Collagen mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties

Abstract

In an effort to elucidate the nature of the collagen platelet interaction, the effects of collagen modification on platelet aggregation have been studied. Purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. Neither of the glycopeptide classes produced or inhibited native human skin insoluble collagen mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosyl glucose (Hyl Gal and Hyl Gal Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet rich plasma showed that, at concentrations greater than 50 nM, Hyl Gal gives apparent binding to platelets, but there was no evidence of Hyl Gal Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl Gal (at 29 μM) nor Hyl Gal Glc (at 18 μM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 μM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Hyl Gal Glc containing 36 residue rat skin soluble collagen α1 (I) cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Human skin insoluble collagen was reacted with periodate and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation. Thus, collagen platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple helical fold or perhaps even for a particular amino acid sequence in the triple helix. It is concluded that under physiological conditions, where the criteria of high specificity and affinity are met, the collagen carbohydrate is not a determining factor in collagen binding to platelets, but, rather, the effect is mediated primarily by the protein moiety.