GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach.

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...

Summary. Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI de®ciency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no speci®c, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a puri®ed system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and FcgRIIA, 9O12.2 Fab fragments completely blocked collageninduced platelet aggregation and secretion from 5 mg mL À1 and fully prevented CRP-induced activation from 1.5 mg mL À1 . 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial¯ow conditions. We thus describe here for the ®rst time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth.

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