Susan L. Cranmer

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 αIIbβ3) 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 αIIbβ3. 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.

Synergistic Adhesive Interactions and Signaling Mechanisms Operating between Platelet Glycoprotein Ib/IX and Integrin αIIbβ3 STUDIES IN HUMAN PLATELETS AND TRANSFECTED CHINESE HAMSTER OVARY CELLS

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. These include the following: 1) examining the sufficiency of GPIb/IX and integrin αIIbβ3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin αIIbβ3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin αIIbβ3, 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 αIIbβ3mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin αIIbβ3. Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin αIIbβ3 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 αIIbβ3 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 αIIbβ3 activation.

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbα cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbα blocking antibody (AK2) and was not observed with CHO cells expressing GPIbβ and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20–200 s−1), GPIb-IX was able to support cell rolling at both venous (150 s−1) and arterial (1500–10,500 s−1) shear rates. Studies with a mutant GPIbα receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000–6000 s−1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbα and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.

High shear―dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIbα-filamin interaction

Platelets have evolved a highly specialized membrane skeleton that provides stability to the plasma membrane and facilitates adhesion under high shear stress. The cytoskeletal anchorage of glycoprotein (GP) Ibα plays an important role in regulating the membrane skeleton. However, its role in regulating membrane stability remains unknown. To investigate this role, we have developed a new mouse model that expresses wild-type human GPIbα (hGPIbα(WT)), or a mutant form of human GPIbα that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIbα(FW)-Phe568Ala and Trp570Ala substitutions). Our study demonstrates that the link between platelet GPIb and the cytoskeleton does not alter the intrinsic ligand binding function of GPIbα or the ability of the receptor to stimulate integrin α(IIb)β(3)-dependent spreading. However, exposure of hGPIbα(FW) platelets to pathologic shear rate levels (5000 to 40,000 s(-1)) leads to the development of unstable membrane tethers, defective platelet adhesion, and loss of membrane integrity, leading to complete disintegration of the platelet cell body. These outcomes suggest that the GPIbα-filamin A interaction not only regulates the architecture of the membrane skeleton, but also maintains the mechanical stability of the plasma membrane under conditions of high shear.

Leucine-rich repeats 2-4 (Leu60-Glu128) of platelet glycoprotein Ibα regulate shear-dependent cell adhesion to von Willebrand factor

Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbα (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbα expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbα-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbα N-terminal domain and GPIbα-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbα with human GPIbα containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbα with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbα antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbα, cells expressing GPIbα, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s-1. Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbα, is essential for GPIbα·VWF-dependent adhesion as hydrodynamic shear increases.

14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function.