Multimerin-1 Binds to Specific Motifs in Vessel Wall Collagens and Contributes to Thrombosis: Novel Insights Regarding the Mechanisms That Support Human and Mouse Platelet Adhesion

Multimerin-1 (MMRN1) is a homopolymeric glycoprotein stored in platelets and vascular endothelial cells for regulated release that enhances platelet adhesion to collagen. We tested Mmrn1 contributions to platelet adhesion ex vivo and in vivo using selective deficient mice and triple-helical collagen mimetic peptides to evaluate a putative Mmrn1-selective binding motif. Selective Mmrn1-deficient (Mmrn1^-/-) mice were generated by crossing Mmrn1^+/- "knockout-first allele" (Mmrn1^flxneo/+ from European Mutant Mouse Archive) with flp deleter mice, and then with transgenic global cre deleter mice. The resultant heterozygous Mmrn1^+/- mice were crossed with wild type (Mmrn1^+/+) mice to remove the cre and flp transgenes. Inbred, gender-mixed, age-matched Mmrn1^+/+ and Mmrn1^-/- mice were then used for in vitro and in vivo assessments of platelet function. The following highly conserved GPAGPOGPX sequences in fibrillar collagens (that are distinct from collagen motifs that bind von Willebrand factor [VWF], platelet integrin alpha2beta1 or platelet GPVI) were identified to bind MMRN1: GPAGPOGPI (type I), GPAGPOGPV (type II), and GPAGPOGPQ (type III). In static adhesion assays with washed human platelets, triple-helical GPAGPOGPX peptides enhanced platelet adhesion when co-presented with the alpha2beta1-binding peptide GFOGER, like the VWF-binding peptide derived from type III collagen which was tested as a positive control. Static platelet adhesion to GPAGPOGPX peptides was also enhanced by agonists that induce MMRN1 release (p<0.02 for each comparison, where family-wise α=0.05, k=7), and co-presentation of GPAGPOGPX peptides with both GFOGER and III-23 maximized platelet adhesion (p<0.02 for each comparison, where family-wise α=0.05, k=5), providing insights on how MMRN1 and VWF synergistically enhance platelet adhesion. In high shear (1500 s^-1) flow assays of platelet adhesion, platelet adhesion to triple-helical GPAGPOGPX peptides that were co-presented with GFOGER was greater than adhesion to GFOGER alone (p=0.015). Mmrn1^-/- mice were viable and fertile, with normal blood cell counts and bleeding times (respective medians [ranges] for Mmrn1^+/+ versus Mmrn1^-/- mice: 300 [60-900] seconds vs. 330 [90-900] seconds, p=0.07), and confirmed Mmrn1 deficiency by Western blot analyses of platelets. In assays of platelet adhesion at high shear (1500 s^-1), platelets from Mmrn1^-/- mice showed a significant impairment in adhesion to full-length fibrillar collagen (% surface area covered by platelets ± SEM for Mmrn1^+/+versus Mmrn1^-/- mice: 37 ± 3% vs. 18 ± 2%, p<0.001) and in collagen-induced aggregation in assays with washed platelets (20-30% reduction in maximal aggregation for Mmrn1^-/- platelets compared to Mmrn1^+/+ platelets, p<0.05). In assays of washed platelet adhesion, Mmrn1^-/- platelets showed minimal static adhesionto the GPAGPOGPX peptide, unlike Mmrn1^+/+ platelets(p<0.0001). Assays of platelet adhesion under conditions of flow indicated that co-presentation of GPAGPOGPX with GFOGER enhanced the adhesion of activated Mmrn1^+/+ but not Mmrn1^-/- platelets. Intravital microscopyexperiments indicated that Mmrn1^-/- mice also had impaired platelet adhesion in arterioles treated with ferric chloride (platelets deposited per minute ± SEM for Mmrn1^+/+ versus Mmrn1^-/- mice: Mmrn1^+/+ : 82 ± 4 vs. Mmrn1^-/-: 42 ± 6, p<0.001), and the thrombi that formed in Mmrn1-deficient mice were smaller and less stable, with delayed vessel occlusion times (respective medians [ranges] for Mmrn1^+/+ versus Mmrn1^-/- mice: 17 [10-35] min vs. 35 [22-40] min, p<0.001). Taken together, our studies illustrate that multimerin-1 has functions in supporting and enhancing platelet adhesive function, in vivo and in vitro, and that fibrillar collagens contain highly conserved GPAGPOGPX motifs that bind Mmrn1 and contribute to platelet adhesion.