Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Sep 18.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2014 Jun 26;34(9):1961–1967. doi: 10.1161/ATVBAHA.114.303851

Direct interaction of kindlin-3 with integrin αIIbβ3 in platelets is required for supporting arterial thrombosis in mice

Zhen Xu 1,2, Xue Chen 3, Huiying Zhi 2, Juan Gao 1, Katarzyna Bialkowska 4, Tatiana V Byzova 4, Elzbieta Pluskota 4, Gilbert C White II 2, Junling Liu 3, Edward F Plow 1,4,‡, Yan-Qing Ma 1,2,‡
PMCID: PMC4167429  NIHMSID: NIHMS627156  PMID: 24969775

Abstract

Objective

Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbβ3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbβ3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbβ3 in platelets in supporting integrin αIIbβ3-mediated platelet functions.

Approach and Results

We generated a strain of kindlin-3 knock-in (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbβ3 on platelets similarly to their wild type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbβ3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis.

Conclusions

These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbβ3 is involved in supporting integrin αIIbβ3 activation and integrin αIIbβ3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation

Keywords: Platelets, Kindlin-3, Integrin αIIbβ3, Thrombosis

INTRODUCTION

Integrin αIIbβ3 on platelets plays a major role in vascular homeostasis by mediating platelet aggregation 1, 2. As expressed on circulating platelets, the integrin αIIbβ3 receptors exist in a quiescent state where they exhibit low affinity for their cognate ligands. However, upon encounter with stimulatory agonists, the integrin receptors undergo a transformation to a state in which they can orchestrate productive functional responses, including formation of platelet aggregates leading to artery thrombus formation. Such integrin activation depends upon two intracellular proteins, talin and kindlin 3–6.

Talin is involved in integrin activation by virtue of its binding to the cytoplasmic tail (CT) of the integrin β subunit 7. In the absence of agonists, talin exists in the cytosol in an autoinhibited state in which its rod domain interacts with and shields the integrin binding site within the F3 subdomain of the talin head domain 8, 9. This auto-inhibition is relieved by multiple mechanisms that encourage talin to interact with membrane and the integrin β CT, thereby disrupting the integrin α/β CT complex 10. The kindlins, consisting of three members in mammals (kindlin-1, -2 and -3), are also capable of binding to the integrin β CT through their own F3 subdomains, but they bind primarily to a different site than talin 11–13. While the essential role of kindlins in supporting integrin activation has been established by in vivo deficits in integrin function associated with their deficiencies 14, 15, their mechanism of action is unclear. Kindlins do not dissociate the integrin α/β CT complex and do not enhance the capacity of talin to dissociate the CT complex 16, events that are necessary for integrin activation.

Kindlin-3 is preferentially expressed in and particularly important for the function of integrins on hematopoietic cells 13, 17. Deficiency of kindlin-3 expression in humans causes type III leukocyte adhesion deficiency (LAD-III), which is associated with an inability to activate integrins on platelets and leukocytes and manifests as susceptibility to bleeding and infections 18–21. To date, multiple distinct mutations in kindlin-3 gene have been identified in LAD-III patients, which all lead to the absence of kindlin-3 protein expression in blood cells, but the severity of symptoms in the patients has been variable 22, 23. For example, only one LAD-III patient was reported to have abnormally shaped red blood cells 24. Genetically modified mice with deficiency of kindlin-3 have been described and do exhibit the defects in platelet and leukocyte functions observed in LAD-III patients 13, 25, that were attributable to an inability to activate multiple integrin subclasses. However, these mice only survived one week 13, and, although their erythrocytes were misshaped, their shape was unlike that seen in the one human patient 24, 26. Also, the kindlin-3 knockout mice displayed altered expression of multiple genes in hematopoietic cells 26, including integrins, suggesting that the integrin dysfunction in kindlin-3-deficient mice might arise from effects unrelated from the direct interaction of kindlin-3 with integrin.

For kindlin-1 and -2, reconstitution experiments have clearly shown that the integrin-binding site in their F3 subdomain is important for integrin activation in model cells 12, 27. Even though kindlin-3 is similar to the other two family members, to assume a similar mechanism of action might be premature. A recent finding showed that ADAP, an adaptor protein restricted to hematopoietic cells, recruits talin and kindlin-3 to integrin αIIbβ3 in platelets and raises the possibility that kindlin-3 activates integrin independent of direct binding 28. Adding further to this uncertainty are the recent observations that kindlin-3 had no effect on integrin activation in nanodiscs in which talin head induced a measurable effect 29 and the in vivo observation that kindlin-2 could exert integrin independent functions 30. These findings bring into question the premise that direct interaction of kindlin-3 and integrin is essential for its regulation of hematopoietic cell responses that are blunted in kindlin-3 deficient mice and human patients.

In this study, we have generated kindlin-3 gene knock-in (K3KI) mice that carry a mutation that disrupts the interaction of kindlin-3 with integrin αIIbβ3. Using this animal model, we demonstrate that the direct contact between kindlin-3 and integrin αIIbβ3 is indeed required to support integrin function in platelets in arterial thrombosis.

MATERIALS AND METHODS

Materials and Methods are available in the online-only Data Supplement.

RESULTS

Identification of an integrin interaction-defective substitution in kindlin-3 protein

The inability of kindlin-3 to influence αIIbβ3 activation in nanodiscs 29; the ability of kindlin-3 to be recruited to integrin together with talin by adaptor proteins 28; and the effects of kindlin-3 deficiency on integrin and many other protein expression levels in mice 26 are among the data that bring into question the role of the direct interaction between kindlin-3 and integrins in controlling the responses of hematopoietic cells. These uncertainties prompted us to initially test if kindlin-3 does indeed share the same integrin-binding properties as kindlin-1 and -2. A double-substitution (QW/AA) in the F3 subdomains of kindlin-1 and -2 has been shown to disrupt the interaction of integrin β CTs with kindlin-1 and -2 11, 12, 27, and the corresponding substitutions were introduced into kindlin-3 (Fig. 1A). Using a previously established flow cytometry-based protein-peptide interaction assay 31, we attached EGFP-kindlin-3 and EGFP-kindlin-3 mutant from transfected cell lysates to microbeads and measured their interaction with the biotinylated integrin β3 CT peptides by flow cytometry. To restrict the involvement of any unrelated proteins in this assay, the EGFP-kindlin-3 coupled microbeads were extensively washed and no unexpected protein band was detected after SDS-PAGE and coomassie blue staining (data not shown). As shown in Fig. 1B, compared to EGFP control, EGFP-kindlin-3 on the microbeads displayed a significant interaction with the integrin β3 CT peptides. However, kindlin-3 with the QW/AA substitution exhibited a dramatically reduced interaction with the integrin β3 CT peptides. These results demonstrate that the QW/AA substitution in kindlin-3 significantly perturbs the interaction between kindlin-3 and integrin β3 CT. To consider whether the QW/AA substitution affected the structural stability of kindlin-3 protein, EGFP-kindlin-3 and the QW/AA mutant expressed in CHO cells were loaded on a native gel and their mobilities on nondenaturing gels were assessed by Western blotting. As shown in Fig. 1C, kindlin-3 protein and the QW/AA mutant migrated identically, suggesting that the kindlin-3 mutant exhibits a similar structural stability with its WT counterpart. We also noted that the expression levels of EGFP-kindlin-3 and the EGFP-fused QW/AA mutant form were similar in several mammalian cell lines, HEL, K562 and RAW cells (data not shown), which is also consistent with similar folding of the two kindlin-3 molecules. Therefore, our results suggest that kindlin-3 shares the same recognition mechanism as kindlin-1 and -2 binding to integrin β CT and the QW/AA substitutions can be used to specifically perturb the interaction between kindlins and integrin.

Figure 1.

Figure 1

Open in a new tab

The QW/AA substitution in kindlin-3 protein blocks integrin β3 CT recognition. (A) The QW residues in the kindlin-3-F3 subdomain were substituted with two alanine residues. (B) Microbeads coated with protein G were used for testing the interaction of kindlin-3 with integrin β3 CT peptide. EGFP-fused kindlin-3 (EGFP-Kindlin-3), EGFP-fused kindlin-3 mutant (EGFP-Kindlin-3-AA) or EGFP alone were transiently expressed in CHO cells and then coupled onto microbeads via an anti-EGFP antibody. EGFP or EGFP-Kindlin-3 loaded microbeads were extensively washed and incubated with the biotinylated integrin β3 CT peptides followed by streptavidin conjugated with Alexa Fluor 633. The loading of EGFP and EGFP-fusions on the microbeads and the binding of integrin β3 CT peptides to EGFP-fused kindlin-3 on the microbeads were analyzed on a BD LSRII Flow Cytometer. (C) EGFP-fused kindlin-3 (EGFP-Kindlin-3) and EGFP-fused kindlin-3 mutant (EGFP-Kindlin-3-AA) carrying the QW/AA substitution were expressed in CHO cells and subjected to native PAGE under non-reducing and non-denaturing conditions. The migration of these two proteins was further evaluated by Western blotting with an anti-EGFP antibody.

Generation of K3KI)mice that carry the QW/AA substitution

To evaluate the functional significance of the direct interaction between kindlin-3 and integrin in vivo, a kindlin-3 knock-in allele in which a double-mutation (QW/AA) was introduced into the kindlin-3 gene locus (Fig. 2, A & B) was used to generate K3KI mice. K3KI mice were born in expected Mendelian ratios and were fertile. Significantly, K3KI mice showed normal survival (mice of > × months of age with no overt signs of ill-health), which is different from kindlin-3−/− mice, which only survive one week postnatally 13. Spontaneous bleeding was not observed in K3KI mice and platelet counts in peripheral blood of K3KI mice were comparable to those in WT mice (Fig. 2C). As with kindlin-3−/− mice 25, K3KI mice exhibited significant leukocytosis (Fig. 2D). In contrast to the kindlin3 knockout mice which were severely anemic 25, red blood cells in peripheral blood of K3KI mice were only slightly suppressed (Fig. 2E). We did note some acanthocytes in blood smears of some of the K3KI mice as was reported in the kindlin-3−/− mice 26, but this varied among the individual K3KI mice and is being further explored.

Figure 2.

Figure 2

Open in a new tab

Generation of kindlin-3 gene knock-in (K3KI) mice that carry the QW/AA mutation. (A) A diagram illustrating the gene-targeting strategy for generating K3KI mice. The numbered rectangles represent kindlin-3 gene exons and the mutated nucleotides (CAATGG to GCCGCC) locate in exon14 (asterisks). Neor represents the neomycin cassette which was used for selecting positive embryonic stem cell clones. The 5′ and 3′ arms for homologous recombination are highlighted. The inserted flippase recognition target (FRT) sequences were used to remove the neomycin cassette. (B) Targeted genomic DNA fragment was amplified by PCR from the selected embryonic stem cell clones and sequenced, and the mutated sites in the kindlin-3 gene were verified. (C) Platelet counts in peripheral blood of WT and K3KI mice were measured and no significant difference was found between them. (D) The counts of leukocytes in peripheral blood of K3KI mice were significantly elevated compared to WT mice. (** p < 0.01) (E) Red blood cell counts in peripheral blood of WT and K3KI mice. (* p < 0.05) (F) Cell surface expression of integrin αIIbβ3 on K3KI platelets and WT counterparts were measured by flow cytometry using a phycoerythrin-conjugated anti-mouse CD41 antibody. (G) Washed platelets from WT and K3KI mice were lysed and the expression of kindlin-3 (Kind-3), talin and Rap1 proteins (Rap1a and Rap1b) in platelets was evaluated by Western blotting. Actin was also measured as a loading control. (H) Integrin αIIbβ3 was immunoprecipitated from the platelet lysates and kindlin-3 and integrin β3 in the precipitates were measured by Western blotting.

We examined the expression of integrin αIIbβ3 on platelets by flow cytometry and found that integrin αIIbβ3 expression levels were identical on WT and K3KI platelets (Fig. 2F). The expression of kindlin-3 protein in washed platelets from WT and K3KI mice were similar as assessed by Western blots (Fig. 2G). In addition, two other proteins with important roles in integrin activation, talin and Rap1 (Rap1a + Rap1b), were also expressed at similar levels in K3KI and WT platelets (Fig. 2G). Significantly, the results of co-immunoprecipitation experiments showed that integrin αIIbβ3 markedly reduced its association with the kindlin-3 mutant in K3KI platelets (~ 85% reduction) compared to the association between integrin αIIbβ3 and kindlin-3 in WT platelets under the same conditions (Fig. 2H). Thus, we successfully generated a strain of mice expressing a kindlin-3 mutant with a significant reduction in integrin binding activity, which can be used to evaluate the functional significance of the interaction between kindlin-3 and integrin in hematopoietic cells.

Interaction between kindlin-3 and integrin αIIbβ3 in platelets facilitates αIIbβ3 activation and is essential for αIIbβ3-mediated platelet spreading

To evaluate if the direct interaction between the kindlin-3 QW sequence and integrin affects hemostasis, tail bleeding assays were performed on WT and K3KI mice. After a small portion of tail-tip was removed, the bleeding time was measured. The results revealed that K3KI mice displayed a significantly prolonged bleeding time compared to their WT counterparts (Fig. 3A). In WT mice, the mean bleeding time was 211 ± 126 sec (n = 8), whereas the bleeding time of all K3KI mice (n= 8) exceeded 600 sec. Thus, the QW/AA substitution in kindlin-3 led to a severe hemostatic defect in K3KI mice. The extended bleeding time from K3KI mice led us to analyze integrin αIIbβ3 signaling in K3KI platelets. Integrin inside-out signaling was evaluated by agonist-induced soluble fibrinogen binding to αIIbβ3 on platelets. As shown in Fig. 3B, integrin αIIbβ3 activation on K3KI platelets was completely inhibited upon stimulation with ADP (20 μM) and partially but significantly inhibited in response to protease-activated receptor 4 agonist peptide (AYPGKF, 150 μM) or collagen related peptide (CRP, 2 μg/ml). As assessed by flow cytometry, the inhibition was ~ 50% in mean fluorescence intensity (MFI). The residual fibrinogen binding to K3KI platelets in presence of PAR-4 agonist peptide or CRP suggests that K3KI platelets still retain some ability to support integrin αIIbβ3 inside-out signaling in response to strong stimuli.

Figure 3.

Figure 3

Open in a new tab

Impaired K3KI platelet function. (A) A 3-mm mouse tail segment was transected and the bleeding time was monitored for 10 min. The time to first cessation in bleeding was recorded for each mouse. (**, p < 0.01) (B) Integrin αIIbβ3 activation on washed platelets from K3KI mice and their WT counterparts was tested using flow cytometry by measuring the binding of Alexa Fluor 647 conjugated fibrinogen in response to ADP (20 μM), protease-activated receptor 4 (PAR-4) agonist peptide (150 μM) and CRP (2 μg/ml). (**, p < 0.01) (C) Washed platelets from K3KI mice and WT counterparts were allowed to adhere and spread on immobilized fibrinogen for 120 min in a tissue culture incubator. After washing, the adherent platelets were fixed, permeabilized and stained with rhodamine-conjugated phalloidin and the images were captured under a fluorescence microscope (100× objective). Spreading areas of adherent platelets were quantified using Metamorph software. (*, p < 0.05) (D) Representative traces to show the aggregation kinetics of WT and K3KI platelets stimulated with different doses of the indicated agonists. (E) Photograph of thrombin-induced clot retraction of PRP containing WT and K3KI platelets.

Next, we measured the spreading of K3KI platelets on immobilized fibrinogen and found that integrin αIIbβ3-mediated K3KI platelet spreading was significantly compromised (Fig. 3C), demonstrating that interaction with kindlin-3 is essential for integrin αIIbβ3 to mediate outside-in signaling. Inspite of the impaired bidirectional signaling of integrin αIIbβ3 on K3KI platelets, degranulation was unaffected; P-selectin translocation to the platelet surface on WT and K3KI platelets in response to stimulation with PAR-4 agonist peptide or CRP was identical (data not shown). In addition, the fibrinogen levels in WT and K3KI platelets were similar as evaluated by Western blots (data not shown).

Interaction between kindlin-3 and integrin αIIbβ3 in platelets supports platelet aggregation and clot retraction

Next, ex vivo platelet aggregation studies were performed in response to different agonists. As shown in Fig. 3D, K3KI platelets showed negligible aggregation in response to ADP and only a weak response to U46619 compared to WT platelets. Notably, partial aggregation of K3KI platelets was observed in response to collagen and thrombin. The capacity of K3KI platelets to retain an ability to aggregate in response to higher doses of strong agonists differs from previous observations on kindlin-3−/− platelets, which had negligible responses to all agonists tested13.

We also evaluated clot retraction induced by platelets from WT and K3KI mice as a response driven by αIIbβ3-mediated outside-in signaling. Using high doses of thrombin, sufficient to induce aggregation, K3KI platelets failed to support clot retraction (Fig 3E) whereas WT platelets retracted well. Taken together, these findings demonstrate that the QW sequence-mediated interaction of kindlin-3 with integrin αIIbβ3 on platelets is important for supporting platelet aggregation and essential for clot retraction.

The interaction between kindlin-3 and integrin is essential for thrombus formation in vitro

To further evaluate the functional significance of the direct interaction between kindlin-3 and integrin in platelets, an in vitro thrombus formation assay was performed under flow conditions using a whole-blood microfluidic perfusion system as previously described 32. Platelets in whole blood were labeled with mepacrine and allowed to flow over a collagen-coated surface at 80 dynes/cm2. The adhesion and aggregation of platelets were visualized as the accumulation of fluorescence on the collagen-coated surface. As shown in Fig. 4A, platelet adhesion was markedly diminished and thrombus formation was severely hampered in blood from K3KI mice in contrast with the strong signal for both platelet adhesion and thrombus formation obtained with blood from WT mice at the same conditions. This experiment was repeated twice and similar results were obtained. The calculated percentage of surface fluorescence coverage or total integrated fluorescence intensity per μm2 area at different time points was increased about 30-fold for blood from WT mice compared to that from K3KI mice, suggesting that a direct interaction between kindlin-3 and integrin in platelets is essential for efficient platelet-mediated thrombus formation in this in vitro assay.

Figure 4.

Figure 4

Open in a new tab

Resistance to arterial thrombus formation in K3KI mice. (A) Whole blood drawn from WT and K3KI mice was labeled with mepacrine and perfused into laminar flow chambers coated with collagen under a flow of 80 dynes/cm2. Time-course images of platelet adhesion and aggregation were acquired using epifluorescence microscopy. (B) The mouse carotid artery was exposed and stimulated with 10% FeCl3. After that, blood flow in the carotid artery was monitored by a transit-time flowmeter. “Zero” on the X-axis represents the time at which filter paper saturated with FeCl3 solution was removed from the surface of the artery. The times to occlusion were measured. (**, p < 0.01)

Crosstalk between kindlin-3 and integrin in platelets is essential for arterial thrombus formation in vivo

The function of K3KI platelets in vivo was measured in a FeCl3-induced arterial thrombosis model. A transit-time perivascular flowmeter was used to monitor blood flow in the carotid artery after a 3-min vascular injury with 10% FeCl3. Data from 5 mice are shown in Fig. 4B. For WT mice, stable occlusion occurred within 15 min. In contrast, occlusion failed to occur in the arteries of K3KI mice within the testing time (45 min). These results show that contact between kindlin-3 and integrin in platelets is required to support arterial thrombus formation in vivo.

DISCUSSION

The pathogenesis of human LAD-III has been attributed to the absence of kindlin-3 protein expression in hematopoietic cells due to heritable mutations in kindlin-3 gene in patients 23. It has been well documented that kindlin-3 is required to support integrin activation in hematopoietic cells 13, 19, 20, 25. However, the molecular mechanisms involved are still poorly understood. Proteomic analysis disclosed that the expression levels of multiple proteins in kindlin-3 deficient murine cells could be either up or down regulated 26, bringing some uncertainty to the cause of integrin dysfunction in hematopoietic cells. Although kindlin-3 has been categorized as an integrin-binding protein, the functional significance of the direct interaction between kindlin-3 and integrin αIIbβ3 in platelets still remains unknown. By mutagenesis, it has been demonstrated that the kindlin-3 binding sites in the integrin β3 CT (NITY759 motif) is critical for supporting integrin αIIbβ3 activation 12, 33. However, the NITY759 motif in the β3 CT is in a region that can mediate interaction with multiple proteins 34, 35 so that mutations in this region could possibly produce off-target effects. In our present study, we generated a kindlin-3 knock-in (K3KI) mouse model harboring a QW/AA substitution in kindlin-3 protein to disconnect kindlin-3 from integrins in hematopoietic cells. The value of the K3KI mouse model include: 1) K3KI mice have normal expression levels of kindlin-3 protein in platelets, which limits possible indirect effects resulting from the absence of kindlin-3 protein; 2) Theoretically, the QW/AA substitution in kindlin-3 protein should be able to disconnect kindlin-3 from multiple integrin members in hematopoietic cells but these very limited and specific point mutations should not perturb non-integrin dependent kindlin functions 30; 3) The interaction of integrin with other integrin CT-binding partners also should not be directly affected in K3KI cells. These features suggest that K3KI mouse provides a unique opportunity to evaluate the integrin-kindlin-3 signaling in vivo. Using this model, we demonstrate that the direct interaction between kindlin-3 and integrin αIIbβ3 in platelets is essential for supporting certain platelet function and arterial thrombus formation, which could not be concluded from the previous studies on LAD-III patients and kindlin-3 null mice, thus representing an important step forward in our understanding kindlin-3’s function in platelets.

Unlike kindlin-3 deficient platelets, which display minimal integrin αIIbβ3 activation and aggregation in response to all agonists 13, we found that high concentrations of agonists such as thrombin and collagen still could induce significant integrin αIIbβ3 activation (Fig. 3B) and aggregation of K3KI platelets (Fig. 3D). Indeed, at a higher thrombin concentration, the extent of aggregation of K3KI platelets nearly approached that of WT platelets. Possible interpretations include: 1) the interaction of the kindlin-3 QW/AA mutant with integrins in K3KI platelets is compromised but not eliminated; a weak interaction could still occur, which might be sufficient, together with talin, to induce platelet aggregation in the face of a strong stimulus; 2) The kindlin-3 QW/AA mutant might employ a mechanism involving the third molecule, such as ADAP, or a signaling pathway to support integrin function, which is independent of the direct interaction between kindlin-3 and integrins and which is operative at high doses of strong agonists 28. Further studies will be required to address these possibilities. Nonetheless, in vivo platelet aggregation in K3KI mice under pathological conditions were significantly suppressed, suggesting that the ex vivo K3KI platelet aggregation under strong stimulations may not have the opportunity to occur in vivo.

Although significant ex vivo platelet aggregation could be induced by thrombin (0.1 ~ 0.2 U/ml) for K3KI platelets (Fig. 3D), the aggregates formed at a high thrombin concentration (0.8 U/ml) failed to support K3KI platelet-mediated clot retraction (Fig. 3E), raising a possibility that kindlin-3 may employ distinct mechanisms in supporting integrin αIIbβ3-mediated platelet aggregation (inside-out signaling dependent) and clot retraction (outside-in signaling dependent). The profound effect of kindlin-3 QW/AA mutation on platelet spreading is consistent with the major importance of kindlin-3 in outside-in signaling across integrin αIIbβ3. Hypothetically, the absence of or a weak interaction between integrin αIIbβ3 and kindlin-3 in K3KI platelets may fail to sustain the high mechanical forces between platelet integrin αIIbβ3 and fibrinogen/fibrin, thus leading to clot retraction defects for K3KI platelets. Alternatively, kindlin-3, like talin 36, may play distinct roles in orchestrating different integrin-dependent functions.

In summary, our findings in this study demonstrate the importance of the direct interaction between kindlin-3 and integrin αIIbβ3 in platelets for supporting integrin αIIbβ3 mediated platelet responses and arterial thrombosis, thus furthering our understanding of the molecular regulation of kindlin-3 signaling in platelets.

Supplementary Material

supplementary data

SIGNIFICANCE.

The direct interaction of kindlin-3 with integrin αIIbβ3 in platelets is a key conduit in arterial thrombus formation and represents a potential therapeutic target for developing novel anti-thrombotic strategies.

Acknowledgments

SOURCES OF FUNDING

This work was supported by grants from Blood Center Research Fund of Blood Center of Wisconsin, National Natural Science Foundation of China (81270579 and 31370748) and the Tarazi Endowment at the Cleveland Clinic.

Footnotes

DISCLOSURES

None

Reference List

  • 1.Coller BS, Shattil SJ. The GPIIb/IIIa (integrin alphaIIbbeta3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood. 2008 Oct 15;112(8):3011–25. doi: 10.1182/blood-2008-06-077891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bledzka K, Smyth SS, Plow EF. Integrin alphaIIbbeta3: from discovery to efficacious therapeutic target. Circ Res. 2013 Apr 12;112(8):1189–200. doi: 10.1161/CIRCRESAHA.112.300570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Shattil SJ. The beta3 integrin cytoplasmic tail: protein scaffold and control freak. J Thromb Haemost. 2009 Jul;7(Suppl 1):210–3. doi: 10.1111/j.1538-7836.2009.03397.x. [DOI] [PubMed] [Google Scholar]
  • 4.Meves A, Stremmle C, Gottschalk K, Fassler R. The Kindlin protein family: new members to the club of focal adhesion proteins. Trends Cell Biol. 2009 Aug 19;19(10) doi: 10.1016/j.tcb.2009.07.006. [DOI] [PubMed] [Google Scholar]
  • 5.Malinin NL, Plow EF, Byzova TV. Kindlins in FERM adhesion. Blood. 2010;115:4011–7. doi: 10.1182/blood-2009-10-239269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ye F, Kim C, Ginsberg MH. Molecular mechanism of inside-out integrin regulation. J Thromb Haemost. 2011 Jul;9(Suppl 1):20–5. doi: 10.1111/j.1538-7836.2011.04355.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg MH, Calderwood DA. Talin binding to integrin β tails: a final common step in integrin activation. Science. 2003;302:103–6. doi: 10.1126/science.1086652. [DOI] [PubMed] [Google Scholar]
  • 8.Goksoy E, Ma Y-Q, Wang X, Kong X, Perera D, Plow EF, Qin J. Structural basis for the autoinhibition of talin in regulating integrin activation. Mol Cell. 2008;31:124–33. doi: 10.1016/j.molcel.2008.06.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Goult BT, Xu XP, Gingras AR, Swift M, Patel B, Bate N, Kopp PM, Barsukov IL, Critchley DR, Volkmann N, Hanein D. Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation. J Struct Biol. 2013 Oct;184(1):21–32. doi: 10.1016/j.jsb.2013.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Das M, Subbayya IS, Qin J, Plow EF. Mechanisms of talin-dependent integrin signaling and crosstalk. Biochim Biophys Acta. 2013 Jul 24; doi: 10.1016/j.bbamem.2013.07.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Shi X, Ma YQ, Tu Y, Chen K, Wu S, Fukuda K, Qin J, Plow EF, Wu C. The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility. J Biol Chem. 2007 Jul 13;282(28):20455–66. doi: 10.1074/jbc.M611680200. [DOI] [PubMed] [Google Scholar]
  • 12.Ma YQ, Qin J, Wu C, Plow EF. Kindlin-2 (Mig-2): a co-activator of beta3 integrins. J Cell Biol. 2008 May 5;181(3):439–46. doi: 10.1083/jcb.200710196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Moser M, Nieswandt B, Ussar S, Pozgajova M, Fassler R. Kindlin-3 is essential for integrin activation and platelet aggregation. Nat Med. 2008 Mar;14(3):325–30. doi: 10.1038/nm1722. [DOI] [PubMed] [Google Scholar]
  • 14.Larjava H, Plow EF, Wu C. Kindlins: essential regulators of integrin signalling and cell-matrix adhesion. EMBO Rep. 2008 Nov 7;9:1203–8. doi: 10.1038/embor.2008.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Plow EF, QJBT Kindling the flame of integrin activation and function with kindlins. 328. Curr Opin Hematol. 2009 Sep 16;16(5):323. doi: 10.1097/MOH.0b013e32832ea389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Bledzka K, Liu J, Xu Z, Perera HD, Yadav SP, Bialkowska K, Qin J, Ma YQ, Plow EF. Spatial coordination of kindlin-2 with talin head domain in interaction with integrin beta cytoplasmic tails. J Biol Chem. 2012 May 30; doi: 10.1074/jbc.M111.336743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Ussar S, Wang HV, Linder S, Fassler R, Moser M. The Kindlins: subcellular localization and expression during murine development. Exp Cell Res. 2006 Oct 1;312(16):3142–51. doi: 10.1016/j.yexcr.2006.06.030. [DOI] [PubMed] [Google Scholar]
  • 18.Mory A, Feigelson SW, Yarali N, Kilic SS, Bayhan GI, Gershoni-Baruch R, Etzioni A, Alon R. Kindlin-3: a new gene involved in the pathogenesis of LAD-III. Blood. 2008 Sep 15;112(6):2591. doi: 10.1182/blood-2008-06-163162. [DOI] [PubMed] [Google Scholar]
  • 19.Malinin NL, Zhang L, Choi J, Ciocea A, Razorenova O, Ma Y-Q, Podrez EA, Tosi M, Lennon DP, Caplin AI, Shurin SB, Plow EF, Byzova TV. A point mutation in kindlin-3 ablates activation of three integrin subfamilies in humans. Nature Med. 2009;15:313–8. doi: 10.1038/nm.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Svensson L, Howarth K, McDowall A, Patzak I, Evans R, Ussar S, Moser M, Metin A, Fried M, Tomlinson I, Hogg N. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation. Nat Med. 2009 Mar;15(3):306–12. doi: 10.1038/nm.1931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kuijpers TW, van d V, Weterman MA, de BM, Tool AT, van den Berg TK, Moser M, Jakobs ME, Seeger K, Sanal O, Unal S, Cetin M, Roos D, Verhoeven AJ, Baas F. LAD-1/variant syndrome is caused by mutations in FERMT3. Blood. 2009 May 7;113(19):4740–6. doi: 10.1182/blood-2008-10-182154. [DOI] [PubMed] [Google Scholar]
  • 22.van d V, Maddalena A, Sanal O, Holland SM, Uzel G, Madkaikar M, de BM, van LK, Koker MY, Parvaneh N, Fischer A, Law SK, Klein N, Tezcan FI, Unal E, Patiroglu T, Belohradsky BH, Schwartz K, Somech R, Kuijpers TW, Roos D. Hematologically important mutations: leukocyte adhesion deficiency (first update) Blood Cells Mol Dis. 2012 Jan 15;48(1):53–61. doi: 10.1016/j.bcmd.2011.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Harris ES, Weyrich AS, Zimmerman GA. Lessons from rare maladies: leukocyte adhesion deficiency syndromes. Curr Opin Hematol. 2013 Jan;20(1):16–25. doi: 10.1097/MOH.0b013e32835a0091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Meller J, Malinin NL, Panigrahi S, Kerr BA, Patil A, Ma Y, Venkateswaran L, Rogozin IB, Mohandas N, Ehlayel MS, Podrez EA, Chinen J, Byzova TV. Novel aspects of Kindlin-3 function in humans based on a new case of leukocyte adhesion deficiency III. J Thromb Haemost. 2012 Jul;10(7):1397–408. doi: 10.1111/j.1538-7836.2012.04768.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Moser M, Bauer M, Schmid S, Ruppert R, Schmidt S, Sixt M, Wang HV, Sperandio M, Fassler R. Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nat Med. 2009 Mar;15(3):300–5. doi: 10.1038/nm.1921. [DOI] [PubMed] [Google Scholar]
  • 26.Kruger M, Moser M, Ussar S, Thievessen I, Luber CA, Forner F, Schmidt S, Zanivan S, Fassler R, Mann M. SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell. 2008 Jul 25;134(2):353–64. doi: 10.1016/j.cell.2008.05.033. [DOI] [PubMed] [Google Scholar]
  • 27.Harburger DS, Bouaouina M, Calderwood DA. Kindlin-1 and -2 directly bind the C-terminal region of beta integrin cytoplasmic tails and exert integrin-specific activation effects. J Biol Chem. 2009 Apr 24;284(17):11485–97. doi: 10.1074/jbc.M809233200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kasirer-Friede A, Kang J, Kahner B, Ye F, Ginsberg MH, Shattil SJ. ADAP interactions with talin and kindlin promote platelet integrin alphaIIbbeta3 activation and stable fibrinogen binding. Blood. 2014 Feb 12; doi: 10.1182/blood-2013-08-520627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ye F, Petrich BG, Anekal P, Lefort CT, Kasirer-Friede A, Shattil SJ, Ruppert R, Moser M, Fassler R, Ginsberg MH. The Mechanism of Kindlin-Mediated Activation of Integrin alphaIIbbeta3. Curr Biol. 2013 Nov 18;23(22):2288–95. doi: 10.1016/j.cub.2013.09.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pluskota E, Ma Y, Bledzka KM, Bialkowska K, Soloviev DA, Szpak D, Podrez EA, Fox PL, Hazen SL, Dowling JJ, Ma YQ, Plow EF. Kindlin-2 regulates hemostasis by controlling endothelial cell surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism. Blood. 2013 Jul 29; doi: 10.1182/blood-2013-04-497669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Xu Z, Gao J, Hong J, Ma YQ. Integrity of kindlin-2 FERM subdomains is required for supporting integrin activation. Biochem Biophys Res Commun. 2013 May 3;434(2):382–7. doi: 10.1016/j.bbrc.2013.03.086. [DOI] [PubMed] [Google Scholar]
  • 32.Zhi H, Rauova L, Hayes V, Gao C, Boylan B, Newman DK, McKenzie SE, Cooley BC, Poncz M, Newman PJ. Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood. 2013 Mar 7;121(10):1858–67. doi: 10.1182/blood-2012-07-443325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Xi X, Bodnar RJ, Li Z, Lam SC, Du X. Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling. J Cell Biol. 2003 Jul 21;162(2):329–39. doi: 10.1083/jcb.200303120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ma YQ, Qin J, Plow EF. Platelet integrin alpha(IIb)beta(3): activation mechanisms. J Thromb Haemost. 2007 Jul;5(7):1345–52. doi: 10.1111/j.1538-7836.2007.02537.x. [DOI] [PubMed] [Google Scholar]
  • 35.Cantor JM, Ginsberg MH, Rose DM. Integrin-associated proteins as potential therapeutic targets. Immunol Rev. 2008 Jun;223:236–51. doi: 10.1111/j.1600-065X.2008.00640.x. [DOI] [PubMed] [Google Scholar]
  • 36.Shen B, Zhao X, O’Brien KA, Stojanovic-Terpo A, Delaney MK, Kim K, Cho J, Lam SC, Du X. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature. 2013 Nov 7;503(7474):131–5. doi: 10.1038/nature12613. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplementary data
NIHMS627156-supplement-supplementary_data.pdf (193.9KB, pdf)

RESOURCES