The antithrombotic potential of selective blockade of talin-dependent integrin alpha IIb beta 3 (platelet GPIIb-IIIa) activation

Abstract

In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(3) (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(3)(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(3)(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Figure 1. Structure-based mutagenesis of the β₃ integrin cytoplasmic domain.

(A) Space-filled models of talin F3 domain (left) or filamin A immunoglobulin–like domain 21 in complex with short internal fragments of the β₃ integrin cytoplasmic domain (shown as sticks, with Tyr747 and Leu746 in yellow and purple, respectively). (B) Affinity chromatography using recombinant WT and mutant β₃ cytoplasmic domain proteins and mouse platelet lysates as described in Methods. Bound platelet proteins were analyzed by Western blotting with the indicated antibodies (left panel) and by Coomassie blue staining (right panel). Quantitation of the amount of talin bound in mutant relative to WT platelets is shown. Similar results were obtained from 2 independent experiments. F, filamin; T, talin.

Figure 2. Generation of β₃(Y747A) and β₃(L746A) mutant mice.

(A) Targeting vector containing 6 kb of the 3ι end of the mouse β₃ integrin gene (exons 14 and 15). Tyr747- and Leu7460-to-alanine mutations were inserted into exon 15. DTA, diphtheria toxin A. (B) Southern blot analysis of R1 ES cell genomic DNA transfected with the β₃(Y747A) targeting vector digested with BamHI (left) or EcoRI (right) using a 5′ probe or 3′ probe, respectively. (C) PCR genotyping of genomic DNA isolated from β₃(Y747A) and β₃(L746A) mouse ear biopsy samples using primers C and D shown in A. H, HindIII; E, EcoRI.

Figure 3. β₃(Y747A) and β₃(L746A) mice are protected from microvascular thrombosis.

(A) Mice were injected intravenously with 800 μg/kg collagen and 60 μg/kg epinephrine and monitored for 30 minutes. *P < 0.005. (B) Representative sections of H&E-stained lungs from a WT mice that died during the assay and β₃(Y747A) and β₃(L746A) mice that survived and were sacrificed immediately following the assay. WT sections show extensive microthrombi throughout the lungs, while β₃ mutant lungs were clear. Scale bars: 200 μm (upper panel); 100 μm (lower panel).

Figure 4. β₃(L746A) mice are protected from arterial thrombosis.

(A) Thrombosis was induced in the carotid artery of mice by a 3-minute application of 5% ferric chloride to the surface of the vessel. The time to complete vessel occlusion was considered the time after injury to zero blood flow as measured with a Doppler flow probe. Representative Doppler flow tracings from a WT and a β₃(L746A) mouse are shown. (B) H&E-stained sections of carotid arteries of WT and β₃(L746A) mice obtained 30 minutes after ferric chloride injury. A section of β₃(L746A) carotid artery distal to the injury is shown for comparison. Original magnification, ×200.

Figure 5. Bleeding diathesis in β₃ mutant mice.

(A) The presence of fecal blood was detected using a guaiac-based hemoccult test. Fecal specimens obtained from the indicated genotypes of mice at 6–12 weeks of age were scored as positive or negative in a blinded manner. (B) Box plot showing hemoglobin concentration measured in peripheral blood. Filled circles represent outliers and were included in statistical analysis. *P < 0.05 compared with WT. (C) Tail bleeding times. β₃(Y747A) and β₃(L746A) homozygotes showed bleeding times of at least 10 minutes (at which time bleeding was stopped by cauterization), while WT mice showed significantly shorter bleeding times.

Figure 6. β₃(Y747A) and β₃(L746A) platelet interactions with fibrinogen.

(A) FITC-labeled fibrinogen binding was measured by flow cytometry. Fibrinogen binding was reduced in β₃ mutant platelets in response to platelet agonists. Specific binding was defined as that which was inhibitable by 2 mM EDTA. Mean fluorescence intensity (MFI) for each agonist treatment was normalized to the MFI obtained with 0.5 mM MnCl₂ treatment. n = 7, 6, and 4 for WT, β₃(Y747A), and β₃(L746A), respectively. Epi, epinephrine. (B) Platelets from both WT mice and β₃ mutants showed increased fibrinogen binding in the presence of 0.5 mM MnCl₂ (left). Surface β₃ integrin expression of platelets obtained from β₃ mutant animals (right). Results are representative of 4 independent experiments and at least 4 mice per genotype. (C) Aggregation of platelets obtained from WT or β₃ mutant mice was measured in response to the addition of 100 μM ADP or 0.5 mM PAR4 peptide. Results are representative of at least 2 independent experiments on at least 3 animals from each genotype. (D) Platelet adhesion to immobilized fibrinogen. Washed platelets were incubated in fibrinogen-coated microtiter wells for 1 hour at room temperature. Results are shown as percentage of adherent cells relative to the total number added to each well. n = 7, 5, and 4 for WT, β₃(Y747A), and β₃(L746A), respectively. Error bars represent SD.

Figure 7. pp125^FAK phosphorylation.

Platelets were incubated in suspension with 250 μg/ml soluble fibrinogen with or without 0.5 mM MnCl₂ and ADP/epinephrine (100 μM each), as indicated, for 20 minutes at room temperature. Platelets were lysed, and pp125^FAK phosphorylation was measured by immunoblotting using an antibody against pp125^FAK (pTyr397). Blots were stripped and reprobed with an antibody to pp125^FAK. Results are shown relative to maximum pp125^FAK phosphorylation signal for each group. *P < 0.05.

Figure 8. Agonist-stimulated platelet spreading.

Platelets were allowed to spread on fibrinogen-coated coverslips (100 μg/ml) in the presence of MnCl₂ (0.5 mM), ADP (100 μM), PMA (200 nM), or PAR4 peptide (1 mM), as indicated, for 45 minutes, fixed, and stained with rhodamine-phalloidin. The results were quantified from 2 independent experiments. *P < 0.05. Error bars represent SD. Original magnification, ×1,260.