Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αβ as Novel Antiplatelet Agents.

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis.

Platelet Spreading and Clot Retraction are Regulated by two Distinct αβ Outside-in Signaling Pathways.

Bi-directional signaling through platelet integrin αβ is essential in hemostasis and thrombosis. In quiescent platelets αβ is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen.

Glutamate Receptor Dysregulation and Platelet Glutamate Dynamics in Alzheimer's and Parkinson's Diseases: Insights into Current Medications.

Two of the most prevalent neurodegenerative disorders (NDDs), Alzheimer's disease (AD) and Parkinson's disease (PD), present significant challenges to healthcare systems worldwide. While the etiologies of AD and PD differ, both diseases share commonalities in synaptic dysfunction, thereby focusing attention on the role of neurotransmitters. The possible functions that platelets may play in neurodegenerative illnesses including PD and AD are becoming more acknowledged.

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling.

Calcium-induced dissociation of CIB1 from ASK1 regulates agonist-induced activation of the p38 MAPK pathway in platelets.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown.

Binding of CIB1 to the αIIb tail of αIIbβ3 is required for FAK recruitment and activation in platelets.

Background: It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood.

Objectives: We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling.

Ask1 regulates murine platelet granule secretion, thromboxane A generation, and thrombus formation.

Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets.

Junctional adhesion molecule-A suppresses platelet integrin αIIbβ3 signaling by recruiting Csk to the integrin-c-Src complex.

Fibrinogen binding to activated integrin induces outside-in signaling that results in stable platelet aggregates and clot retraction. How integrin αIIbβ3 is discouraged from spontaneous activation is not known. We have recently shown that junctional adhesion molecule-A (JAM-A) renders protection from thrombosis by suppressing integrin outside-in signaling.

Effects of JAM-A deficiency or blocking antibodies on neutrophil migration and lung injury in a murine model of ALI.

Transmigration of neutrophils (PMNs) from the vasculature into inflamed tissues, mediated by interactions between PMNs and adhesion molecules on endothelial cells, is an essential aspect of inflammation. The crucial adhesion molecules include junctional adhesion molecule (JAM)-A. Investigation of the role of this molecule in models of inflammatory disease has been limited, however, and results in different disease models have varied.

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets.

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)β(3). Once platelet activation has occurred, integrin α(IIb)β(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling.

Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration.

Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1(-/-) mouse model.

CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.

Contra-regulation of calcium- and integrin-binding protein 1-induced cell migration on fibronectin by PAK1 and MAP kinase signaling.

Calcium- and integrin-binding protein 1 (CIB1) has been shown to be involved in cell spreading and migration. The signaling events regulated by CIB1 during cell migration are poorly understood. Here we found that accumulation of CIB1 at the tip of the filopodia requires an intact cytoskeleton.

Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner.

Calcium-dependent inhibition of polo-like kinase 3 activity by CIB1 in breast cancer cells.

Members of the polo-like kinases (Plk1, Plk2, Plk3 and Plk4) are involved in the regulation of various stages of the cell cycle and have been implicated in cancer progression. Unlike its other family members, the expression of Plk3 remains steady during cell cycle progression, suggesting that its activity may be spatiotemporally regulated. However, the mechanism of regulation of Plk3 activity is not well understood.

Putting the brakes on cancer cell migration: JAM-A restrains integrin activation.

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion.

Attenuation of junctional adhesion molecule-A is a contributing factor for breast cancer cell invasion.

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion.

Junctional adhesion molecules in angiogenesis.

The process of new blood vessel formation from pre-existing vessels is known as angiogenesis. This process is important, both during physiological processes such as development and during wound healing, as well as during pathological processes, such as cancer and arthritis. Understanding the molecular mechanism of the regulation of angiogenesis and the identification of the key players involved in this process may help identify new therapeutic targets to combat and control angiogenesis and hence arthritis and cancer.

Fibroblast growth factor-2 failed to induce angiogenesis in junctional adhesion molecule-A-deficient mice.

Objective: We have previously shown that JAM-A regulates fibroblast growth factor-2 (FGF-2)-induced endothelial cell morphology, proliferation, and migration. Whether JAM-A is involved in FGF-2-induced angiogenesis in vivo is not known. We used JAM-A null mice to conclusively determine the role of JAM-A in FGF-2-induced neovascularization.

Junctional adhesion molecule-A-induced endothelial cell migration on vitronectin is integrin alpha v beta 3 specific.

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin superfamily, and is mainly expressed in the tight junctions of both epithelial and endothelial cells. We have recently shown that JAM-A is involved in basic fibroblast growth factor (bFGF)-induced angiogenesis. Here, we show that, when ectopically expressed in human umbilical vein endothelial cells (HUVECs), JAM-A induced enhanced cell migration on vitronectin, but had no effect on fibronectin.

Essential role of junctional adhesion molecule-1 in basic fibroblast growth factor-induced endothelial cell migration.

Objective: Recently, we have shown that blocking of junctional adhesion molecule-1/A (JAM-1/A) inhibits basic fibroblast growth factor (bFGF)-induced angiogenesis. Because the process of endothelial cell proliferation is a key initial step of neovascularization, we studied the effect of functional knockdown of JAM-1 on human umbilical vein endothelial cell (HUVEC) adhesion and migration induced by bFGF.

Methods And Results: We introduced small interfering RNAs specific to JAM-1 in HUVECs, stimulated them with bFGF, and studied the resultant adhesion and migration of these cells on vitronectin and fibronectin.

Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen.

Platelet spreading on the subendothelium in response to vascular injury is fundamental to the regulation of physiologic hemostasis. Previously, we have shown that, when bound to glycoprotein IIb (GPIIb), calcium- and integrin-binding protein (CIB) regulates platelet spreading on immobilized fibrinogen (Fg). In this study, we investigated the signaling events that occur downstream of CIB in the absence of signaling that occurs as a result of granular secretion.

Signaling through JAM-1 and alphavbeta3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and alphavbeta3 complex.

Growth factor-induced neovascularization has received a great deal of attention because it is fundamental to the growth and metastasis of solid tumors. This multistep process requires extensive signaling through growth factor receptors and integrins. Among the integrins involved in this process, integrin alphavbeta3 is specific to basic fibroblast growth factor (bFGF)-induced angiogenesis.

Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen.

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets.