No abstract

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

In vitro studies indicate that binding of talin to the β 3 integrin cytoplasmic domain (tail) results in integrin α IIb β 3 (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the β 3 tail. We introduced a β 3 (Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a β 3 (L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate α IIb β 3 require binding of talin to the β 3 tail. β 3 (L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of β 3 (Y747A) and virtually all β 3 -null animals examined. Remarkably, less than 5% of β 3 (L746A) animals exhibited this form of bleeding. These results establish that α IIb β 3 activation in vivo is dependent on the interaction of talin with the β 3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of β 3 integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Increased ligand binding to cellular integrins (“activation”) plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis and hemostasis and thrombosis[1-5]. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering[6] and depends on integrin-talin interactions[5]. Loss of kindlins results in reduced activation of integrins[7-13]. Kindlins might promote talin binding to integrins through a cooperative mechanism[5, 14-16]; however, kindlins do not increase talin association with integrins[17]. Here we report that, unlike talin head domain (THD), kindlin-3 caused little effect on the affinity of purified monomeric αIIbβ3, and it didn’t enhance activation by THD. Furthermore, studies with ligands of varying valency showed that kindlins primarily increased cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduced αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduced the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence (TIRF) and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to <100 microliters per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin αIIbβ3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.