Deep vein thrombosis (DVT) is one of the major causes of morbidity and mortality worldwide. DVT is associated with multiple risk factors and occurs when a blood clot forms in the deep veins. Significantly, recent studies have revealed that neutrophils are essentially involved in DVT formation, indicating that DVT is a complex, coordinated dysfunction of both the coagulation cascade and the innate immune system. Studies in animal DVT models have demonstrated that initial neutrophil recruitment and subsequent release of neutrophil extracellular traps (NETs) promote thrombus growth; in addition, later neutrophil infiltration facilitates thrombus resolution, suggesting that neutrophils may play dual functional roles in DVT. Kindlin-3 is a recently identified integrin ? cytoplasmic tail binding protein in blood cells and required for supporting integrin-mediated leukocyte recruitment and infiltration. Patients with kindlin-3 deficiency suffer from Type-III leukocyte adhesion deficiency, a rare genetic disorder characterized by recurrent infections and severe bleeding due to integrin dysfunction in both leukocytes and platelets. Importantly, our preliminary observations have suggested that kindlin-3 also carries out a number of integrin-independent functions in neutrophils, including suppressing NET release. Therefore, we hypothesize that kindlin-3 plays multiple roles in regulating the kinetics of DVT. The overall goal of this competitive application is to examine both integrin-dependent and integrin-independent functions of kindlin-3 in neutrophils in DVT formation, and to mechanistically interpret the novel function of kindlin-3 that regulates NET release.
Two Specific Aims are proposed: (1) Specific Aim 1 will determine the importance of kindlin-3/integrin interactions in neutrophils in DVT formation. We will examine the kinetics of DVT formation in a standard inferior vena cava (IVC) ligature model in mice that carry an integrin-interaction disrupting mutation in kindlin-3 or a kindlin-3 deletion specifically in neutrophils. The kinetics and extent of neutrophil recruitment, infiltration in the IVC, NET release, thrombus growth and resolution will be quantified in these mice using a combination of both intravital microscopy and histological analysis. We expect that kindlin-3 plays important roles in both initial neutrophil recruitment to inflamed IVC where venous thromboemboli develop and later neutrophil infiltration into the formed thrombi that promotes DVT resolution. (2) Specific Aim 2 will determine the molecular mechanism by which neutrophil kindlin-3 regulates NET release in DVT. The key subdomain(s) and residue(s) in kindlin-3 responsible for regulating NET release will be mapped in informative model systems. The signaling intermediates between kindlin-3 and NET release will be identified by established biochemical approaches. Further, the contribution of kindlin-3/NETs signaling pathway to DVT formation will be evaluated in mice. Taken together, these studies will redefine our understanding of the complex mechanisms by which neutrophil kindlin-3 regulates the kinetics of DVT, thus providing opportunities for designing novel and targeted therapeutic strategies for prevention and treatment of DVT.
The goal of this research project is to produce mechanistic insight into the molecular basis of kindlin-3 signaling in deep vein thrombosis (DVT), a major cause of morbidity and mortality worldwide. This study will generate valuable information which will expand our understanding of DVT formation, thus providing opportunities for developing novel and safer therapeutic strategies to prevent and treat DVT.
|Xu, Zhen; Ni, Beiwen; Cao, Zhongyuan et al. (2018) Kindlin-3 negatively regulates the release of neutrophil extracellular traps. J Leukoc Biol 104:597-602|
|Gao, Juan; Huang, Ming; Lai, Jingjing et al. (2017) Kindlin supports platelet integrin ?IIb?3 activation by interacting with paxillin. J Cell Sci 130:3764-3775|