Ana Kasirer-Friede

Platelet integrins and immunoreceptors

Summary:  Stable platelet adhesion to extracellular matrices and the formation of a hemostatic or pathological thrombus are dependent on integrin αIIbβ3, also known as GPIIb–IIIa. However, maximal platelet responses to vascular injury may involve the participation of other integrins expressed in platelets (αVβ3, α2β1, α5β1, and α6β1). Platelet membrane ‘immunoreceptors’ contain at least one subunit with an extracellular immunoglobulin superfamily domain and/or an intracellular stimulatory immunoreceptor tyrosine‐based activation motif (ITAM) or immunoreceptor tyrosine‐based inhibitory motif (ITIM). Platelet ITAM receptors, such as FcγRIIA and the GPVI–FcRγ complex, promote activation of integrins, while ITIM receptors, such as platelet–endothelial cell adhesion molecule‐1, may promote their inhibition. This review summarizes the structure and function of platelet integrins and immunoreceptors, the emerging functional relationships between these receptor classes, and the consequences of their interaction for platelet function in hemostasis and thrombosis.

Microfluidic devices for studies of shear-dependent platelet adhesion

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 microl 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 alphaIIbbeta3, 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.

The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase-associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin-regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3.

Role for ADAP in shear flow-induced platelet mechanotransduction.

Binding of platelets to fibrinogen via integrin alphaIIbbeta3 stimulates cytoskeletal reorganization and spreading. These responses depend on tyrosine phosphorylation of multiple proteins by Src family members and Syk. Among Src substrates in platelets is adhesion- and degranulation-promoting adapter protein (ADAP), an adapter with potential binding partners: SLP-76, VASP, and SKAP-HOM. During studies of platelet function under shear flow, we discovered that ADAP(-/-) mouse platelets, unlike ADAP+/+ platelets, formed unstable thrombi in response to carotid artery injury. Moreover, fibrinogen-adherent ADAP(-/-) platelets in shear flow ex vivo showed reduced spreading and smaller zones of contact with the matrix. These abnormalities were not observed under static conditions, and they could not be rescued by stimulating platelets with a PAR4 receptor agonist or by direct alphaIIbbeta3 activation with MnCl2, consistent with a defect in outside-in alphaIIbbeta3 signaling. ADAP+/+ platelets subjected to shear flow assembled F-actin-rich structures that colocalized with SLP-76 and the Rac1 exchange factor, phospho-Vav1. In contrast, platelets deficient in ADAP, but not those deficient in VASP or SKAP-HOM, failed to form these structures. These results establish that ADAP is an essential component of alphaIIbbeta3-mediated platelet mechanotransduction that promotes F-actin assembly and enables platelet spreading and thrombus stabilization under fluid shear stress.

What is vinculin needed for in platelets

Summary.  Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F‐actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, αIIbβ3. Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4–Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin‐deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on αIIbβ3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin‐deficient platelets displayed normal agonist‐induced fibrinogen binding to αIIbβ3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin‐deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin‐deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild‐type littermates in response to carotid artery injury with FeCl3. Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to αIIbβ3, vinculin is not required for the traditional functions of αIIbβ3 or the platelet actin cytoskeleton.

Regulation of Platelet Adhesion Receptors

Platelet adhesion to subendothelial matrices and cohesion to other platelets is essential for hemostasis, but recent studies have revealed that platelet adhesive interactions may also generate cross-talk with other cell types, including leukocytes, tumor cells, and microbial pathogens. In so doing, these adhesive interactions may impact a variety of protective or disease-promoting processes, and they are mediated by platelet adhesion receptors from several gene families, most notably integrins, as exemplified by the platelet-restricted integrin, αIIbβ3. During hemostasis, multiple brakes and controls on platelet adhesion receptors prevent pathological thrombosis. Thus, threshold levels of excitatory stimuli must be surpassed and sufficient to drive intracellular signaling for full activation of integrin receptors. Stimuli for primary hemostasis derive from released or generated soluble agonists, vascular matrices exposed by vessel injury, or by hydrodynamic shear stress. While the non-integrin adhesion receptor, GPIb-V-IX, is critical for platelet adhesion to matrix von Willebrand factor (VWF) during hemostasis and is a mediator of platelet aggregation through interactions with VWF at pathologically high shear stresses in atherosclerotic arteries, finely regulated interactions of αIIbβ3 with fibrinogen and other Arg-Gly-Asp (RGD)-containing ligands are required for platelet aggregation in hemostatic thrombi. In this chapter we present an overview of classical and nonclassical regulation of platelet adhesion receptors and associated signaling pathways, with a principle focus on platelet signaling pathways that converge on and regulate αIIbβ3.

Optogenetic interrogation of integrin αVβ3 function in endothelial cells

ABSTRACT The integrin αVβ3 is reported to promote angiogenesis in some model systems but not in others. Here, we used optogenetics to study the effects of αVβ3 interaction with the intracellular adapter kindlin-2 (Fermt2) on endothelial cell functions potentially relevant to angiogenesis. Because interaction of kindlin-2 with αVβ3 requires the C-terminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to β3ΔRGT–GFP and mCherry–kindlin-2, respectively, and expressed in β3 integrin-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVβ3 as assessed by immunofluorescence and total internal reflection fluorescence (TIRF) microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVβ3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with β3 integrin and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology. Highlighted Article: Optogenetics show that the interaction of kindlin-2 with β3 integrin and other binding partners supports endothelial cell functions relevant to angiogenesis, including sprouting and podosome formation.

Glycoprotein Ib-IX-mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion: Receptor clustering and VWF adhesion

Summary.  The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to αIIbβ3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib–IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF–GP Ib–IX and fibrinogen–αIIbβ3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506‐binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib–IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell‐permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma‐derived VWF or the VWF A1 domain and GP Ib–IX(FKBP)2, and those between fibrinogen‐coated beads and αIIbβ3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib–IX(FKBP)2 and A1 or plasma‐derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and αIIbβ3 was not changed by the clustering agent; however, the strength of single fibrinogen–αIIbβ3 bonds increased significantly after ligation of GP Ib–IX(FKBP)2 by A1. These results demonstrate that GP Ib–IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib–IX can activate αIIbβ3, thereby increasing the strength of its interaction with fibrinogen.