A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin ␣ 2 ␤ 1 . The capacity of the peptides to support Mg 2؉ -dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant ␣ 2 I-domain, ␣ 2 ␤ 1 purified from platelet membranes, and recombinant soluble ␣ 2 ␤ 1 expressed as an ␣ 2 -Fos/␤ 1 -Jun heterodimer) bound well to only three peptides, two containing GXXGER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXXGEN motifs (GLKGEN and GLOGEN). Two mutant ␣ 2 I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-␣ 2 monoclonal antibody 6F1 and by chelation of Mg 2؉ . We describe two novel high affinity integrinbinding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

The essential event in platelet adhesion to the injured blood vessel wall is the binding to subendothelial collagen of plasma von Willebrand factor (VWF), a protein that interacts transiently with platelet glycoprotein Ib␣ (GPIb␣), slowing circulating platelets to facilitate firm adhesion through collagen receptors, including integrin ␣2␤1 and GpVI. To locate the site in collagen that binds VWF, we IntroductionThe interaction of collagen with von Willebrand factor (VWF) requires unique structural properties in both proteins. Optimal hemostatic function requires multimerization of up to 50 VWF monomers in circulating plasma; higher-order multimers bind collagen more tightly than smaller assemblies of VWF. 1 Several collagens occur in the vessel wall, of which collagens I and III are considered most important in supporting platelet adhesion to the damaged vasculature. 2 We have identified the residues in the VWF A3 domain that bind collagen III, using site-directed mutagenesis guided by the crystal structure of the VWF A3 domain in complex with a monoclonal antibody (RU5) that inhibits its interaction with collagen. 3,4 Nishida et al mapped the collagen-binding mode of the A3 domain by nuclear magnetic resonance and confirmed results by site-directed mutagenesis. 5 However, the VWF-binding site(s) in collagen is unknown, although progress in understanding how collagen interacts with integrin ␣2␤1 and GpVI has been made using short synthetic triple-helical peptide analogues of collagen, 6,7 including the Collagen III Toolkit. 8 We used the same approach to identify the high-affinity VWF-binding site in human collagen III, information that may help to develop the collagen-VWF interaction as an antithrombotic target. 9,10 Materials and methods Peptide synthesisThe synthesis and characterization of the 57 overlapping triple-helical peptides of the Collagen III Toolkit (Table S1, available at the Blood website; see the Supplemental Materials link at the top of the online article) is detailed elsewhere. 8 The same approach was used to synthesize and verify derivative peptides (Table S2). The sequence of peptide no. 23 is GPC-(GPP) 5 -GPOGPSGPRGQOGVMGFOGP-KGNDGAO-(GPP) 5 -GPC-NH 2 , and of the minimal VWF-binding derivative peptide, GPC-(GPP) 5 -GPRGQOGVMGFO-(GPP) 5 -GPC-NH 2 . Static platelet-binding assayBlood was obtained from the antecubital vein of informed volunteers, in accordance with the Helsinki protocol, into 0.105-M citrate Vacutainers (Becton Dickinson, Oxford, United Kingdom). Platelet-rich plasma was prepared after 2 spins for 1 minute at 1200g. Then, 10% (vol/vol) of ACD buffer (39 mM citric acid, 75 mM trisodium citrate, 135 mM D-glucose, pH 4.5) and prostaglandin E 1 (280-nM final concentration) were added, and the platelets were pelleted for 12 minutes at 700g, then resuspended in 6 mL buffer (5.5 mM D-glucose, 128 mM NaCl, 4.26 mM Na 2 HPO 4 , 7.46 mM NaH 2 PO 4 , 4.77 mM trisodium citrate, 2.35 mM citric acid, 0.35% bovine serum albumin [BSA], pH 6.5). Prostaglandin E 1 was added similarly, and the ...

Summary The platelet receptors glycoprotein (Gp)VI, integrin α 2 β 1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca 2+ ([Ca 2+ ] i ) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca 2+ ] i measurements using confocal imaging. All three collagen receptors coupled to [Ca 2+ ] i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca 2+ ] i signals leading to realtime increases in integrins α 2 β 1 − and α IIb β 3 -mediated platelet adhesion. α IIb β 3 -dependent platelet aggregation was dependent on P 2 Y 12 signalling. Co-engagement of α 2 β 1 and GpIb/V/IX generated transient [Ca 2+ ] i spikes and low amplitude [Ca 2+ ] i responses that potentiated GpVI-dependent [Ca 2+ ] i signalling. Therefore α 2 β 1 GpIb/V/IX and GpVI synergise to generate [Ca 2+ ] i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for α IIb β 3 in stable platelet adhesion and aggregation. Supplementary Material to this article is available online at .

Human Domain Antibodies (dAbs) that bind to and inhibit the function of platelet glycoprotein VI (GPVI) have been isolated from phage display libraries and their efficacy demonstrated using in vitro models of platelet activation. Here we describe the properties of one such antibody, BLO8-1, which has been shown to specifically inhibit the binding of recombinant human GPVI to cross-linked collagen related peptide (CRP-XL) in vitro. BLO8-1 specifically binds to the platelet cell surface and prevents CRP-XL induced platelet aggregation in platelet-rich plasma, as well as inhibiting thrombus formation in whole blood under arterial shear conditions. Using a series of mutant GPVI molecules, BLO8-1 was shown to recognize an epitope within the collagen binding domain of GPVI, therefore the anti-thrombotic effect of this dAb is predicted to be due to direct blocking of the collagen-GPVI interaction. These data, together with the desirable properties of Domain Antibodies, show that dAbs could potentially be used to generate novel biopharmaceuticals with anti-thrombotic properties.