Direct Interaction of Kindlin-3 With Integrin αIIbβ3 in Platelets Is Required for Supporting Arterial Thrombosis in Mice

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

Interaction of kindlin-3 and β2-integrins differentially regulates neutrophil recruitment and NET release in mice

“…18 Inbred wild-type (WT) littermates were used as controls to confront comparative studies. All the mice used in this study were 8 to 12 weeks of age.…”

“…12,13 Because kindlin-3 can interact with multiple signaling molecules in addition to integrins, 14,15 and knockout of kindlin-3 can alter the expression of multiple molecules in blood cells, 16 it is obvious that the functional consequence of the crosstalk between kindlin-3 and b 2 -integrins in neutrophils could not be simply concluded from the studies of either LAD-III patients or kindlin-3 knockout mice, 17 and needs to be further delineated with more targeted methods. Here, using the established kindlin-3 knock-in (K3KI) mice, 18 we pinpoint the differential involvement of the interaction between kindlin-3 and b 2 -integrins in regulating neutrophil recruitment and NET release, and establish a novel strategy for selectively restricting NET release in innate immune response.…”

Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor

“…22,23,58 However, agonist-induced fibrinogen binding to aIIbb3 in b3DRGT murine platelets is only minimally impaired, 13 and similar observations were made in knock-in platelets expressing a b3 integrin binding-defective kindlin-3 mutant. 59 WOW-1 Fab binding to b3(DiY.F) ECs stimulated by VEGF is reportedly impaired. 7 However, unlike aVb3DRGT, interactions of b3(DiY.F) with both talin and kindlin-2 are likely to be impaired.…”

“…13 It is consistent with the observation that knock-in mice expressing a kindlin-3 mutant with impaired binding to b3 exhibit reduced thrombus formation. 59 Expression of kindlin-3 has been reported in some types of human ECs 61 ;…”

Kindlin supports platelet integrin αIIbβ3 activation by interacting with paxillin

“…Both TH and kindlins are FERM domain-containing proteins, consisting of typical FERM subdomains (F 1 , F 2 and F 3 ) and an extra N-terminal F 0 subdomain (Larjava et al, 2008). The F 3 subdomains in TH and kindlins are responsible for binding to the integrin β CTs, but their binding sites in the β CTs are distinct: an NPLY/F motif in the middle of β CTs for TH and an NxxY/F motif at the C-termini of β CTs for kindlins Ma et al, 2008;Moser et al, 2008;Shi et al, 2007;Xu et al, 2015Xu et al, , 2014. Meanwhile, the F 3 subdomain of TH also recognizes the membrane-proximal residues of the integrin β CTs and is thus capable of unclasping the integrin α/β CT complex and triggering integrin activation (Wegener et al, 2007); in addition, other subdomains in TH are functionally supportive by facilitating membrane attachment (Goult et al, 2010).…”