Mechanisms of Static and Shear-Induced Platelet Adhesion: Differences in the Adhesion Supported by MMRN1 Comparisons with Other β3 Integrin Ligands.

Abstract Platelet adhesion and aggregation at the site of vascular injury are key events in hemostasis and thrombosis. These processes are supported by interactions between platelet glycoprotein (GP) receptors (including integrin αIIbβ3, GP Ib-IX-V and GPVI) and ligands that include von Willebrand factor (VWF), collagen, and fibrinogen (Fg). Recently, the polymeric protein multimerin 1 (MMRN1) was identified to bind β3 integrins. Normally, MMRN1 is sequestered within secretion granules of platelets, megakaryocytes, and endothelium until its release. In static adhesion assays, MMRN1 supports platelet adhesion to integrins αIIbβ3 and αvβ3 by an RGD-dependent mechanism. Study goals and methods: To further determine the mechanisms of MMRN1 binding to platelets, we investigated (i) the importance of platelet activation in MMRN1 binding to platelets, (ii) the ability of MMRN1 to support platelet adhesion compared to other adhesive ligands (Fg, VWF), and (iii) the role of β3 integrins in MMRN1 binding to platelets at low and high shear. Results: The binding of secreted platelet MMRN1 to thrombin activated platelets was significantly reduced by antibody inhibitors of ligand binding to αIIbβ3 and αvβ3. Thrombasthenic platelets (GT), deficient in αIIbβ3 and αvβ3 integrins, stored normal quantities of MMRN1 but after activation, they retained 34% less MMRN1 than normal platelets. Platelet adhesion experiments confirmed that αIIbβ3/αvβ3 and other binding sites supported MMRN1 binding to platelets. MMRN1 supported platelet adhesion at both low (150 s-1) and high (1500 s-1) shear rates. Like platelet adhesion to VWF, platelet adhesion to MMRN1 was greater at high shear rates. Platelet stimulation by agonists was essential to induce platelet binding to MMRN1 in static and shear adhesion assays, but not to induce platelet adhesion to Fg and vWF. While platelets activated by ADP and TRAP showed similar adhesion to Fg and VWF, TRAP induced more platelet adhesion to MMRN1 at high shear rates than ADP. Inhibitors of ligand binding to αIIbβ3 and αvβ3 had greater effects on high compared to low shear platelet adhesion to MMRN1. Conclusions: These data indicate interesting differences in the mechanisms that support platelet adhesion to MMRN1 and other β3 ligands, which could be important for molecular events in hemostasis and thrombosis. The activation-dependent binding of MMRN1 to platelets, augmented by high shear flow, may reflect the unique recognition properties of platelet β3 integrins and/or exposure of other binding sites (e.g. phosphatidylserine) on activated platelets that promote adhesive interactions with MMRN1.