THE STRUCTURE OF PLATELET-REACTIVE SITES IN COLLAGENS
Collagen-induced platelet aggregation is an essential step in haemostasis and may be important in thrombosis, particularly that associated with the atherosclerotic plaque. We have located platelet-binding sites in collagen by fragmentation of the molecule with cyanogen bromide (CB) and measurement of the platelet aggregatory activity of the fragments following their renaturation to restore triple-helical configuration and polymerisation to introduce quaternary structure. We have found a high1y-reactive site in collagen type III located in the peptide α1(III)CB4 which was active at a concentration of less than 0.5μg/ml. The equivalent peptide from type I collagen α1(I)CB3 occurring in precisely the same location in the respective parent molecule (residues 403-551) and exhibiting a structure highly homologous to that of α1(III)CB4 was active only at concentrations higher than 200μg/ml. α1(I)CB7, the most active of the type I peptides, was able to cause platelet aggregation at around 5μg/ml whilst the equivalent type III peptide μ1(III)CB5 was inactive. Modification of specific amino acid residues indicated the importance of lysine in the activity of μ1(III)CB4 and of arginine in that of α1(I)CB7. Comparison of the structure of these peptides leads us to conclude that a reactive site comprises two basic residues, a specific distance apart, the conformation of one to the other dictated by collagen triple-helical configuration. One residue occurs in the sequence GlyPro(orHyp)LYS(orARG)GlyGlu, the other in GlyLYS(orARG)Pro(orHyp)GlyGlu. The lower reactivity of α1(I)CB7 relative to α1(III)CB4 can be attributed to the presence of two arginyl rather than lysyl residues and because the spacing of the two in CB4(G1y-LYS-Y-G1y-X-Y-G1y-X-LYS) represents a more favourable one than in CB7(Gly-X-ARG-Gly-X-Y-Gly-X-Y-Gly-ARG-Y).