title: Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation.
abstract: We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was alpha(IIb)beta(3)-dependent, since it was blocked by alpha(IIb)beta(3)-specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 microm) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.
authors.affiliation: U348 INSERM and IFR6 Circulation-Lariboisière, Hôpital Lariboisière, 41 Boulevard de la Chapelle, 75475 Paris Cedex 10, France.
authors.forename: S
authors.initials: S
authors.lastname: Osdoit
authors.forename: J P
authors.initials: JP
authors.lastname: Rosa
mesh_terms: D001792:Blood Platelets / Q000201:enzymology / Q000502:physiology*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D015638:Cytochalasin D; D014443:Tyrosine; D005337:Fibrin; D017027:Protein Tyrosine Phosphatases