Multimerin 1 (MMRN1) is a massive, homopolymeric platelet and endothelial cell protein with functions that are emerging to support platelet adhesive processes. MMRN1 supports platelet adhesion under arterial flow conditions by a mechanism dependent on interactions with von Willebrand factor (VWF). The goals of this thesis were to further define the platelet adhesive functions of MMRN1 by: 1) characterizing the molecular mechanisms of VWF interactions with MMRN1; and 2) investigating if multimerin 1 is important for platelet adhesive functions using mice with and without a selective multimerin 1 (Mmrn1) deficiency. Studies of the mechanism of MMRN1-VWF binding indicated that MMRN1 bound to shear exposed VWF, and that MMRN1 interacted with the A1 and A3 domains in the VWF A1A2A3 region. VWF A1A2A3 also bound to MMRN1 with a physiologically relevant binding affinity, and supported platelet adhesion to MMRN1 at a high shear rate. The selective loss of Mmrn1 in mice had limited effects on tail bleeding times, although it impaired collagen-induced aggregation of washed platelets, as well as high shear platelet adhesion of whole blood on collagen surfaces, in vitro. Additionally, the selective loss of Mmrn1 in mice was associated with impaired and delayed platelet-rich thrombus formation in vivo, in arterioles treated with ferric chloride. These findings provide new insights on platelet adhesive, haemostatic functions at arterial shear rates, and the involvement of the platelet and endothelial cell protein, multimerin 1, to support these processes. / Thesis / Doctor of Philosophy (PhD)

Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a large homopolymeric glycoprotein that is synthesized and stored by platelets and endothelial cells until activation-induced release. MMRN1 is able to support platelet adhesion through mechanisms involving von Willebrand factor (VWF) and glycoprotein (GP)Ibα, and β3 integrins on activated platelets, and it enhances platelet adhesion to fibrillar collagen, potentially by binding to putative MMRN1-specific GPAGPOGPX (where O is hydroxyproline and X is valine or glutamine) motifs in fibrillar collagens. Using mice with and without selective Mmrn1 deficiency, the goals of this thesis were: 1) further characterize the ability of Mmrn1 to enhance platelet adhesion to collagen, 2) explore the role of fluid shear stress in the ability of Mmrn1 to enhance platelet adhesion, and 3) test the specificity of the GPAGPOGPX motif for Mmrn1 and the ability of GPAGPOGPX to support or enhance platelet adhesion. Mmrn1-deficient (Mmrn1-/-) mouse platelets showed impaired aggregate formation on fibrillar collagen surfaces under high (1500 s-1) and low (300 s-1) shear flow compared to wild-type (Mmrn1+/+) mouse platelets, which was due to reduced initial adhesion and a slower rate of platelet accumulation onto collagen surfaces. Similarly, Mmrn1-/- platelets formed smaller aggregates on immobilized recombinant (r)Vwf surfaces compared to wild-type platelets, and Mmrn1-/- platelets had impaired adhesion and aggregate formation on immobilized murine fibrinogen, but not fibrin, when platelets were pre-activated to release Mmrn1. Type I fibrillar collagen was found to contain a variant of the GPAGPOGPX motif (GPAGPOGPI), and GPAGPOGPX motifs supported adhesion of wild-type, but not Mmrn1-/-, platelets. When presented with the VWF-binding GPRGQOGVMGFO motif and the integrin α2β1-binding GFOGER motif present in fibrillar collagens, the GPAGPOGPX motifs synergistically enhanced platelet adhesion. These findings expand upon the known adhesive functions of platelet multimerin 1 and update knowledge of the motifs that support platelet adhesion to fibrillar collagens. / Dissertation / Doctor of Philosophy (Medical Science)