Elvin Harper

Animal collagenases: Specificity of action, and structures of the substrate cleavage site☆

Abstract Two highly purified animal collagenases, one derived from homogenates of the rabbit V 2 ascites cell carcinoma growing in muscle and the second isolated from the media of tadpole tissue cultures cleaved isolated non helical α chains from chick and rat skin collagen, and the CNBr peptide CB7 from chick skin α1 chains at one, and the same peptide bond. Although two other Gly-Ile bonds exist elsewhere in the α1 chain they were not cleaved.

Collagenase, procollagenase and activator relationships in tadpole tissue cultures☆

Abstract In cultures of tadpole tissues which produce collagenase, an activator of procollagenase appears in the medium simultaneously with active enzyme and a precursor product relationship exists between the amounts of zymogen and enzyme liberated into the medium as a function of incubation time. The initial two day lag period before enzyme appears is characterized by high zymogen levels and the absence of activator and enzyme inhibitors.

Human Platelet Collagenase

The presence of proteolytic enzymes such as cathepsin and elastase in platelets and the important role of collagen in platelet aggregation suggested that collagenase might be present in platelets. Epinephrine, ADP, and collagen liberate collagenase from platelets in plasma as measured by the hydrolysis of [(14)C]glycine-labeled collagen fibrils. The collagenase activity appeared in an early phase of platelet aggregation and was not a part of the release reaction. However, only 50% of the total collagenase could be liberated by the aggregating agents used. Sucrose density gradient analysis of platelet homogenates using appropriate sub-cellular markers indicated that collagenase appeared in both the granule and membrane fractions. Gel-filtered platelets failed to show collagenase activity before exposure to aggregating agents but released more collagenolytic activity than was found in platelet-rich plasma. This observation was explained by the finding that collagenolytic activity was inhibited by normal human plasma. One of the inhibitors is alpha(1)-antitrypsin as demonstrated by decreased inhibition in plasma from a patient with homozygous alpha(1)-antitrypsin deficiency. Platelet collagenase activity could also be demonstrated by its ability to decrease the viscosity of collagen solutions and to produce collagen fragments similar to those produced by other mammalian collagenases on disk gel electrophoresis. The observation that partially purified platelet collagenase could destroy the platelet-aggregating activity of collagen suggests that the enzyme might function in a negative feedback mechanism limiting thrombus formation.

Critical Role of the Carbohydrate Side Chains of Collagen in Platelet Aggregation

The reaction between human platelet membrane glucosyl transferase and collagen has recently been proposed as the mechanism for pletelet-collagen adhesion. Collagen contains glucosyl-galactose and galactose side chains linked through the galactose to hydroxylysine. Oxidation of the 6-hydroxymethyl position of the galactosyl residue to aldehydes with galactose oxidase completely abolishes platelet aggregation. This enzymatic modification of collagen can be fully reversed by reduction of the aldehydes formed by NaBH(4) with complete restoration of platelet aggregating ability. Limited digestion with bacterial collagenase abolishes the ability of collagen to aggregate platelets. Removal of the N-terminal telopeptides from collagen with trypsin does not affect platelet aggregation. Tertiary structure of soluble collagen is essential for platelet aggregation. Normal collagen is less effective than lathyritic collagen, which contains only a small number of cross-links. The decreased number of aldehyde groups in the lathyritic collagen are not responsible for the increase in aggregating ability, since reduction with NaBH(4) does not alter platelet aggregation. These results suggest that integrity and accessibility of the galactose receptor site may be crucial for the formation of a ternary collagenenzyme-platelet membrane complex which must precede platelet aggregation.

Studies on the specificity of bacterial collagenase

Abstract The specificity of Clostridium histolyticum Collagenases A and B has been investigated employing a cyanogen bromide peptide of known sequence obtained from chick skin collagen. The enzyme A cleaves only between the X and Gly bond in the sequence-Pro-X-Gly-Pro-Y-.

The effect of the conformation of collagen on its ability to aggregate platelets

Abstract The interaction between platelets and collagen, which marks the initiation of hemostasis, was examined as a function of the conformation and the multimerization of soluble guinea pig skin collagen. Both the rate and extent of collagen multimerization, as measured by the intensity of scattered light, are dependent on the time of incubation and on the temperature of the system. Compounds such as arginine or penicillamine, which at low concentrations ( 2 × 10−2 M) decrease the ability of collagen to form multimers and hence to initiate platelet aggregation. By these techniques we have been able to correlate the initiation of platelet aggregation by collagen with the extent of preformed multimers, the lag time approaching zero as the multimerization of the initiating collagen increases.

The effect of chemical or enzymatic modifications upon the ability of collagen to form multimers and to initiate platelet aggregation.

Abstract The previous paper (1) presented some correlations between collagen multimer formation and platelet aggregation. The present study investigates the effect of chemical or enzymic modification upon its melting temperature and rate of multimerization, and therefore upon platelet aggregation. Digestion of collagen with tadpole or bacterial collagenase led to a decreased melting point of the collagen fragments and prevented their initiation of platelet aggregation. In contrast, the absence of interchain crosslinks, in lathyritic collagen, had no effect on these parameters and thus these crosslinks are not required for collagen multimer formation and induction of platelet aggregation. Oxidation of the galactoses alone with galactose oxidase, or of all the sugars by periodate, led to a perturbed melting curve featuring two melting points, ∼ 28° and 37°. These treated collagen preparations were thus unable to form multimers at platelet aggregation (> 29°) temperatures, and they could not initiate the aggregation of platelets. Reduction with sodium borohydride regenerated a normal melting curve, multimerization, and platelet aggregation initiation. Thus the carbohydrate residues, and a majority of the intact tropocollagen chains, are required for the maintenance of native structure and of multimerization at 37° which, in turn, are necessary for initiation of platelet aggregation by guinea pig skin collagen.