Biomedical subjects
Publications and source records attributed to E R Simons.
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The rates of collagen fibril formation are, under otherwise identical conditions, a function of the age of the acid-soluble rat tail tendon collagen utilized. The rate is most rapid with young adult (165 day) collagen, which exhibits no induction period before collagen multimerization begins. In contrast, 21-day collagen exhibits both a 30% slower rate and an induction period. Collagens from older animals also have age-dependent fibril formation rates going from a maximum at 165 days to 80% of that for 1,100-day-old material. These age-related differences in collagen fibril formation kinetics appear to be related to the degree of cross-linking of the collagen involved.
Rat tail tendon collagen-initiated platelet aggregation exhibits a collagen age-dependent lag time. This lag time is an inverse function of the previously determined rate of fibril formation of collagen, and corresponds to the elapsed time necessary to form a collagen fibril of requisite size under the platelet aggregation conditions chosen. Such fibers exhibit native spacing and appear to be 45 to 90 A in diameter. Fibers preformed to that size (less than 2 min for 21- to 1,100-day-old collagen), no matter what the age of the collagen, give rise to identical platelet aggregations. Fibers formed after more prolonged incubation, greater than or equal to 20 min, have impaired platelet aggregating ability.
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A noncovalent fluorescent probe that responded to changes in transmembrane potential was used to study the response of washed human platelets to aggregating agents. Concentration-dependent changes in the fluorescence were observed in response to ADP and to thrombin. No such changes were observed in response to collagen fibrils. Thus there was an indication that platelet membrane potential changed in response to aggregating stimuli, supporting the hypothesis that the mechanisms of platelet aggregation resembled the mechanisms of other systems that show stimulus-response coupling (e.g., muscle, adrenal chromaffin cells). The different responses to specific agents indicate that the agents may trigger platelet aggregation through different mechanisms.
The conformational changes upon anaerobic carbamyl phosphate addition to dilute hemoglobin solutions have been studied by means of circular dichroism. Freshly prepared carbamyl phosphate stabilizes the conformation of pure deoxyhemoglobin without detectable NH2-terminal carbamylation. Addition of preincubated (i.e. partially converted to cyanate) carbamyl phosphate, however, results in such carbamylation and in the formation of the conformationally different carbamyldeoxyhemoglobin which exhibits enhanced oxygen affinity. Fresh carbamyl phosphate carbamylates hemoglobin in deoxygenated hemolysates containing erythrocyte phosphatases. Therefore the reversal of SS erythrocyte sickling by carbamyl phosphate is attributable to carbamylation by the carbamyl phosphate hydrolysis product, cyanate.
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Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying identical conformations about the heme groups of the respective proteins. The spectra of the oxygenated forms change little upon addition of KCNO, which is known to carbamoylate the NH2 terminals of the individual chains (Cerami and Manning, 1971). The spectra of the deoxygenated forms, on the other hand, are markedly altered. The decreased magnitude of the 430-nm extremum with increased cyanate concentration can be used to calculate an addition curve which becomes asymptotic at a cyanate:heme molar ratio of approximately 10(3). This conformational change occurs in the absence of O2 and has been predicted (Njikam et al.,1973); it can also be demonstrated by difference spectroscopy techniques, whereby a comparable addition curve can be constructed from changes in the 555-nm absorption, while the 541-nm absorption remains invariant. The change described corresponds to the formation of a new conformation, corresponding to carbamoyldeoxyhemoglobin, carrying one carbamoyl group per chain. In the presence of a small quantity of oxygen, however, the above reported changes in CD are accompanied by a concomitant rise in the 415-nm peak-corresponding to the formation of oxyhemoglobin-while those in the difference spectra reflect not only a change in the 555-nm band but also a parallel one at 541 nm, confirming the formation of oxyhemoglobin. Thus the conformation achieved upon carbamoylation of deoxyhemoglobin has the higher oxygen affinity predicted by Nigen et al. (1974) for carbamoyldeoxyhemoglobin. Cyanate has been used (Cerami and Manning, 1971) as an antisickling reagent in vivo and in vitro, but, although it has been shown that it binds covalently to the NH2-terminal residues of hemoglobin (Lee and Manning, 1973), its effect on hemoglobin conformation has not been previously shown nor has its mechanism of action been fully clarified. The results presented here show that the effect of cyanate on hemoglobin is the formation of a new conformation with heightened oxygen affinity. Since oxyHbS does not aggregate while deoxyHbS does, in a temperature-dependent fashion, the formation of carbamoyldeoxyHbS interferes with such aggregation in vitro in deoxygenated samples. In vivo, where there are generally low residual concentrations of O2, the formation of oxyHb is favored by the higher O2 affinity of carbamoyldeoxyHbS, and aggregation with concomitant red cell sickling is therefore disfavored.
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Noncovalently bound fluorescent probes have been used to study changes in the platelet which may occur during platelet aggregation. Platelets were exposed to either N-phenyl-naphthylamine (NPN) or 8-anilino-1-naphthalene-sulfonic acid (ANS). Both dyes were bound by the platelet, and platelet aggregation by collagen or thrombin was unaffected by the presence of the label. No change in fluorescence intensity or wavelength of maximum intensity was observed during platelet aggregation.
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