Loss of talin1 in platelets abrogates integrin activation, platelet aggregation, and thrombus formation in vitro and in vivo

Abstract

Platelet adhesion and aggregation at sites of vascular injury are essential for normal hemostasis but may also lead to pathological thrombus formation, causing diseases such as myocardial infarction or stroke. Heterodimeric receptors of the integrin family play a central role in the adhesion and aggregation of platelets. In resting platelets, integrins exhibit a low affinity state for their ligands, and they shift to a high affinity state at sites of vascular injury. It has been proposed that direct binding of the cytoskeletal protein talin1 to the cytoplasmic domain of the integrin beta subunits is necessary and sufficient to trigger the activation of integrins to this high affinity state, but direct in vivo evidence in support of this hypothesis is still lacking. Here, we show that platelets from mice lacking talin1 are unable to activate integrins in response to all known major platelet agonists while other cellular functions are still preserved. As a consequence, mice with talin-deficient platelets display a severe hemostatic defect and are completely resistant to arterial thrombosis. Collectively, these experiments demonstrate that talin is required for inside-out activation of platelet integrins in hemostasis and thrombosis.

Figure 1.

Prolonged bleeding times in Tln^−/− mice. (A) Scheme of the targeted alleles. In the conditional allele (fl) exons 1–4 are flanked with loxP sites. Exposure of the conditional (fl) allele to Cre results in the removal of the Tln1 floxed region (exons 1–4) and generates the deleted allele (−). RI, EcoRI. (B) Western blot analyses of platelet lysates from control Tln1 (+/+) and Tln1^−/− (−/−) mice. (C) Tail bleeding times in wild-type and Tln1^−/− chimeras. (D) Peripheral platelet counts in control and Tln1^−/− mice.

Figure 2.

Impaired platelet function in Tln1^−/− mice. (A) Platelet aggregation assay reveals impaired aggregation of Tln1^−/− platelets (gray lines) in response to ADP, U46619, thrombin, CRP, and collagen when compared with control platelets (black lines). Arrows denote the addition of agonist. (B) Wild-type (black bars), but not Tln1^−/− platelets (gray bars), bind fibrinogen in response to 10 μM ADP, 10 μM ADP plus 3 μM U46619, or 10 μg/ml CRP. Treatment with 3 mM MnCl₂ triggers comparable binding. (C and D) Tln1^−/− platelets (gray bars) reveal a complete block in activation of αIIbβ3 after stimulation with 10 μM ADP, 10 μg/ml CRP, and different thrombin concentrations (0.001–0.1 U/ml), whereas platelet degranulation measured by the surface expression of P-selectin is not or mildly affected. Wild-type platelets (black bars) were used as a control. MFI, mean fluorescence intensity.

Figure 3.

Defective adhesion and spreading of Tln1^−/− platelets. Whole blood from control and talin1^−/− mice was perfused over a collagen-coated surface at a wall shear rate of 1,000 s⁻¹. Representative phase contrast images (A) and surface area coverage ± SD for four mice per group (B) taken at the end of the perfusion period (4 min). Bar, 30 μm. (C) Washed wild-type and Tln1^−/− platelets were stimulated with 0.01 U/ml thrombin, and then allowed to adhere to immobilized fibrinogen for 45 min. The differential interference contrast images shown are representative of four individual experiments. Bar, 3 μm.

Figure 4.

In vivo analysis of Tln1^−/− platelets in thrombosis. (A) Mesenteric arterioles were injured by FeCl₃, and the number of attached fluorescently labeled platelets per mm² was measured 5 min later. In parallel, the onset of thrombus formation (B) and the time when occlusion occurred (C) were determined. (D) In vivo video microscopy reveals the beginning of thrombus formation after ∼7 min, which then leads to vessel occlusion at ∼15 min. In contrast to wild-type platelets, Tln1^−/− platelets did not adhere to the vessel wall and thombus formation did not occur. Bar, 30 μm.