Talin-dependent integrin activation is required for fibrin clot retraction by platelets

Abstract

Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.

Figure 1

Talin1(L325R) binds to integrins but is defective in agonist-induced platelet integrin activation. (A) Affinity chromatography with the use of recombinant β1, β3, and αIIb cytoplasmic domains and platelet lysates from Tln1^(wt/fl),Cre⁺ (indicated as wt) or Tln1^(L325R/fl),Cre⁺ (indicated as L325R) mice. Bound talin protein was visualized by Western blotting with anti-talin 8d4 antibody, and equal loading of integrins was verified by Coomassie stain. Densitometric quantitation of talin bound was normalized to integrin loading. The value shown indicates the amount of talin1(L325R) bound to the integrin relative to talin1 (wt). Data are representative of 2 independent experiments. (B) Specific binding of fluorescein isothiocyanate-fibrinogen to platelets was measured by the use of flow cytometry by subtracting the nonspecific fibrinogen binding that occurred in the presence of 5mM ethylenediaminetetraacetic acid in each condition. *P < .05, **P < .005. (C) Activation of β1 integrin was assessed by measuring the binding of the conformation-sensitive antibody 9EG7 relative to binding of the conformation-insensitive β1 integrin antibody HMβ1-1. **P < .005.

Figure 2

Fibrin clot retraction is impaired in talin1-deficient and talin1(L325R) mutant platelets. (A) Clot retraction of PRP from Tln1^(wt/fl),Cre⁺ or Tln1^(L325R/fl),Cre⁺, or Tln1^(fl/fl),Cre⁺ mice was initiated by 1.0 U/mL thrombin and photographed after 2 hours. As indicated, 0.5mM MnCl₂, 10μM cytochalasin D, or 50μM eptifibatide was added to PRP 5 minutes before the addition of thrombin. Efficient clot retraction, visible as a consolidated clot and transparent clot liquor, was observed in PRP from Tln1^(wt/fl),Cre⁺ mice and Tln1^(L325R/fl),Cre⁺ mice in the presence of manganese. (B) Fibrin clot retraction was quantified by calculating the weight of the serum extruded from the clot relative to the initial clot weight and expressed as a percentage. n = 3. *P < .005.