Abstract

Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke¹. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel². The shift is mediated through binding of the cytoskeletal protein Talin to the subunit cytoplasmic tail^{3, 4, 5}. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of -integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

Introduction

Cellular control of integrin activation is essential for virtually all cells, including platelets, which seal injured blood vessels and stop bleeding. At sites of injury, the platelet receptors GPIb and GPVI bind to von Willebrand factor (vWF) and collagen, respectively^{1, 2, 3}, which together with locally produced thrombin trigger activation of integrin _IIb3 and the release of soluble platelet agonists including ADP and thromboxane A2 (TxA2). Activated _IIb3 integrins bind fibrinogen, vWF and fibronectin, thus allowing firm platelet adhesion and platelet aggregation. The central role of integrin activation in platelet adhesion and aggregation sparked the search for critical integrin tail-binding proteins that control integrin affinity for ligands. Irrespective of the platelet-activating stimulus and signaling pathways, Talin binding to the -integrin tails was shown to be the final common step in _IIb3 integrin activation and ligand binding^{4, 6, 7}. Talin, a major cytoskeletal protein at integrin adhesion sites, consists of a large C-terminal rod-like domain and an N-terminal FERM (protein 4.1, ezrin, radixin, moesin) domain with three subdomains: F1, F2 and F3 (ref. 8). The phosphotyrosine-binding (PTB) subdomain in the F3 domain sequentially binds to two distinct regions in the cytoplasmic tails and is sufficient for integrin activation in vitro^{8, 9}.

In addition to Talin, other FERM domain–containing proteins, including the Kindlins, interact with integrin tails¹⁰. The Kindlin protein family consist of three members (Kindlin-1, Kindlin-2 and Kindlin-3), all of which localize to integrin adhesion sites^{11, 12, 13}. In contrast to the widely expressed Kindlin-1 and Kindlin-2, Kindlin-3 is restricted to hematopoietic cells and is particularly abundant in megakaryocytes and platelets¹². The structural hallmark of Kindlins is a FERM domain whose F2 subdomain is split by a pleckstrin homology (PH) domain. In a comparison of FERM-domain proteins, the F3 subdomains of Kindlins have been found to share highest homology with the F3 domain of Talin¹⁰.

To address the function of Kindlin-3 in vivo, we disrupted the Kind3 (also called Fermt3) gene in mice (Supplementary Fig. 1 online). Mice heterozygous for the Kindlin-3–null mutation (Kind3^+/-) were normal, whereas mice lacking Kindlin-3 (Kind3^-/-; Fig. 1a) died within a week of birth and showed a pronounced osteopetrosis (unpublished data) and severe hemorrhages in the gastrointestinal tract, skin, brain and bladder, which were already apparent during development (Fig. 1b and data not shown). To test whether the severe bleeding of Kindlin-3–deficient mice was due to impaired platelet production and/or function, we generated fetal liver cell chimeras by transferring either Kind3^-/- or wild-type fetal liver cells into lethally irradiated wild-type recipient mice. Tail-bleed assays revealed that Kind3^-/- chimeras suffer from a pronounced hemostatic defect like that of Kindlin-3–deficient mice. After the tail-tip cut, bleeding in control mice arrested within 10 min (mean of 5.4 4.3 min), whereas Kind3^-/- chimeras bled for longer than 15 min, suggesting severe platelet dysfunction (Fig. 1c).

Figure 1: Kindlin-3–deficient animals show severe hemorrhages.

(a) Western blot analyses from spleen and platelet lysates of wild-type (Kind3^+/+), heterozygous (Kind3^+/-) and Kindlin-3–deficient (Kind3^-/-) mice. (b) E15.5 embryos reveal severe bleeding. Postnatally, Kind3^-/- mice show skin (arrowhead) and intestinal (arrows) bleeding. All scale bars, 1 mm. (c) Tail-bleeding times in wild-type and Kind3^-/- mice. (d) Peripheral platelet counts in wild-type and Kind3^-/- chimeras.

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Kind3^-/- chimeras showed platelet counts similar to those of wild-type chimeras (Fig. 1d), ruling out an essential role for Kindlin-3 in platelet formation. Analysis of glycoprotein abundance on platelets revealed elevated levels of the vWF receptor complex GPIb-IX in Kindlin-3–deficient as compared to wild-type platelets, whereas levels of other glycoproteins, including GPVI, CD9, and ₁ and ₃ integrins, were reduced (Supplementary Table 1 online). Thus Kindlin-3 has an apparent yet undefined role in the expression of several glycoproteins. However, the reduced expression of integrin _IIb3 does not account for the hemostasis defect, as mice carrying a heterozygous null mutation in the ₃ integrin express even less _IIb3 integrin on their platelets (50% of wild-type) without developing a bleeding defect¹⁴.

To determine the mechanism of the platelet defect, we performed ex vivo platelet aggregation studies. Wild-type platelets aggregated in response to ADP, the TxA2 analog U46619, thrombin, collagen and the GPVI-activating collagen-related peptide (CRP), whereas none of the agonists induced aggregation of Kind3^-/- platelets (Fig. 2a). Notably, all agonists induced a comparable activation-dependent change from discoid to spherical shape in control and Kind3^-/- platelets, which can be seen in aggregometry as a short decrease in light transmission following the addition of agonists. This suggests a selective defect in _IIb3-dependent aggregation rather than a general impairment of signaling pathways in Kind3^-/- platelets.

Figure 2: Impaired platelet function in Kind3^-/- mice.

(a) Platelet aggregation assay reveals impaired aggregation of Kind3^-/- platelets (gray lines) in response to ADP, U46619, thrombin, CRP and collagen when compared with control platelets (black lines). Arrows denote addition of agonist. (b) Wild-type (Kind^+/+) platelets (black bars), but not Kind3^-/- platelets (gray bars), bind fibrinogen in response to ADP (10 M), ADP (10 M) plus U46619 (3 M) or CRP (10 g/ml). Treatment with MnCl₂ (Mn²⁺; 3 mM) triggers comparable binding. Resting (rest.) platelets were used as a control. (c,d) Kind3^-/- platelets (gray bars) show a complete block in activation of integrin-_IIb3 after stimulation with ADP (10 M), CRP (10 g/ml) and different concentrations (0.001–0.1 U/ml) of thrombin (c), whereas platelet degranulation measured by the surface expression of P-selectin is largely intact after the same treatments (d). Wild-type platelets (black bars) were used as a control. At the intermediate thrombin concentration, moderately but significantly reduced degranulation was observed with mutant platelets (^**P < 0.01). MFI, mean fluorescence intensity. (e) Kind3^-/- platelets in whole blood failed to form thrombi when perfused over a collagen-coated (0.25 mg/ml) surface at a wall shear rate of 1,000 s^-1. Scale bar, 30 m. (f) Mesenteric arterioles were injured with FeCl₃, and adhesion and thrombus formation of fluorescently labeled platelets were monitored by in vivo video microscopy. Representative images (left) and time to vessel occlusion (right) are shown. Each symbol represents one individual. Scale bar, 30 m.

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To test whether activation of _IIb3 integrin is indeed abrogated in Kind3^-/- platelets, we determined their ability to bind fibrinogen using flow cytometry. Wild-type platelets showed robust fibrinogen binding in response to ADP, ADP plus U46619 and CRP, whereas Kind3^-/- platelets failed to bind fibrinogen upon agonist treatment (Fig. 2b). The antibody JON/A-PE, which specifically detects the activated form of mouse _IIb3 integrin¹⁴, likewise did not bind stimulated Kind3^-/- platelets (Fig. 2c). When cellular activation of _IIb3 integrin was bypassed by the addition of MnCl₂, comparable fibrinogen binding to Kind3^-/- and wild-type platelets occurred (Fig. 2b). Together, these findings indicate that loss of Kindlin-3 expression prevents energy-dependent conformational rearrangements required for integrin-_IIb3 activation.

Resting platelets store P-selectin in -granules, which fuse with the plasma membrane during agonist-induced platelet activation¹. Both CRP and thrombin induced P-selectin translocation on wild-type and Kind3^-/- platelets, although a significant reduction was consistently observed at intermediate concentrations of thrombin (P < 0.001; Fig. 2d). As expected, the weak agonist ADP did not induce P-selectin surface expression in wild-type and Kind3^-/- platelets. Thus, although loss of Kindlin-3 specifically disables integrin activation, it still permits agonist-induced P-selectin translocation.

Integrin-_IIb3 is also involved in adhesion to immobilized ligands, including collagen-bound vWF, where it acts in concert with the collagen-binding ₂₁ integrin^{2, 15}. We analyzed the ability of Kind3^-/- platelets to interact with fibrous collagen in a whole-blood perfusion assay. Under high- (1,000 s^-1, Fig. 2e) or low-shear (150 s^-1, data not shown) flow conditions, wild-type platelets adhered to the collagen fibers and rapidly built stable three-dimensional aggregates (Fig. 2e). In contrast, Kind3^-/- platelets never established stable adhesions and either detached immediately or translocated along the fibers for a few seconds, resulting in virtually no platelets attaching to the collagen-coated surface at the end of the 4-min perfusion time (Fig. 2e). Furthermore, agonist-stimulated Kind3^-/- platelets failed to bind the monoclonal anti-₁ integrin antibody 9EG7, which specifically recognizes the activated form of ₁ integrins¹⁶, and also failed to adhere to soluble, pepsin-digested collagen type I under static conditions (Supplementary Fig. 2 online), a process known to be mediated exclusively by ₂₁ integrins¹⁷. Together these findings indicate that Kind3^-/- platelets can establish initial contacts with vWF/collagen via GPIb and probably GPVI, but are unable to adhere firmly as a result of defective activation of _IIb3 and ₂₁ integrins.

As platelet aggregation may lead to pathological occlusive thrombus formation, we examined whether lack of Kindlin-3 is protective against ischemia and infarction after mesenteric arteriole injury. This injury was induced by ferric chloride and assessed by in vivo fluorescence microscopy. Five minutes after injury, numerous platelets adhered firmly to the denuded vessel wall in control chimeras (5,380 2,465/mm²); after approximately 10 min, the first thrombi were observed; and after 18–39 min, the vessels were occluded (mean occlusion time: 27.6 8.1 min; Fig. 2f). In contrast, a few Kind3^-/- platelets transiently (<5 s) attached to the injured vessel wall, but virtually none adhered firmly throughout the 40-min observation period (50 24 / mm²). Furthermore, no thrombi formed in the injured vessels of Kind3^-/- chimeras, and blood flow was maintained in all vessels tested (Fig. 2f). These results confirm the ex vivo results and underscore the pivotal function of Kindlin-3 in integrin-mediated platelet adhesion to injured vessels in vivo.

The requirement for Kindlin-3 to trigger agonist-induced integrin activation on platelets, integrin-mediated platelet adhesion and thrombus formation suggests that it may be a downstream target of cellular signaling pathways that activate integrins. To test whether Kindlin-3 is able to activate integrins, we overexpressed Kindlin-3 in integrin-_IIb3–overexpressing CHO cells. In these cells Kindlin-3 was unable to trigger integrin activation, likely because the hematopoietic Kindlin-3 is not recruited to integrin containing focal adhesions¹³. Kindlin-3 overexpression in the mouse macrophage cell line RAW 264.7 (RAW), however, yielded a 2.2-fold increase in binding of the Cy5-labeled fibronectin repeat 7-10 (FN7-10), which harbors the integrin-binding RGD motif (Fig. 3a). Enhanced green fluorescent protein (EGFP)-transfected RAW cells showed virtually no increase in FN7-10 binding as compared to untransfected cells, whereas Talin overexpression and treatment with Mn²⁺ induced a 2.5-fold increase. Notably, overexpression of a Kindlin-3 variant with a point mutation in the PTB-like domain (Q597A), which in Talin (R358) reduces binding to tails⁴, did not trigger FN7-10 binding. These data indicate that Kindlin-3 is capable of activating FN binding integrins and that this activity requires an intact PTB-like domain.

Figure 3: Biochemical analyses of Kind3^-/- platelets.

(a) Binding of Cy-5–labeled fibronectin III 7-10 fragments (FNIII7-10; FN) to RAW 264.7 cells transfected with EGFP, Talin, EGFP-Kindlin-3 or EGFP-Kindlin-3(Q597A). Binding of Cy-5–labeled fibronectin III 7-10 lacking the RGD binding loop (FNIII7-10RGD) was used to estimate nonspecific binding, and binding in the presence of 5 mM Mn²⁺ served as a positive control. Data shown are mean s.e.m. of five independent experiments, normalized to FNIII7-10 binding to EGFP-transfected cells and with the background binding of FNIII7-10RGD subtracted. P values were obtained by unpaired t-test. (b) Western blot analysis of lysates from wild-type (+/+) control and Kind3^-/- (-/-) platelets. (c) Pull-down experiment with His-tagged IIb, ₁ and ₃ integrin tails incubated with 100 g platelet lysate. (d) Protein-protein interaction assay of recombinant GST–Kindlin-3 F3 domain (GST-K3F3) incubated with His-tagged IIb and ₃ integrin tails reveals direct binding between the F3 domain of Kindlin-3 and the ₃ tail. GST pull-down experiments were performed with recombinant GST-₁A (wild-type), GST-₁Y788A, GST-₁Y800A, GST-₁W780A and GST-₁TT793/794AA (e); with GST-₃ (wild-type), GST-₃Y747A, GST-₃Y759A, GST-₃W739A and GST-₃ST752/753AA (f); and with GST-₃ (wild-type) and GST-₃S752A (g) integrin cytoplasmic domains after incubation with 100 g platelet lysate. GST protein and GST-IIb were used as controls. 5% of the platelet lysate used for the pull-down experiment is shown as input control. Bound Talin and Kindlin-3 proteins were detected by western blotting. Coomassie blue staining showed that equal amount of GST fusion proteins were used. Shown are results from pull-down assays representative of a minimum of seven experiments.

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How does Kindlin-3 activate integrins? As reduced Talin expression did not account for the defect (Fig. 3b), we investigated whether the FERM domain of Kindlin-3, which is similar to that of Talin, might play a direct role in integrin activation. As previously shown, recombinant Talin bound wild-type ₁ and ₃ integrin tails, and this binding was significantly reduced when alanine mutations were introduced into the membrane-proximal tryptophan residue or NPxY motif (₃W739A, ₃Y747A, ₁W780A and ₁Y788A)¹⁸. Kindlin-3 was also able to interact with the wild-type ₁ and ₃ integrin tails (Fig. 3c), in the presence and absence of Talin1 (Supplementary Fig. 3 online), and the F3 subdomain of Kindlin-3 was sufficient for this interaction and this interaction occurred in a direct manner (Fig. 3d). However, specific point mutations within the ₁ and ₃ integrin cytoplasmic tails revealed that the binding properties of Kindlin-3 were different from those of Talin, as the former still bound to ₃W739A, ₃Y747A, ₁W780A and ₁Y788A tails, although less efficiently than to wild-type tails (Fig. 3e,f). Mutations to the membrane distal NxxY motif of the ₁ and ₃ tails (₁Y800A, ₃Y759A) and the ₁TT793/794AA and ₃ST752/753AA, however, abolished Kindlin-3 but not Talin binding (Fig. 3e,f). Moreover, the ₃S752P mutation found in a subset of individuals with Glanzmann's thrombasthenia also abolished Kindlin-3 binding¹⁹ (Fig. 3g). These data indicate that Talin primarily requires membrane-proximal residues for binding, whereas Kindlin-3 requires membrane-distal residues for binding to the ₁ and ₃ tails.

Ligand-occupied integrins transduce signals that lead to the activation of Src family kinases, resulting in cell spreading (outside-in signaling). We tested the role of Kindlin-3 in outside-in signaling by analyzing the adhesion of washed control and Kind3^-/- platelets to fibrinogen under static conditions. As mouse platelets, in contrast to human platelets, do not spread well on immobilized fibrinogen without cellular activation²⁰, we performed the experiments in the presence of 0.01 U/ml thrombin, with and without added Mn²⁺. Comparable adhesion of control and Kind3^-/- platelets to the fibrinogen matrix occurred, confirming a previous observation that integrin-_IIb3 activation is not required for static adhesion of platelets to fibrinogen²¹. However, whereas control platelets readily formed lamellipodia and spread within 10–15 min, Kind3^-/- platelets only formed filopodia, with occasional transient small lamellipodia, and completely failed to spread for up to 45 min (Fig. 4a,b). We obtained similar results when we carried out adhesion in the presence of Mn²⁺ (Fig. 4c). Thus, Kindlin-3 is also required for integrin _IIb3-dependent outside-in signaling.

Figure 4: Defective adhesion and spreading of Kind3^-/- platelets.

(a) Washed wild-type and Kind3^-/- platelets were stimulated with 0.01 U/ml thrombin and then allowed to adhere to immobilized fibrinogen for 15 min. Scale bar, 5 m. (b) Scanning electron microscopy of wild-type and Kind3^-/- platelets after thrombin stimulation and adhesion to fibrinogen for 30 min. Scale bars, 1 m. (c) Washed platelets were allowed to adhere to immobilized fibrinogen in the presence of 3 mM Mn²⁺ for 30 min. Left, representative DIC images. Scale bar, 5 m. Right, number of lamellipodia-forming platelets (% of adherent platelets; mean s.d. of four experiments per group).

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Our study demonstrates that Kindlin-3 is essential for platelet integrin activation and subsequent integrin outside-in signaling. Furthermore, we found that Kindlin-3 regulates activation of both ₃ (_IIb3) and ₁ (₂₁) integrins, suggesting that Kindlin-3, like Talin, is a general regulator of integrin activation. We propose that this regulatory mechanism is mediated through a direct interaction between the PTB site of the F3 domain in Kindlin-3 and the integrin ₃ and ₁ tails, including their distal NxxY motifs. Because Talin binding requires an intact proximal NPxY motif, our findings raise questions regarding the roles of Kindlin-3 and Talin in integrin activation and the hierarchy of their binding to the integrin tails. Future studies on the structure of the Kindlin-3–integrin complex are required to examine the relative roles of Kindlin-3 and Talin interactions with integrin tails so as to fully understand how these receptors become activated. Finally, we show that elimination of Kindlin-3 prevents the formation of pathological thrombi. As Kindlin-3 is selectively expressed in cells of hematopoietic origin, it may serve as a potential target for the design of therapeutics aimed at specifically disrupting integrin activation in platelets.

Acknowledgments

We thank D. Calderwood (Yale University) and I. Campbell (Oxford University) for recombinant integrin tails and integrin tail expression vectors and help with pull-down assays, G. Wanner for imaging of platelets by scanning electron microscopy, M. Sixt and M. Boesl for mouse manipulation experiments, and R. Zent, A. Pozzi, M. Humphries and M. Schwartz for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Max Planck Society.

Received 11 October 2007; Accepted 4 January 2008; Published online 17 February 2008.

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1. Department of Molecular Medicine, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
2. University of Würzburg, Rudolf Virchow Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, Zinklesweg 10, 97080 Würzburg, Germany.
3. Institute of Clinical Biochemistry and Pathobiochemisty, Josef-Schneider-Str. 2, 97078 Würzburg, Germany.

Correspondence to: Reinhard Fässler¹ e-mail: faessler@biochem.mpg.de

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