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

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 β 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.


BRIEF DEFINITIVE REPORT
Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix, which triggers adhesion and aggregation of platelets ( 1,2 ). The consequence of this process is the formation of a thrombus that prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels. Integrins play a central role in adhesion and aggregation of platelets ( 3 ). Integrins are heterodimeric transmembrane receptors composed of an ␣ and a ␤ subunit that are expressed in a low affi nity state in resting platelets. After activation, mediated by other platelet receptors, integrins shift to a high affi nity state and effi ciently bind their ligands ( 3 ).
Cellular control of integrin activation, which is crucial for numerous biological processes such as cell adhesion, motility, diff erentiation, and apoptosis ( 4 -8 ), requires transmission of a signal from the small cytoplasmic tails to the large extracellular domains ( 9 ). Most of the seminal work on integrin activation was performed on ␣ IIb ␤ 3 integrin, which is the principal integrin expressed on platelets and whose major ligands are fi brinogen, fi bronectin, and von Willebrand factor. The affi nity of this integrin for ligands is highly modulatable, and upon activation, it mediates platelet adhesion, aggregation, and spreading on the exposed extracellular matrix of injured vessel walls ( 3 ) as well as pathological thrombus formation ( 1 ). Because of the importance of ␣ IIb ␤ 3 integrin in platelet aggregation, it has become an attractive pharmacological target for the prevention of ischemic cardiovascular events. Strategies to inhibit its function include antibodies (abciximab), cyclic peptides adapted from a snake venom disintegrin (eptifi bitide), and nonpeptide analogues of an RGD [Arg-Gly-Asp] peptide (tirofi ban and lamifi ban) that inhibit ligand binding ( 10 ). Although these inhibitors are benefi cial for patients undergoing percutaneous coronary intervention, they do not have widespread clinical use because of their side eff ect of unwanted bleeding ( 10 ). Therefore, considerable eff ort has been made to identify the molecular mechanisms that regulate ␣ IIb ␤ 3 activation to identify potential new targets to inhibit platelet aggregation.
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 affi nity state for their ligands, and they shift to a high affi nity 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 ␤ subunits is necessary and suffi cient to trigger the activation of integrins to this high affi nity 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 talindefi cient 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.
To test whether the hemostatic disorder was due to defective platelet formation, we assessed platelet counts, which were similar to controls ( Fig. 1 D ). Flow cytometric analysis demonstrated normal expression levels of prominent surface receptors in mutant platelets, except for ␣ IIb ␤ 3 integrin, which was reduced by ‫ف‬ 15% ( Table I ). 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 diff erentiation and platelet production but regulates the expression of some platelet surface receptors.
To determine whether the severe hemostatic defect is caused by impaired inside-out activation of ␣ IIb ␤ 3, we induced platelet aggregation using diff erent agonists. As shown in Fig. 2 A , Tln1 Ϫ / Ϫ platelets failed to aggregate in response to high concentrations of thrombin, ADP, the stable thromboxane A 2 analogue U46619, collagen, and the GPVI-specifi c agonist collagen-related peptide (CRP). Interestingly, all agonists induced a comparable activation-dependent change from discoid to spherical shape in control and Tln1 Ϫ / Ϫ 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 ␣ IIb ␤ 3-dependent aggregation rather than a general impairment of activation of signaling pathways in Tln1 Ϫ / Ϫ platelets.
To assess integrin ␣ IIb ␤ 3 function more directly, we measured agonist-induced binding of Alexa Fluor 488 -tagged fi brinogen by fl ow cytometry. Control platelets bound fi brinogen, which was inhibited by an ␣ IIb ␤ 3-blocking antibody (not depicted), in response to all agonists tested, whereas At sites of vascular injury, platelet activation is triggered by exposed subendothelial collagens, thromboxane A 2 and ADP released from activated platelets, and thrombin generated by the coagulation cascade ( 2,11 ). Although these different agonists activate diff erent signaling pathways, it has been proposed that the fi nal common step for ␣ IIb ␤ 3 integrin activation is the activation and binding of the cytoskeletal protein talin1 to integrin ␤ tails ( 12 ).
Talin1 is an ‫ف‬ 270-kD ( 13 ) elongated dimeric actin-binding protein and is the only talin isoform expressed in hematopoietic cells ( 14 ). Talin1 colocalizes with activated integrins, is a component of focal adhesions, and links integrins with the actin cytoskeleton ( 15,16 ). The best evidence for the requirement of talin for integrin ␣ IIb ␤ 3 activation came from studies with talin1-depleted, mouse embryonic stem cellderived megakaryocytes, which revealed that diff erent agonists such as ADP or thrombin were unable to induce ␣ IIb ␤ 3 activation ( 17 ). Another study demonstrated that mice expressing the L746A mutation of ␤ 3 integrin, which is believed to be unable to bind talin, display impaired inside-out activation of ␣ IIb ␤ 3 resulting in protection from thrombosis and a relatively mild bleeding phenotype ( 18 ). Although these observations suggest an essential role for talin1 in the activation of ␣ IIb ␤ 3 in platelets, direct in vivo evidence for this hypothesis is still lacking.

RESULTS AND DISCUSSION
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 fl anked by loxP sites ( Tln1 fl ; Fig. 1 A ) and crossed them with transgenic mice expressing the Cre recombinase under the control of the inducible interferon-sensitive Mx promoter ( 20 ) ( Tln1 fl /fl /Cre + ; referred to as Tln1 Ϫ / Ϫ mice) to induce effi cient and permanent Tln1 gene deletion in all hematopoietic cells including megakaryocytes by intraperitoneal injection of polyinosinic-polycytidylic acid (pI-pC) ( 21 ). Tln1 fl /fl mice were used as controls and treated identically. 10 d after the last injection, the effi ciency of talin deletion was confi rmed with Western blots from platelet lysates also showing that the expression levels of other proteins, including fi lamin A, integrin linked kinase, ␤ 1 integrin, c-src, and actin, were unaltered ( Fig. 1 B ).
To investigate the consequence of talin defi ciency 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 Tln1 fl /fl /Cre + or Tln1 fl /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 confi rmed by fl ow cytometry (not depicted). Although control chimeras arrested bleeding within 7.3 ± 2.2 min, all of the Tln1 Ϫ / Ϫ chimeras bled longer than 15 min ( Fig. 1 C ). Thus, talin is essential to halt bleeding upon vessel injury.

BRIEF DEFINITIVE REPORT
low thrombin concentrations and the weaker agonist, CRP ( Fig. 2 D ). Collectively, these data show that the inside-out activation of ␣ IIb ␤ 3 integrin is abrogated in Tln1 Ϫ / Ϫ platelets, but other cellular functions such as shape change and degranulation are either not or slightly aff ected.
␣ IIb ␤ 3 integrin is also important for fi rm platelet adhesion to the extracellular matrix, where it acts in concert with ␤ 1 integrins, most notably the collagen-binding ␣ 2 ␤ 1 integrin ( 21,25 ). 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 Ϫ 1 , respectively). Control platelets readily established fi rm adhesions on the collagen fi bers and rapidly built stable three-dimensional aggregates, both at high and low shear ( Fig. 3, A and B, and not depicted), whereas virtually all Tln1 Ϫ / Ϫ platelets either detached within a few seconds or translocated along the fi bers before they were released. As a consequence, virtually no Tln1 Ϫ / Ϫ 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 ( Fig. 3, A and B ). Furthermore, Tln1 Ϫ / Ϫ platelets failed to adhere to soluble type I collagen under static conditions, a process known to be mediated exclusively by ␣ 2 ␤ 1 integrin (unpublished data) ( 2,21 ). Thus, activation of both ␣ IIb ␤ 3 as well as ␣ 2 ␤ 1 is abrogated in Tln1 Ϫ / Ϫ platelets ( 21,25 ).
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 Ϫ / Ϫ platelets to fi brinogen under static conditions. As mouse platelets, in contrast to human platelets, do not spread well on immobilized fi brinogen without cellular activation ( 26 ), the experiments were Tln1 Ϫ / Ϫ platelets were unable to bind fi brinogen ( Fig. 2 B ). This defect was due to impaired inside-out activation of ␣ IIb ␤ 3 as Mn 2+ , known to exogenously activate ␤ 1 and ␤ 3 integrins ( 22,23 ), induced fi brinogen binding in both control and Tln1 Ϫ / Ϫ platelets ( Fig. 2 B ). 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 ␣ IIb ␤ 3 in those cells (Table I). Similar results to those obtained with fi brinogen were seen with the JON/A-PE antibody, which selectively binds to activated ␣ IIb ␤ 3 integrins on mouse platelets ( Fig. 2 C ) ( 24 ). These results demonstrate that ␣ IIb ␤ 3 activation is abolished in Tln1 Ϫ / Ϫ 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 Ϫ / Ϫ platelets in response to intermediate and high thrombin concentrations, a signifi cantly reduced response was seen with The data shown are mean ± SD fl uorescence intensities of six mice per group. 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 ␤ 1 and ␤ 3 integrins ( 28 ). 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 ␣ IIb ␤ 3 and ␣ 2 ␤ 1 integrins in mammals. This was not certain despite recent reports showing that mutations of critical residues in the talin binding site of the ␤ 1 and ␤ 3 integrin tails, respectively, abrogate integrin activation and lead to severe developmental and hemostatic defects in vivo ( 8,18,29 ). Integrin ␤ tails bind a large number of focal adhesion proteins, and it was therefore impossible to clarify whether the mutations aff ect talin only or additional tail-binding proteins that contribute to integrin activation.
Interestingly, Mx-cre -induced loss of talin1 did not signifi cantly aff ect peripheral platelet counts. Because talin2, which is very similar to talin1, is not expressed in signifi cant amounts in hematopoietic cells ( 14 ), our fi ndings suggest that talin is dispensable for megakaryocyte maturation, pro-platelet formation, and platelet shedding. In addition, fl ow cytometric forward scatter/side scatter profi les of control and Tln1 Ϫ / Ϫ platelets are indistinguishable, indicating that size and shape of the mutant cells are largely normal as is the expression profi le of prominent surface glycoproteins. The only exception is an ‫ف‬ 15% reduction in surface expression of integrin ␣ IIb ␤ 3, performed in the presence of 0.01 U/ml thrombin. Comparable adhesion of control and Tln1 Ϫ / Ϫ platelets to the fi brinogen matrix occurred, confi rming the previous observation that ␣ IIb ␤ 3 activation is not required for static adhesion of platelets to fi brinogen ( 27 ). However, although control platelets readily formed lamellipodia and spread within 10 -15 min, Tln1 Ϫ / Ϫ platelets only formed fi lopodia, with occasional transient small lamellipodia, and completely failed to spread for up to 45 min ( Fig. 3 C ). Thus, talin1 is also required for ␣ IIb ␤ 3-dependent outside-in signaling. These results diff er from observations made with platelets expressing a talin binding -defi cient ␤ 3 (L746A) integrin mutant, which spread on fi brinogen in the presence of agonists ( 18 ). A possible explanation for this discrepancy is that the ␤ 3(L746A) mutation only partially disrupts the ␤ 3 and talin interaction.
Pathological thrombus formation in vivo was determined by intravital microscopy of injured mesenteric arterioles in bone marrow chimeric Tln1 Ϫ / Ϫ mice. For this, platelets were fl uorescently labeled in vivo and injury was induced by topical application of 20% FeCl 3 . In control mice, platelets rapidly interacted with the injured vessel wall and were fi rmly attached 5 min after injury ( Fig. 4, A and D ), after which additional platelets were recruited from the circulation resulting in aggregate formation ( Fig. 4 B ) and complete vessel occlusion in all mice by 20 min (mean occlusion time, 15.3 ± 2.0 min; Fig. 4, C and D ). In contrast, in Tln1 Ϫ / Ϫ mice platelets only transiently attached to the site of injury, no thrombi were formed, and blood fl ow was maintained throughout the observation  (A) Mesenteric arterioles were injured by FeCl 3 , and the number of attached fl uorescently labeled platelets per mm 2 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 ‫ف‬ 7 min, which then leads to vessel occlusion at ‫ف‬ 15 min. In contrast to wild-type platelets, Tln1 Ϫ / Ϫ platelets did not adhere to the vessel wall and thombus formation did not occur. Bar, 30 μ m.
To generate bone marrow chimeras, 4 × 10 6 cells from the bone marrow of talin1 fl /fl -Mx-Cre or talin1 fl /fl mice were injected into the tail vein of lethally irradiated (10 Gray) recipient C57BL/6 mice. 3 -4 wk after transfer, a single intraperitoneal injection of pI-pC (GE Healthcare) was performed to induce the knockout. Platelets were isolated from whole blood collected from the retro-orbital plexus.
Mice were housed in the animal facility of the Max-Planck Institute of Biochemistry. Mouse breeding and all experimental procedures were approved by the Regierung von Oberbayern and the Regierung von Unterfranken.

Aggregometry.
To determine platelet aggregation, light transmission was measured using washed platelets (200 μ l with 0.5 × 10 6 platelets/ μ l) in the presence of 70 μ g/ml human fi brinogen. Transmission was recorded on a Fibrintimer 4 channel aggregometer (APACT Laborger ä te und Analysensysteme) over 10 min and was expressed as arbitrary units with 100% transmission adjusted with buff er. Adhesion under fl ow conditions. Rectangular coverslips (24 × 60 mm) were coated with 0.25 mg/ml fi brillar type I collagen (Nycomed) for 1 h at 37 ° C and blocked with 1% BSA. Perfusion of heparinized whole blood was performed as described previously ( 21 ). In brief, transparent fl ow chambers with a slit depth of 50 μ m and equipped with the collagen-coated coverslips were rinsed with Hepes buff er and connected to a syringe fi lled with the anticoagulated blood. Perfusion was performed at room temperature using a pulse-free pump at low (150 s Ϫ 1 ) and high shear stress (1,000 s Ϫ 1 ). During perfusion, microscopic phase-contrast images were real-time recorded. Thereafter, the chambers were rinsed by a 10-min perfusion with Hepes buff er, pH 7.45, at the same shear stress, and phase-contrast pictures were recorded from at least fi ve diff erent microscopic fi elds ( × 63). Image analysis was performed off -line using Metamorph software (Visitron). Thrombus formation results are expressed as the mean percentage of total area covered by thrombi.
Analysis of bleeding time. Mice were anesthetized, and a 3-mm segment of the tail tip was cut off with a scalpel. Tail bleeding was monitored by gently absorbing the bead of blood with a fi lter paper without contacting the wound site. When no blood was observed on the paper after 15-s intervals, bleeding was determined to have ceased. The experiment was stopped after 15 min. which, however, does not account for the hemostasis defect because mice carrying a heterozygous-null mutation in the ␤ 3 integrin express even less (50%) ␣ IIb ␤ 3 integrin on their platelets without developing a bleeding defect ( 30 ).
This indicates that talin does neither make a signifi cant 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 Ϫ / Ϫ platelets displayed reduced degranulation ( Fig. 2 D ), indicating that talin defi ciency either directly or indirectly impairs granule release. Further studies will be required to address this question.
Both our ex vivo fl ow 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 ␣ IIb ␤ 3 integrin, but also the activation of ␤ 1 integrins, which are known to contribute to platelet adhesion to the extracellular matrix in vitro and in vivo ( 25,28 ). 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 infi nite bleeding from a tail wound ( Fig. 1 D ). We did, however, not observe spontaneous intestinal or subcutaneous bleeding in Tln1 Ϫ / Ϫ chimeric mice, as recently reported in mice with a mutation in the ␤ 3 integrin subunit ( ␤ 3 [Y747A]) that abrogates interaction with several intracellular binding partners, including talin and fi lamin ( 18 ). This indicates that the loss of platelet integrin function alone is not suffi cient to cause such a severe phenotype in mice, at least not when the defi ciency is induced in adult animals. It is possible that the severe spontaneous bleeding observed in ␤ 3 (Y747A) mice is caused by a combined defect in platelets and other cells of the vascular system that express ␤ 3, such as endothelial cells. Interestingly, another mutation ( ␤ 3 [L746A]) that is thought to only disrupt talin binding also abrogated inside-out activation of ␣ IIb ␤ 3 in platelets but did not produce severe spontaneous bleedings ( 18 ). Interestingly, these platelets exhibited only partial defects in their ability to spread on fi brinogen in the presence of agonists or Mn 2 + , indicating that outside-in signaling through ␣ IIb ␤ 3 is intact in those cells. We found that Tln1 Ϫ / Ϫ platelets were completely unable to spread on fi brinogen, suggesting that talin is essential for outside-in signaling. An explanation why outside-in signaling was not completely disrupted in ␤ 3 (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 fi ndings have important implications for the development of novel agents to prevent or treat ischemic cerebro-and cardiovascular diseases.

Mice.
Conditional talin1-defi cient mice were generated by introducing loxP sites fl anking coding exons 1 -4 by gene targeting. Targeting of the Tln1 locus was confi rmed by Southern blot of EcoR1-digested genomic