We previously showed that disruption of the talin1 (Tln1
) gene in mice results in embryonic lethality around gastrulation excluding the usage of Tln1
-null animals for studies on platelet function (19
). Therefore, we generated mice carrying a Tln1
gene flanked by loxP sites (Tln1fl
; ) and crossed them with transgenic mice expressing the Cre recombinase under the control of the inducible interferon-sensitive Mx promoter (20
; referred to as Tln1−/−
mice) to induce efficient and permanent Tln1
gene deletion in all hematopoietic cells including megakaryocytes by intraperitoneal injection of polyinosinic-polycytidylic acid (pI-pC) (21
mice were used as controls and treated identically. 10 d after the last injection, the efficiency of talin deletion was confirmed with Western blots from platelet lysates also showing that the expression levels of other proteins, including filamin A, integrin linked kinase, β1 integrin, c-src, and actin, were unaltered ().
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 (more ...)
To investigate the consequence of talin deficiency on hemostasis, we performed tail bleeding experiments. To restrict the Cre-induced deletion of the Tln1 gene to the hematopoietic system, bone marrow derived from either Tln1fl/fl/Cre+ or Tln1fl/fl mice was transferred into irradiated normal recipient mice. 3 wk after transfer, the Tln1 gene was deleted by pI-pC injections and the functional absence of the protein in circulating platelets was confirmed by flow cytometry (not depicted). Although control chimeras arrested bleeding within 7.3 ± 2.2 min, all of the Tln1−/− chimeras bled longer than 15 min (). Thus, talin is essential to halt bleeding upon vessel injury.
To test whether the hemostatic disorder was due to defective platelet formation, we assessed platelet counts, which were similar to controls (). Flow cytometric analysis demonstrated normal expression levels of prominent surface receptors in mutant platelets, except for αIIbβ3 integrin, which was reduced by ~15% (). Forward scatter/side scatter characteristics were the same as control, suggesting normal size and shape of the mutant platelets. Thus, talin is not required for megakaryocyte differentiation and platelet production but regulates the expression of some platelet surface receptors.
Glycoprotein expression on Tln1−/− platelets
To determine whether the severe hemostatic defect is caused by impaired inside-out activation of αIIbβ3, we induced platelet aggregation using different agonists. As shown in , Tln1−/− platelets failed to aggregate in response to high concentrations of thrombin, ADP, the stable thromboxane A2 analogue U46619, collagen, and the GPVI-specific agonist collagen-related peptide (CRP). Interestingly, all agonists induced a comparable activation-dependent change from discoid to spherical shape in control and Tln1−/− platelets, which can be seen in aggregometry as a short decrease in light transmission after the addition of agonists. This suggests a selective defect in αIIbβ3-dependent aggregation rather than a general impairment of activation of signaling pathways in Tln1−/− platelets.
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 (more ...)
To assess integrin αIIbβ3 function more directly, we measured agonist-induced binding of Alexa Fluor 488–tagged fibrinogen by flow cytometry. Control platelets bound fibrinogen, which was inhibited by an αIIbβ3-blocking antibody (not depicted), in response to all agonists tested, whereas Tln1−/−
platelets were unable to bind fibrinogen (). This defect was due to impaired inside-out activation of αIIbβ3 as Mn2+
, known to exogenously activate β1 and β3 integrins (22
), induced fibrinogen binding in both control and Tln1−/−
platelets (). This binding was 24.1% lower in the mutant platelets as compared with the control (P < 0.01), which corresponds well to the reduced surface expression of αIIbβ3 in those cells (). Similar results to those obtained with fibrinogen were seen with the JON/A-PE antibody, which selectively binds to activated αIIbβ3 integrins on mouse platelets () (24
). These results demonstrate that αIIbβ3 activation is abolished in Tln1−/−
platelets, even at high agonist concentrations. Surface expression of P-selectin was determined as a measure of agonist-induced degranulation. Although normal degranulation was observed in Tln1−/−
platelets in response to intermediate and high thrombin concentrations, a significantly reduced response was seen with low thrombin concentrations and the weaker agonist, CRP (). Collectively, these data show that the inside-out activation of αIIbβ3 integrin is abrogated in Tln1−/−
platelets, but other cellular functions such as shape change and degranulation are either not or slightly affected.
αIIbβ3 integrin is also important for firm platelet adhesion to the extracellular matrix, where it acts in concert with β1 integrins, most notably the collagen-binding α2β1 integrin (21
). Therefore, platelet adhesion to a collagen-coated surface was tested in whole blood perfusion assays under conditions of low and high shear stress (150 and 1,000 s−1
, respectively). Control platelets readily established firm adhesions on the collagen fibers and rapidly built stable three-dimensional aggregates, both at high and low shear (, and not depicted), whereas virtually all Tln1−/−
platelets either detached within a few seconds or translocated along the fibers before they were released. As a consequence, virtually no Tln1−/−
platelets were attached to the collagen surface at the end of the experiment, whereas control platelets covered 53.3 ± 8.6% of the surface area (). Furthermore, Tln1−/−
platelets failed to adhere to soluble type I collagen under static conditions, a process known to be mediated exclusively by α2β1 integrin (unpublished data) (2
). Thus, activation of both αIIbβ3 as well as α2β1 is abrogated in Tln1−/−
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 (more ...)
Ligand-occupied integrins transduce signals leading to the activation of Src family kinases resulting in cell spreading. The role of talin in this process was tested by analyzing the adhesion of washed control and Tln1−/−
platelets to fibrinogen under static conditions. As mouse platelets, in contrast to human platelets, do not spread well on immobilized fibrinogen without cellular activation (26
), the experiments were performed in the presence of 0.01 U/ml thrombin. Comparable adhesion of control and Tln1−/−
platelets to the fibrinogen matrix occurred, confirming the previous observation that αIIbβ3 activation is not required for static adhesion of platelets to fibrinogen (27
). However, although control platelets readily formed lamellipodia and spread within 10–15 min, Tln1−/−
platelets only formed filopodia, with occasional transient small lamellipodia, and completely failed to spread for up to 45 min (). Thus, talin1 is also required for αIIbβ3-dependent outside-in signaling. These results differ from observations made with platelets expressing a talin binding–deficient β3 (L746A) integrin mutant, which spread on fibrinogen in the presence of agonists (18
). A possible explanation for this discrepancy is that the β3(L746A) mutation only partially disrupts the β3 and talin interaction.
Pathological thrombus formation in vivo was determined by intravital microscopy of injured mesenteric arterioles in bone marrow chimeric Tln1−/−
mice. For this, platelets were fluorescently labeled in vivo and injury was induced by topical application of 20% FeCl3
. In control mice, platelets rapidly interacted with the injured vessel wall and were firmly attached 5 min after injury (), after which additional platelets were recruited from the circulation resulting in aggregate formation () and complete vessel occlusion in all mice by 20 min (mean occlusion time, 15.3 ± 2.0 min; ). In contrast, in Tln1−/−
mice platelets only transiently attached to the site of injury, no thrombi were formed, and blood flow was maintained throughout the observation period in all analyzed vessels. Thus, talin is essential for platelet attachment to the injured vessel wall, a process mediated by the concerted action of β1 and β3 integrins (28
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 (more ...)
The molecular mechanisms regulating the inside-out activation of integrins have been studied intensively during the last two decades. One central result of these studies was the proposal that the cytoskeletal protein talin might be a central regulator of this process. Our study now provides unambiguous in vivo evidence for the unique and essential role of talin for the activation of αIIbβ3 and α2β1 integrins in mammals. This was not certain despite recent reports showing that mutations of critical residues in the talin binding site of the β1 and β3 integrin tails, respectively, abrogate integrin activation and lead to severe developmental and hemostatic defects in vivo (8
). Integrin β tails bind a large number of focal adhesion proteins, and it was therefore impossible to clarify whether the mutations affect talin only or additional tail-binding proteins that contribute to integrin activation.
Interestingly, Mx-cre–induced loss of talin1 did not significantly affect peripheral platelet counts. Because talin2, which is very similar to talin1, is not expressed in significant amounts in hematopoietic cells (14
), our findings suggest that talin is dispensable for megakaryocyte maturation, pro-platelet formation, and platelet shedding. In addition, flow cytometric forward scatter/side scatter profiles of control and Tln1−/−
platelets are indistinguishable, indicating that size and shape of the mutant cells are largely normal as is the expression profile of prominent surface glycoproteins. The only exception is an ~15% reduction in surface expression of integrin αIIbβ3, which, however, does not account for the hemostasis defect because mice carrying a heterozygous-null mutation in the β3 integrin express even less (50%) αIIbβ3 integrin on their platelets without developing a bleeding defect (30
This indicates that talin does neither make a significant contribution to platelet shape, which has been proposed to depend on the integrity of the cortical cytoskeleton, nor to cytoskeletal rearrangements after cellular activation. However, at low thrombin concentrations or in response to the weaker agonist CRP, Tln1−/− platelets displayed reduced degranulation (), indicating that talin deficiency either directly or indirectly impairs granule release. Further studies will be required to address this question.
Both our ex vivo flow adhesion studies as well as the in vivo analysis of platelet adhesion to the injured arterial wall indicate that loss of talin abrogates not only the activation of αIIbβ3 integrin, but also the activation of β1 integrins, which are known to contribute to platelet adhesion to the extracellular matrix in vitro and in vivo (25
). This complete abolishment of integrin function resulted in a profound protection from arterial thrombosis but also in a complete loss of primary hemostasis as indicated by infinite bleeding from a tail wound (). We did, however, not observe spontaneous intestinal or subcutaneous bleeding in Tln1−/−
chimeric mice, as recently reported in mice with a mutation in the β3 integrin subunit (β3 [Y747A]) that abrogates interaction with several intracellular binding partners, including talin and filamin (18
). This indicates that the loss of platelet integrin function alone is not sufficient to cause such a severe phenotype in mice, at least not when the deficiency is induced in adult animals. It is possible that the severe spontaneous bleeding observed in β3 (Y747A) mice is caused by a combined defect in platelets and other cells of the vascular system that express β3, such as endothelial cells. Interestingly, another mutation (β3 [L746A]) that is thought to only disrupt talin binding also abrogated inside-out activation of αIIbβ3 in platelets but did not produce severe spontaneous bleedings (18
). Interestingly, these platelets exhibited only partial defects in their ability to spread on fibrinogen in the presence of agonists or Mn2 +
, indicating that outside-in signaling through αIIbβ3 is intact in those cells. We found that Tln1−/−
platelets were completely unable to spread on fibrinogen, suggesting that talin is essential for outside-in signaling. An explanation why outside-in signaling was not completely disrupted in β3 (L746A) platelets could be that talin may still be able to weakly interact with talin. Thus, although reducing the interactions of talin and integrin tails may have promise as a therapeutic modality to reduce thrombus formation in patients with thrombotic disorders (18
), complete inhibition of talin1 function results in catastrophic bleeding. These findings have important implications for the development of novel agents to prevent or treat ischemic cerebro- and cardiovascular diseases.