Fibrinogen solutions were irradiated with UVC (254 nm) to inactivate contaminating viruses. In order to protect fibrinogen during UVC irradiation, 0.5 mM rutin was added prior to UVC exposure and subsequently removed during processing. Viral kill by 0.1 J/cm2 UVC resulted in the following inactivation values (log 10): non-lipid-enveloped viruses: Parvo > or = 5.5; encephalomyocarditis virus > or = 6.5; hepatitis A virus > or = 6.5: lipid-enveloped viruses: human immunodeficiency virus > or = 5.7; vesicular stomatitis virus > or = 5.7. Fibrinogen irradiated with 0.5 mM rutin did not significantly differ from unirradiated material in terms of clot time and breaking strength. In the absence of rutin, UVC irradiation of fibrinogen at similar fluence led to loss of solubility, increased clot time and the cleavage of fibrino-peptides that reacted with dinitrophenyl hydrazine as a test for ketonic carbonyl groups. High-performance liquid chromatography and mass spectrometry data showed that rutin exposed to UVC formed numerous breakdown, oxidation and combinational products. Experiments with 3H-rutin showed that after UVC irradiation, subsequent processing by a C18 resin and alcohol precipitation removed > 99% rutin, representing < 10 ppm rutin in the final fibrinogen preparations. Residual 3H-rutin was not covalently bonded to the fibrinogen. Immunochemical studies with rabbit antisera to UVC irradiated (with rutin) fibrinogen showed the absence of neoimmungens. By all measures, rutin prevents fibrinogen degradation during virucidal UVC irradiation.

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Human neutrophils labeled with chlortetracycline (CTC), commonly used as a probe of membrane-bound calcium, release lysosomal enzymes and exhibit a rapid decrease in fluorescence when exposed to the chemotactic peptide fMet-Leu-Phe or the lectin Con A. This decrease has been attributed to the release of calcium from a membrane-associated "trigger pool." The nature of this putative pool has been further characterized by examining the effects of various inhibitors on the CTC fluorescence response and lysosomal enzyme release from stimulated neutrophils. These agents included inhibitors of glycolysis (2-deoxyglucose and iodoacetate), an uncoupler of oxidative- phosphorylation (KCN), and a sulfhydryl inhibitor (N-ethylmaleimide). Resting neutrophils labelled with CTC demonstrated an enhanced decay of baseline fluorescence when exposed to 2-deoxyglucose or iodoacetate. This suggested that the pool of membrane-bound calcium labelled by this probe was maintained by glycolytic metabolism. Furthermore, 2-deoxyglucose and iodoacetate inhibited both the stimulated decrease in CTC fluorescence and lysosomal enzyme release induced by fMet-Leu-Phe and Con A in a time-dependent manner. KCN did not inhibit either response to stimulation, but did retard the recovery of CTC fluorescence observed when fMet-Leu-Phe was used as the stimulus. High concentrations of N-ethylmaleimide (100 microM) completely inhibited both the CTC fluorescence response and lysosomal enzyme release almost immediately; low concentrations of N-ethylmaleimide (30 microM) inhibited lysosomal enzyme release in a time-dependent manner without significantly affecting changes in CTC fluorescence. These results are consistent with the hypothesis that CTC serves as a probe of membrane-bound "trigger" calcium, the release of which is dependent upon intact glycolysis and is a requirement for lysosomal enzyme release.

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