T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling Ltype calcium channels to the sarcoplasmic reticulum calciumrelease channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.calcium handling ͉ heart failure ͉ morphology ͉ T-tubule

access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available. Copies of full items can be used for personal research or study, educational, or not-forprofit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVIspecific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled. IntroductionDamage to blood vessels exposes circulating platelets to the extracellular matrix. Here, collagen supports adhesion and stimulates platelet activation by acting as a ligand for a number of platelet receptors. 1 Platelets are first tethered by the interaction of glycoprotein (GP) Ib␣ with the A1 domain of von Willebrand factor (VWF), 2 a plasma protein that binds to exposed collagen. 3 Firm platelet adhesion results from the concerted action of the collagen receptor GPIaIIa (integrin ␣2␤1) and the fibrinogen receptor GPIIbIIIa (␣IIb␤3) which also binds immobilized VWF. 4 Collagen-mediated activation of platelets is dependent on the engagement and clustering of GPVI, 1,5 an immunoglobulin (Ig) superfamily member with homology to killer cell Ig-like receptors (KIRs) and the Fc␣RI. 6 We and others have described the ligandbinding sites of ␣2 integrin 7 and GPIb␣ 2 at the structural level.Recent work has demonstrated the key role played by GPVI in arterial thrombus formation in mice and provides a clear basis for the development of potentially therapeutic GPVI inhibitors. 8 Indeed, blockade of GPVI is attractive for several reasons. First, the expression of GPVI appears to be restricted to platelets and megakaryocytes. 9 Second, collagen is required for prothrombotic "COAT" platelet formation, 10 for which blockade of GPVI may provide a specific control point. Third, patients with congenital or acquired autoantibody-mediated GPVI deficiency have only a mild bleeding disorder, despite having a significantly ...