Howard B. Bensusan

The Role of Collagen Quaternary Structure in the Platelet:Collagen Interaction

WE HAVE INVESTIGATED WHETHER COLLAGEN QUETERNARY STRUCTURE IS REQUIRED FOR THE PLATELET: collagen interaction. Quaternary structure refers to the assembly of collagen monomers (tropocollagen) into polymers (native-type fibrils). Purified monomeric collagen was prepared from acetic acid extracts of fetal calfskin. Polymeric collagen was prepared by dispersion of bovine Achilles tendon collagen and by incubation of monomeric collagen at 37 degrees C and pH 7.4. The state of polymerization was confirmed by electron microscopy. Release of platelet serotonin in the absence of platelet aggregation was used to determine the effectiveness of the platelet: collagen interaction. All forms of collagen produced serotonin release only after a lag period, but polymeric collagen gave a shorter lag period than did monomeric collagen. Monomeric collagen was also quanidinated selectively to convert collagen lysine groups to homoarginine, while leaving the arrangement of polar groups intact. Guanidination of monomeric collagen increased the rate of polymerization and reduced the lag time in serotonin release. Glucosamine (17 mM) retarded polymerization and inhibited the release of platelet serotonin by monomeric collagen but had little effect on release produced by thrombin or polymeric collagen. At the same concentration, glucosamine did not reduce the sensitivity of platelets to stimulation by collagen or block the platelet: collagen interaction. The only effect of glucosamine was on the collagen: collagen interaction. Galactosamine had a similar effect, but glucose, galactose, and N-acetylglycosamine had no effect. We conclude from this data that collagen monomers cannot effectively interact with platelets and that, therefore, collagen quaternary structure has a role in the recognition of collagen by platelets.

On the role of the collagen carbohydrate residues in the platelet: Collagen interaction

It has been proposed that the platelet : collagen interaction is mediated in part by the collagen carbohydrate residues. To test this hypothesis we have oxidized monomeric and polymeric collagen with sodium periodate under conditions specifically designed to minimize destruction of periodate-susceptible bonds other than in the carbohydrate residues. Oxidation of the collagen significantly reduced its ability to interact with platelets. The extent of inhibition paralleled the extent of carbohydrate destruction. Oxidation with periodate also delayed the polymerization of the monomeric collagen, but even after polymerization the oxidized collagen failed to initiate the release reaction. These observations suggest that the collagen carbohydrate residues may be either near to or part of the site(s) on the collagen molecule required for platelet adhesion.

The isolation of crosslinked peptides of collagen involving α1-CB6

Abstract On cooling a solution of the peptides from a CNBr digest of insoluble steer-corium collagen a precipitate was formed which represented about 20% of the total protein. No such precipitate formed when reconstituted soluble collagen was similarly treated. Amino acid analysis of the washed precipitate showed that there was an approximate equivalence between the content of 3-hydroxyproline and homoserine, indicating a mixture of α1-CB6 and other peptides. Three fractions were isolated from the precipitate. One was analyzed to be two α1-CB6 peptides linked to one α1-CB(0,1)′, another was one α1-CB6 plus one α1-CB(0,1). The third was a combination of α1-CB6 with a small CNBr peptide containing homoserine.

An improved enzymic hydrolysis of collagen

Abstract A peptidase mixture was prepared from hog-kidney microsomes which contained aminopeptidase, prolidase, prolinase, carboxypeptidase-P, and X-prolyl-aminopeptidase activity. The sequential addition of collagenase, papain, and the peptidase mixture to collagen samples resulted in the hydrolysis of 90% of the peptide bonds. The unhydrolyzed fraction was accounted for by the recovery of 40 residues of prolyl-hydroxyproline, 1.6 residues of hydroxylysine-carbohydrate compounds, and 4–5 residues of α-linked glutamyl-hydroxyproline per 1000 total residues. Correcting the amino acid analysis of the enzyme hydrolysate for these unhydrolyzed residues resulted in an amino acid analysis equivalent to an acid hydrolysate of the enzymic hydrolysate which, in turn, is equivalent to an acid hydrolysate of the original collagen sample. These data indicate that there is no detectable contamination by the enzymes. A non-destructive hydrolysis is now available for use in the search for unusual linkages in collagen.

Protein crosslinkages. I. Effect of iodination on the time-dependent solubility changes of reconstituted collagen fibrils.

It has been shown that the iodination of tropocollagen leads to a more rapid formation of intermoleoular crosslinks of the reconstituted fibrils as judged by the inability to dissolve the fibrils in urea-HOAc solution. Iodination of the tropocollagen without subsequent fibril formation results in the formation of some intramolecular crosslinks as determined by ultracentrifugation at 40° and acrylamide gel electrophoresis and by the melting point and formation of segmented-long-spaced structures of the cooled gelatins derived therefrom. Control experiments and aminoacid analyses have shown that possible oxidation reactions cannot account for the changes. It is postulated that the iodination of histidine and/or tyrosine residues increases the rate of a normally occurring crosslinking reaction.

The effects of platelet secretion inhibitors on protein phosphorylation

Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.

Investigation of crosslinks in collagen following an enzymic hydrolysis.

Abstract Several crosslinks in collagen have previously been isolated by others after hydrolysis by strong acid or base at elevated temperatures. This research was undertaken to determine whether or not the identified crosslinks could have arisen by the fragmentation of some more complex structure under the extreme conditions of hydrolysis. Various samples of collagen which had been reduced by NaB 3 H 4 were subjected to the enzymic hydrolysis developed in this laboratory. All of the reduced crosslinks which had been identified in acid-soluble skin collagen and the reconstituted fibers from acid-soluble skin collagen were located in the enzymic hydrolysates. In addition, the results confirm and supplement a previous observation that there is a steady decrease in the amount of reducible crosslinks in reconstituted fibers during an in vitro incubation. However, denaturation of the fibrils prior to reduction causes a partial reversal of this trend.

Release of reducible crosslinks of collagen by total enzymic hydrolysis.

Abstract Enzymic hydrolysis of cow skin insoluble collagen was done to determine if previously identified reducible Schiff base crosslinks were artifacts of the hydrolytic conditions used. Cow skin insoluble collagen was reduced with NaB 3 H 4 , and samples were subjected to acidic, alkaline and enzymic hydrolysis. In addition, portions of the enzymic hydrolysate were subjected to a second hydrolysis using acid or alkali. The radioactive elution profiles were studied for changes in the concentrations of known crosslink compounds. The results of these experiments show that enzymic hydrolysis does not appear to release the crosslinkages completely, although all of the major ones are found to be present. Alkaline hydrolysis was done on samples of aldol histidine and histidinohydroxymerodesmosine which had been isolated from acid hydrolysates. Synthetic hydroxylysinonorleucine was hydrolyzed with varying concentrations of HCl and with 5 M NH 4 OH for 24, 48 and 72 h. Alkaline hydrolysis results in 60% destruction of the crosslinks, aldol histidine and histidinohydroxymerodesmosine. Synthetic hydroxylysinonorleucine is not affected by acid hydrolysis but it is destroyed in direct proportion to the length of time hydrolyzed in 5 M NH 4 OH.

Purification and characterization of proteins, including procollagen type III, in salt extracts of fetal bovine skin

Abstract The nature of high-molecular-weight proteins in salt extracts of fetal bovine skin was investigated. A series of DEAE cellulose ion-exchange columns separated the mature collagen from the high molecular weight proteins and also separated the high molecular weight proteins from each other. The following proteins were isolated: (a) a very high molecular weight protein which appears to be aggregated mature collagen; (b) two high molecular weight proteins of slightly faster mobility on SDS polyacrylamide gels, one of which is collagen-like and one of which is not; and (c) a type III procollagen, purer than those previously reported in the literature. These latter three proteins were characterized by amino acid analysis, SDS polyacrylamide gel electrophoretic mobility, collagenase sensitivity, and CNBr peptide patterns from SDS-PAGE.

Demonstration of lag phase in the sol-gel transformation of deoxygenated S hemoglobin without temperature alteration.

Summary 1). During the sol to gel transformation of deoxygenated sickle hemoglobin, a time-dependent process preceding gel formation (lag phase) was demonstrated that was inversely proportional to a function of the hemoglobin concentration and that occurred without alteration in temperature, pH, or oxygen tension. 2). As determined by the Schachman modification of the capillary viscometer, preparations of oxyhemoglobin S and A and deoxyhemoglobin A were indistinguishable when compared over a wide range of concentrations. Up to the concentration at which gelling occurred, deoxyhemoglobin S exhibited the same viscosity behavior. The viscosity of deoxygenated hemoglobin S within the lower gelling concentration range was normal during the lag phase and became abnormally high only at the time of gelation.