Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.

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 activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.

Activation of alphaIIbbeta3 is a sufficient but also an imperative prerequisite for activation of alpha2beta1 on platelets.

Platelet integrins alpha2beta1 and alphaIIbbeta3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin alphaIIbbeta3 is not only sufficient, but also a prerequisite for alpha2beta1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of alpha2beta1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of alphaIIbbeta3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful alpha2beta1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of alphaIIbbeta3 for proper alpha2beta1 activation on platelets was demonstrated by using the alphaIIbbeta3 antagonist aggrastat, which was able to completely abolish alpha2beta1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking alphaIIbbeta3 confirmed the indispensability of alphaIIbbeta3 for alpha2beta1 activation.

Platelet integrin alpha2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display.

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.

Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation.

OBJECTIVE: High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of alpha2beta1 and alphaIIbbeta3 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 alpha2beta1 and alphaIIbbeta3, 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 alpha2beta1 and alphaIIbbeta3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and alpha2beta1, indicating that the integrin can support platelet-collagen binding in the absence of its activation. Blockade or absence of GPIbalpha only moderately influenced integrin activation and adhesion unless GPVI was inhibited. CONCLUSIONS: GPVI- and autocrine-released ADP induce affinity changes of alpha2beta1 and alphaIIbbeta3 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. Integrin activation during thrombus formation on collagen was studied using fluorescent-labeled antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of alpha2beta1 and alphaIIbbeta3 integrin, respectively. Glycoprotein VI blockade by 9O12 antibody or P2Y ADP receptors reduced integrin activation along with aggregate formation and fibrinogen binding but not alpha2beta1-dependent adhesion.

Monoclonal antibody IAC-1 is specific for activated alpha2beta1 and binds to amino acids 199 to 201 of the integrin alpha2 I-domain.

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 alpha(2)beta(1). 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 alpha(2) 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 alpha(2)beta(1); (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 alpha(2)beta(1) activation under dynamic and physiologically relevant conditions.