Biomedical subjects

S M Dopheide

Publications and source records attributed to S M Dopheide.

8 recordsLinked to original sources

PubMed2001

Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions.

The functional importance of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) in platelets is unclear. Because PECAM-1 represents a newly assigned immunoglobulin-ITIM superfamily member expressed on the surface of platelets, it was hypothesized that it may play an important regulatory role in modulating ITAM-bearing receptors such as collagen (GP)VI receptor and FcgammaRIIA. To examine the functional role of PECAM-1 in regulating platelet-collagen interactions, 2 different approaches were applied using recombinant human PECAM-1-immunoglobulin chimeras and platelets derived from PECAM-1-deficient mice. Stimulation of platelets by collagen-, (GP)VI-selective agonist, collagen-related peptide (CRP)-, and PECAM-1-immunoglobulin chimera induced tyrosine phosphorylation of PECAM-1 in a time- and dose-dependent manner. Activation of PECAM-1 directly through the addition of soluble wild-type PECAM-1-immunoglobulin chimera, but not mutant K89A PECAM-1-immunoglobulin chimera that prevents homophilic binding, was found to inhibit collagen- and CRP-induced platelet aggregation. PECAM-1-deficient platelets displayed enhanced platelet aggregation and secretion responses on stimulation with collagen and CRP, though the response to thrombin was unaffected. Under conditions of flow, human platelet thrombus formation on a collagen matrix was reduced in a dose-dependent manner by human PECAM-1-immunoglobulin chimera. Platelets derived from PECAM-1-deficient mice form larger thrombi when perfused over a collagen matrix under flow at a shear rate of 1800 seconds(-1) compared to wild-type mice. Collectively, these results indicate that PECAM-1 serves as a physiological negative regulator of platelet-collagen interactions that may function to negatively limit growth of platelet thrombi on collagen surfaces.

Animals↗

PubMed2001

The vascular biology of the glycoprotein Ib-IX-V complex.

It has long been recognized that binding of von Willebrand factor (vWf) by the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates the cascade of events leading to thrombosis and haemostasis. In the last several years, however, it has become apparent that the GP Ib-IX-V complex plays a much broader role in vascular biology, a recognition in part due to the identification of novel adhesive ligands for GP Ib-IX-V such as P-selectin and Mac-1. Binding of vWf by the GP Ib-IX-V complex is also important in formation of the developing thrombus. Platelets can utilize the GP Ib-IX-V complex and PSGL-1 to roll on activated endothelium via surface-expressed endothelial P-selectin. The GP Ib-IX-V complex can bind the I domain of Mac-1, a binding interaction potentially relevant to transmigration of macrophages through mural thrombus, a process required for vessel remodeling post angioplasty. This review will focus on recent advances in our understanding of the structure and function of this important platelet receptor, with particular emphasis on insights made within the last two years.

Endothelium, Vascular↗

PubMed2000

Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets ans transfected Chinese hamster ovary cells.

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin alpha(IIb)beta(3). These include the following: 1) examining the sufficiency of GPIb/IX and integrin alpha(IIb)beta(3) to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin alpha(IIb)beta(3) activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin alpha(IIb)beta(3), we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin alpha(IIb)beta(3) mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin alpha(IIb)beta(3). Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150-1800 s(-)(1)). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin alpha(IIb)beta(3) activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin alpha(IIb)beta(3) activation.

Adenosine Diphosphate↗

PubMed2000

Cytoskeletal regulation of the platelet glycoprotein Ib/V/IX-von willebrand factor interaction.

Shear-induced binding of von Willebrand factor (vWf) to the platelet glycoprotein (GP) Ib/V/IX complex plays a key role in initiating platelet adhesion and aggregation at sites of vascular injury. This study demonstrated that pretreating human platelets with inhibitors of actin polymerization, cytochalasin D or latrunculin B, dramatically enhances platelet aggregation induced by vWf. The effects of these inhibitors were specific to the vWf-GPIbalpha interaction because they enhanced vWf-induced aggregation of Glanzmann thrombasthenic platelets and Chinese hamster ovary (CHO) cells transfected with GPIb/V/IX. Moreover, cytochalasin D enhanced the extent of platelet aggregation induced by high shear stress (5000 s(-1)) and also lowered the shear threshold required to induce aggregation from 3000 s(-1) to as low as 500 s(-1). Studies of CHO cells expressing GPIbalpha cytoplasmic tail truncation mutants that failed to bind actin-binding protein-280 (deletion of residues 569-610 or 535-568) demonstrated that the linkage between GPIb and actin-binding protein-280 was not required for vWf-induced actin polymerization, but was critical for the enhancing effects of cytochalasin D on vWf-induced cell aggregation. Taken together, these studies suggest a fundamentally important role for the cytoskeleton in regulating the adhesive function of GPIb/V/IX.

Actin Cytoskeleton↗

PubMed2000

A revised model of platelet aggregation.

In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin alpha(IIb)beta(3) demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin alpha(IIb)beta(3) mediated cell arrest. Platelet aggregation under flow appears to be a multistep process involving: (a) exposure of vWf on the surface of immobilized platelets; (b) a reversible phase of platelet aggregation mediated by the binding of GPIbalpha on the surface of free-flowing platelets to vWf on the surface of immobilized platelets; and (c) an irreversible phase of aggregation dependent on integrin alpha(IIb)beta(3). Studies of platelet thrombus formation in vivo demonstrate that this multistep adhesion mechanism is indispensable for platelet aggregation in arterioles and also appears to promote platelet aggregate formation in venules. Together, our studies demonstrate an important role for platelet vWf in initiating the platelet aggregation process under flow and challenge the currently accepted view that the vWf-GPIbalpha interaction is exclusively involved in initiating platelet aggregation at elevated shear rates.

Animals↗

PubMed1999

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells.

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.

Actins↗

PubMed1997

Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction.

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alphaIIbbeta3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin alphaIIbbeta3) and signaling enzymes (pp60c-src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin alphaIIbbeta3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alphaIIbbeta3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alphaIIbbeta3, PI 3-kinase, FAK, pp60(c-)src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alphaIIbbeta3 in vWf-induced signal transduction.

Blood Platelets↗

PubMedDate not supplied

Dynamic aspects of platelet adhesion under flow.

1. Cell-cell and cell-matrix adhesive interactions are critical for a wide range of physiological processes, including embryogenesis, inflammation, immunity and haemostasis. 2. The ability of circulating blood cells, such as platelets and leucocytes, to adhere to sites of vascular injury is complicated by the presence of blood flow, which imposes hydrodynamic forces on adhesion contacts. 3. To overcome this problem, platelets and leucocytes have evolved specific adhesion receptors with unique biomechanical properties that enable these cells to adhere to the vessel wall under flow conditions. 4. Platelet adhesion in the normal circulation appears to be a multiple-step process involving an initial reversible interaction between the platelet adhesion receptor glycoprotein Ib-IX-V and the vascular adhesion protein von Willebrand factor. Once tethered to the vessel wall, platelets form irreversible adhesion contacts through the binding of one or more platelet integrins to specific subendothelial matrix proteins. 5. There is now a wealth of evidence demonstrating that these receptors not only mediate platelet adhesion, but also transduce signals leading to platelet activation. 6. In the present review, we will briefly discuss the current understanding of the specific roles of individual platelet receptors in supporting the haemostatic function of platelets and discuss mechanisms by which these receptors induce platelet activation.

Animals↗