Abstract
Abstract 3256
Background.
Integrin a2b1-mediated adhesion to collagens supports cellular attachment, while decorin binding by a2b1 often attenuates adhesion. Collagen I binds to the a2 I-domain via the triple-helical sequence GFOGER, but the decorin binding site is not within the a2 I-domain and has not yet been identified. A single nucleotide polymorphism in the a2 gene ITGA2 (rs1801106) (G1600A) resulting in the amino acid substitution glutamate-534 to lysine-534 (E534K) is the basis for one of the most important human platelet alloantigen (HPA) systems, HPA-5, yet HPA-5 alleles do not influence the binding of a2b1 to collagen I, and the effect of HPA-5 on platelet function, if any, has not been determined. We sought to determine whether the minor allele HPA-5b (534K) might influence the a2b1-mediated adhesion of platelets to a physiologically relevant ligand other than collagen I. One such alternative ligand is decorin, an extracellular matrix small leucine (Leu)-rich proteoglycan (SLRP). Methods/Results. Two groups of normal subjects were compared. The first group (GG) consisted of ten donors homozygous for the major allele rs1801106G. The second group (GA+AA) included one donor homozygous for the minor allele rs1801106A and nine donors heterozygous for these alleles. Aside from this, there were no significant differences between the two groups with respect to platelet count, mean platelet volume, surface expression of four selected platelet receptors, GPIba, GPVI, aIIbb3 or a2b1, or the allelic distribution of five receptor genes, ITGA2B rs5911, ITGB3 rs5918, GP1BA rs6065, GP6 rs1613662 and P2RY1 rs1065776. In direct platelet adhesion assays, we determined that platelets from GG donors bound more strongly to decorin than those from GA+AA donors (p < 0.01), while platelets from either group bound equally well to collagen I (p = 0.73). Using platelets from donors homozygous for the major allele rs1801106G, adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by GFOGER or an a2-specific monoclonal antibody 6F1 known to inhibit cell adhesion to collagen I. Conversely, GFOGER and 6F1 inhibited adhesion to collagen I but not decorin. Conclusions. The simplest explanation of our findings is that 534E, located in the a2 b-propeller domain, is necessary for maximal binding of a2b1 to decorin but not collagen I. These results suggest that 534E contributes to the decorin binding site and that this amino acid represents a potential target for interventions that can modify cell adhesion to decorin and perhaps other proteoglycans. This also represents the first instance of a functional difference attributable to the HPA-5 alleles. These results expand our understanding of the decorin and collagen I interactions described in murine and human studies of cell adhesion and cancer biology, where decorin and collagen I often have opposite effects that are both mediated by a2b1. Decorin knockout (Dcn(−/−)) murine embryonic fibroblasts exhibit greater adhesion to collagen and greater migration on collagen substrates, while decorin attenuates the aggressiveness and metastasis of tumor cells with diverse histologic backgrounds. Further studies of the relationship between the collagen I and decorin binding sites on integrin a2 are warranted, particularly regarding its influence on thrombosis and hemostasis.
Disclosures:
No relevant conflicts of interest to declare.
Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions
