Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent IntroductionThe successive adhesion, activation, and aggregation of platelets are key processes in arterial thrombus formation after endothelial damage. 1,2 Both rupture of atherosclerotic plaques as well as surgical interventions to treat atherosclerosis (eg, percutaneous coronary intervention [PCI]) may cause exposure of the subendothelium and subsequent clot formation. Eventually, this can result in the occlusion of arteries, leading to ischemia, myocardial infarcts, or stroke. Given the central role of platelets in thrombosis, a substantial number of currently marketed antithrombotic drugs, such as aspirin, clopidogrel, and abciximab, target different steps involved in platelet activation and aggregation. 1,2 Thanks to their complementary mechanisms of action, the combination of these agents inhibits platelet aggregation to a greater extent than either agent alone. 3 However, the use of these antiplatelet drugs is hampered by an increased bleeding risk 1,2 and the occurrence of treatment resistance in some patients. 4 Moreover, the irreversible nature of their action can complicate the staunching of bleeding. 1,2 Inhibition of the initial adhesion of platelets to subendothelial collagen provides an alternative strategy to prevent unwanted clot formation. The plasma glycoprotein VWF plays a pivotal role in this adhesion via binding to exposed collagen on the one hand, and the interaction of its A1 domain with the gpIb-IX-V receptor complex on the surface of platelets on the other hand. [5][6][7] Interestingly, the VWF A1 domain is only exposed under high-shear conditions, 8,9 so VWF only acts as a bridging molecule between collagen and platelets in small or stenosed arteries. Therefore, it is expected that drugs inhibiting this interaction between VWF and platelets show an improved safety profile with respect to bleeding tendency. Indeed, the antithrombotic effect of several compounds targeting the gpIb-VWF-A1-axis, like aurintricarboxylic acid, 10-12 recombinant VWF fragments, 10,13-16 a recombinant gpIb chimeric protein, 17,18 anti-VWF mAbs, [19][20][21][22][23][24][25][26][27] and an anti-VWF aptamer 28 has been demonstrated in vitro and in vivo, without increasing the bleeding risk. 13,[16][17][18]21,23,25,28,29 Nevertheless, until now only 3 drug candidates have been evaluated in humans, including ALX-0081. [30][31][32][33] We developed ALX-0081, a bivalent humanized Nanobody directed against the A1 domain of VWF. Nanobodies are therapeutic proteins derived from the heavy-chain variable domains (VHH) that occur naturally in heavy-chain-only Igs of Camelidae. 34,35 Here we...

Objective-High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of ␣2␤1 and ␣IIb␤3 integrin activation during thrombus formation on collagen. Methods and Results-Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of ␣2␤1 and ␣IIb␤3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin ␣2␤1 and ␣IIb␤3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and ␣2␤1, indicating that the integrin can support platelet-collagen binding in the absence of its activation. Blockade or absence of GPIb␣ only moderately influenced integrin activation and adhesion unless GPVI was inhibited. Conclusions-GPVI-and autocrine-released ADP induce affinity changes of ␣2␤1 and ␣IIb␤3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet-collagen interactions and thereby collagen-induced platelet activation. Key Words: ADP Ⅲ collagen Ⅲ glycoprotein VI Ⅲ integrins Ⅲ platelets Ⅲ thrombus P latelet integrins are critical in hemostasis. Abundantly expressed at the platelet surface, integrins are required for platelet interactions with subendothelial matrix components and for platelet-platelet interactions leading to aggregate and thrombus formation. 1 Integrin ␣2␤1 plays a role in platelet adhesion to collagen under static 2,3 and flow conditions. 4,5 Integrin ␣IIb␤3 allows platelets to bind to fibrinogen and von Willebrand factor (vWF) present on collagen and other platelets. 6 This leads to stable platelet adhesion and aggregate formation. 7 On resting platelets, these integrins are considered to be present in a low-affinity state. Intracellular signaling or ligand binding results in conformational changes of the integrins with a switch to higher-affinity states. 6 Agonists such as thrombin, collagen, ADP, and vWF induce ␣IIb␤3 activation and platelet aggregation. 8,9 Full integrin activation with ADP requires the P2Y 1 and P2Y 12 purinergic receptors. 10,11 Recent studies show that integrin ␣2␤1 can also be activated by inside-out signaling. 12,13 Thrombin and collagen turn this integrin into a high-affinity form, whereas ADP changes it to intermediate affinity. 13 Although much is known of the affinity and avidity changes of ␣IIb␤3 on isolated platelets especially, 8,14 regulation of integrin activation during thrombus formation is incompletely understood.In vivo studies as well as ex vivo experiments, in which blood was allowed to flow over collagen under arterial shear conditions, have indicated that glycop...

In this study we describe the first monoclonal antibody, integrin activated conformation-1 (IAC-1), to recognize the active form of the platelet-collagen receptor, the integrin α2β1. IAC-1 has the following properties: (1) IAC-1 fails to bind to resting platelets but readily interacts with platelets stimulated by the glycoprotein VI-specific agonist, convulxin, and by other agonists; (2) similar concentration response relationships for binding of IAC-1 and soluble collagen were observed in convulxin-stimulated platelets; (3) the epitope for IAC-1 is T199Y200K201, which is located at the opposite site of the metal ion-dependent adhesion site in a region not involved in the I-domain “shifts” that occur upon ligand binding; (4) IAC-1 strongly binds to recombinant α2 I-domain, therefore suggesting that the neo-epitope appears to be exposed by an “unmasking” of I-domain-covering regions upon activation; (5) IAC-1 binds to platelets during adhesion to collagen under shear conditions, demonstrating activation of α2β1; (6) as IAC-1 does not interfere with platelet-collagen binding, it defines a new class of antibodies that is distinct from those belonging to the “cation- and ligand-induced binding sites” (CLIBSs) and the “ligand mimetic” group. These characteristics make IAC-1 a very powerful tool to study α2β1 activation under dynamic and physiologically relevant conditions. (Blood. 2004;104:390-396)