Joel S. Bennett

Use of Nonsteroidal Antiinflammatory Drugs An Update for Clinicians: A Scientific Statement From the American Heart Association

Clinical trial data have prompted questions about the degree to which patients and their physicians should consider an increased risk of cardiovascular or cerebrovascular events when selecting medications for pain relief. Since the 2005 publication of a Science Advisory on the use of nonsteroidal antiinflammatory drugs (NSAIDs) by the American Heart Association,1 several important events have occurred that have served as the catalyst for this update for clinicians. (1) Additional data from randomized controlled trials of cyclooxygenase (COX)-2–selective agents have been reported and summarized in meta-analyses, which has reinforced the concern about cardiovascular events with COX-2 inhibitors (coxibs; Figure 1). (2) Several reports have appeared that have identified an increased risk of cardiovascular events even with the nonselective NSAIDs, which has raised concern about the use of those agents as well (Table). (3) Regulatory authorities in several regions of the world have introduced warning statements and advisories to both healthcare professionals and the lay public about the use of various NSAIDs (Figures 2 and 3⇓). Figure 1. Comparison of effects of different selective COX-2 inhibitors vs placebo on myocardial infarction. Event numbers and person-years of exposure, with corresponding mean annual event rates in parentheses, are presented for patients allocated to selective COX-2 inhibitor or placebo. Event rate ratios for pooled data with 95% CIs are indicated by a diamond; rate ratios for individual selective COX-2 inhibitors, with 99% CIs, are indicated by a square and horizontal line. Diamonds to the right of the solid line indicate hazard with a selective COX-2 inhibitor compared with placebo. As noted, there was a significant increase in the rate ratio for myocardial infarction with COX-2 inhibitors compared with placebo. Similar analyses (data not shown) include rate ratios of 1.42 (1.13 to 1.78; P =0.003) for vascular events, 1.02 (0.71 to 1.47; P …

Exposure of platelet fibrinogen receptors by ADP and epinephrine.

The role of fibrinogen as a cofactor for platelet aggregation was examined by measuring the binding of 125I-labeled human fibrinogen to gel-filtered human platelets both before and after platelet stimulation by ADP and epinephrine. Platelet stimulation by ADP resulted in the rapid, reversible binding of fibrinogen to receptors on the platelet surface. Fibrinogen binding increased as the concentration of ADP was increased from 0.1 to 2 microM, reaching a plateau at higher ADP concentrations. Binding occurred only after platelet stimulation and in the presence of divalent cations. However, fibrinogen binding did not occur to ADP-stimulated platelets from three patients with Glanzmanns thrombasthenia. Analysis of fibrinogen binding as a function of increasing fibrinogen concentration demonstrated that maximal platelet stimulation exposed approximately or equal to 45,000 binding sites per platelet with a dissociation constant of 80--170 nM. These fibrinogen binding parameters were essentially the same whether ADP or epinephrine was the platelet-stimulating agent. Thus, these studies demonstrate that platelet stimulation by ADP and epinephrine exposes a limited number of fibrinogen receptors on the platelet surface. Furthermore, these data suggest that the fibrinogen molecules bound to the platelet as a consequence of platelet stimulation are directly involved in the platelet aggregation response.

Computational Design of Peptides That Target Transmembrane Helices

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (αIIbβ3 and αvβ3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Structure and function of the platelet integrin αIIbβ3

The platelet integrin alpha(IIb)beta(3) is required for platelet aggregation. Like other integrins, alpha(IIb)beta(3) resides on cell surfaces in an equilibrium between inactive and active conformations. Recent experiments suggest that the shift between these conformations involves a global reorganization of the alpha(IIb)beta(3) molecule and disruption of constraints imposed by the heteromeric association of the alpha(IIb) and beta(3) transmembrane and cytoplasmic domains. The biochemical, biophysical, and ultrastructural results that support this conclusion are discussed in this Review.

Oligomerization of the integrin alphaIIbbeta3: roles of the transmembrane and cytoplasmic domains.

Integrins are a family of α/β heterodimeric membrane proteins, which mediate cell–cell and cell–matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the α and β subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of αIIbβ3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571–6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the α and β subunits of αIIbβ3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the α- and β-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.

Dimerization of the Transmembrane Domain of Integrin αIIb Subunit in Cell Membranes

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin α and β subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type αIIb TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type αIIb TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the αIIb TM domain oligomerizes in biological membranes. Mutating each of the αIIb TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of αIIb TM helix. These data were used to generate an atomic model of the αIIb homodimer, revealing a family of structures with right-handed crossing angles of 40° to 60°, consistent with a 4.0-residue periodicity, and with an interface rotated by 50° relative to glycophorin A. Thus, although the αIIb TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Binding strength and activation state of single fibrinogen-integrin pairs on living cells

Integrin activation states determine the ability of these receptors to mediate cell–matrix and cell–cell interactions. The prototypic example of this phenomenon is the platelet integrin, αIIbβ3. In unstimulated platelets, αIIbβ3 is inactive, whereas exposing platelets to an agonist such as ADP or thrombin enables αIIbβ3 to bind ligands such as fibrinogen and von Willebrand factor. To study the regulation of integrin activation states at the level of single molecules, we developed a model system based on laser tweezers, enabling us to determine the rupture forces required to separate single ligand-receptor pairs by using either purified proteins or intact living cells. Here, we show that rupture forces of individual fibrinogen molecules and either purified αIIbβ3 or αIIbβ3 on the surface of living platelets were 60 to 150 pN with a peak yield strength of 80–100 pN. Platelet stimulation using either ADP or the thrombin receptor-activating peptide enhanced the accessibility but not the adhesion strength of single αIIbβ3 molecules, indicating that there are only two states of αIIbβ3 activation. Thus, we found it possible to use laser tweezers to measure the regulation of forces between individual ligand-receptor pairs on living cells. This methodology can be applied to the study of other regulated cell membrane receptors using the ligand-receptor yield strength as a direct measure of receptor activation/inactivation state.

The structure and function of platelet integrins

Summary.  Integrins are a ubiquitous family of non‐covalently associated α/β transmembrane heterodimers linking extracellular ligands to intracellular signaling pathways [ 1 ] [Cell, 2002; 110: 673]. Platelets contain five integrins, three β1 integrins that mediate platelet adhesion to the matrix proteins collagen, fibronectin and laminin, and the β3 integrins αvβ3 and αIIbβ3 [ 2 ] [J Clin Invest, 2005; 115: 3363]. While there are only several hundred αvβ3 molecules per platelet, αvβ3 mediates platelet adhesion to osteopontin and vitronectin in vitro [ 3 ] [J Biol Chem, 1997; 272: 8137]; whether this occurs in vivo remains unknown. By contrast, the 80 000 αIIbβ3 molecules on agonist‐stimulated platelets bind fibrinogen, von Willebrand factor, and fibronectin, mediating platelet aggregation when the bound proteins crosslink adjacent platelets [ 2 ] [J Clin Invest, 2005; 115: 3363]. Although platelet integrins are poised to shift from resting to active conformations, tight regulation of their activity is essential to prevent the formation of intravascular thrombi. This review focuses on the structure and function of the intensively studied β3 integrins, in particular αIIbβ3, but reference will be made to other integrins where relevant.

Platelet-fibrinogen interactions.

Abstract: Binding of fibrinogen to GPIIb‐IIIa on agonist‐stimulated platelets results in platelet aggregation, presumably by crosslinking adjacent activated platelets. Although unactivated platelets express numerous copies of GPIIb‐IIIa on their surface, spontaneous, and potentially deleterious, platelet aggregation is prevented by tightly regulating the fibrinogen binding activity of GPIIb‐IIIa. Preliminary evidence suggests that it is the submembranous actin or actin‐associated proteins that constrains GPIIb‐IIIa in a low affinity state and that relief of this constraint by initiating actin filament turnover enables GPIIb‐IIIa to bind fibrinogen. Two regions of the fibrinogen α chain that contain an RGD motif, as well as the carboxyl‐terminus of the fibrinogen γ chain, represent potential binding sites for GPIIb‐IIIa in the fibrinogen molecule. However, ultrastructural studies using purified fibrinogen and GPIIb‐IIIa, and studies using recombinant fibrinogen in which the RGD and relevant γ chain motifs were mutated indicate that sequences located at the carboxyl‐terminal end of the γ chain mediates fibrinogen binding to GPIIb‐IIIa. There is evidence that fibrinogen itself binds to regions in the amino terminal portions of both GPIIb and GPIIIa and that the sites interacting with the fibrinogen γ chain and with RGD‐containing peptides are spatially distinct. Nonetheless, there appears to be allosteric linkage between these sites, accounting for the ability of RGD‐containing peptides to inhibit platelet aggregation and arterial thrombosis.

Agonist-activated αvβ3 on Platelets and Lymphocytes Binds to the Matrix Protein Osteopontin

The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by αvβ3. αvβ3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an αvβ3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both αvβ3 and αIIbβ3 confirmed that activated αvβ3 not activated αIIbβ3 was responsible for the cellular adherence we measured. These studies indicate that αvβ3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated αvβ3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting αvβ3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.