzmann's thrombasthenia and two variants with abnormal platelet aggregation associated with subnormal levels of GP IIb-IIIa complexes. Triton X-100 lysates were prepared in the presence of leupeptin or EDTA to avoid endogenous calcium-dependent protease activation during the solubilization procedure. Platelet fibrinogen was not detected in one patient with type I Glanzmann's thrombasthenia; it was reduced to 5-10% of normal values in two other type I patients and to 65% of normal values in one type II patient. It was normal in patient R.P., a variant of Glanzmann's thrombasthenia with 60% of GP IIb-IIIa complexes but decreased in patient A.P. a newly described variant with 35% of GP IIb-IIIa complexes. These findings support a role for GP IIb-IIIa complexes in the packaging of fibrinogen into alpha-granules. Normal or subnormal amounts of thrombospondin were measured in thrombasthenic platelets. Patient A.P., who was investigated on two different occasions, demonstrated variable levels of thrombospondin. This underlines the need for quantifying this protein when evaluating its expression in this disorder.
authors.affiliation: Laboratoire d'Hémostase et de Thrombose Expérimentale, Unité 150 INSERM-Unité Associée 334 CNRS, Hôpital Lariboisière, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Disdier
authors.forename: C
authors.initials: C
authors.lastname: Legrand
authors.forename: C
authors.initials: C
authors.lastname: Bouillot
authors.forename: V
authors.initials: V
authors.lastname: Dubernard
authors.forename: D
authors.initials: D
authors.lastname: Pidard
authors.forename: A T
authors.initials: AT
authors.lastname: Nurden
mesh_terms: D001791:Blood Platelet Disorders / Q000097:blood*; D001792:Blood Platelets / Q000032:analysis*; D003902:Detergents; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D007122:Immunoelectrophoresis; D007976:Leupeptins; D008562:Membrane Glycoproteins / Q000097:blood*; D017830:Octoxynol; D011092:Polyethylene Glycols; D013915:Thrombasthenia / Q000097:blood*; D019699:Thrombospondins
chemical_list: D003902:Detergents; D007976:Leupeptins; D008562:Membrane Glycoproteins; D019699:Thrombospondins; D011092:Polyethylene Glycols; D005340:Fibrinogen; D017830:Octoxynol; D004492:Edetic Acid; C032854:leupeptin