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163- Cl INHIBITOR (CIINH) TARGET PROTEASE SPE- CIFICITY. Rana Zahedi, Alvin E. Davis 111. Center for Blood Research, Boston, MA.

Study of a P2 Ala +Val mutant CIINH indicated a role for P; in specificity determination. To better define this role and that 01 other residues, a number of different amino acids were introducec at P2, as well as at P6 and P8’19’. P6 Ala +Val is a naturally occurring mutant; previous data suggested involvement of P8’iP9 in complex formation with Cls (He & Whaley, FEBS Let 412:506,1997). Thermal denaturation profiles indicated no majo! conformational changes. Reactivity of the inhibitors toward targel and non-target proteases were studied. Recombinant proteins wert incubated with proteases (I h, 37’C), then subjected tc immunoprecipitation and SDS-PAGE. The table summarizes thf amount of stable complex formation between the engineerec mutants and proteases in comparison with the wild type ClINH.

Cls Clr Kall FXIIa Try PI Th Val &

: cf f) P t t*

Asn J $ 1 * Asp & J J 0 en:,“, Gly cs c) tf tf c) 0 J Leo tf * * tf t t* Ser * c c) nd nd Thr tf i+ : t) oo++

Symbols:, Complex formation equal to (ct), greater than (t) less than (&), that with the WT; *, increased reactive center cleaved inhibitor; 0, no complex formation; nd, not determined.

With each protease tested, recombinant P6 Ala +Val ant P8’19’Gln -+Ala CIINH have the same profile as the wild typt protein on SDS-PAGE. Studies in progress will measure the inhibitory activity of the recombinant proteins by inhibition o cleavage of synthetic chromogenic substrates by Cls.

164- MODULATION OF THE ALTERNATIVE COMPLE- MENT PATHWAY BY Cl INHIBITOR (Cl INH) Eric Wagner, Haixiang Jiang and Michael M. Frank Department of Pediatrics, Duke University Medical Center, Durham, NC

C I INH inhibits many plasma serine proteases including Cl. We examined whether Cl MH regulates the alternative complement pathway. Cl INH was forther purified from material prepared by Baxter Health Care Corporation using jacalin-agarose chromatography followed by size exclusion chromatography. This highly purified preparation inhibited, in a dose-dependent manner, the Iysis of rabbit erythmcytes by human serum absorbed with rabbit erythmcytes in Mg2+-EGTA buffer. Furthermore, Cl INH inhibited the lysis of PNH erythmcytes in acidified human serum. The effect of Cl INH on alternative pathway function was examined. To EAC4b3b were added purified factors B, D, and properdin in either 0.065~ VBS or Mg*‘-EGTA buffer. Lytic sites were developed with guinea pig serum diluted in EDTA buffer. With both factors B and D in limiting concentrations (-1 lytic site&II), Cl INH inhibited lysis of EAC4b3b via the alternative pathway in a dose-dependent manner. C 1 INH accelerated the decay of the preformed alternative pathway C3 convertax in a dose-dependent fashion (rate of decay: O.O24/min with 0.375mg/ml Cl INH vs O.lZ/min with 0.375mg/ml human serum albumin), further suggesting an effect on factor B. The rate of formation of the alternative pathway C3 convertase (T max) was not significantly affected by C 1 INH but the number of lytic sites was markedly reduced when compared with a control protein (human serum albumin). These data suggest that Cl INH has regulatory activity for the alternative pathway, as well as the classical and MBLectin pathways, presumably by interaction with factor B.

165- Cl-INH ABUSE: WHIM OR NECESSITY G. Amo&o, S. Neri”, M Cicardi*, A Riela, M Bmi

Transfusion Center, University Pale-; “IntemaI Medicine, University Catania; *lntemal Medicine, University Milano; Italy

Cl-INH concentrate is the mainstay of therapy for hereditary aogicedcma laryngeal attacks. Scanty patients resistant to classical thempy and needing periodical infusions of Cl-INH have been reported. Use of Cl-INH has been suggested in pathologies accompanied by generalized activation of enzymatic cascades Qxmcreatitis, septic shock), but cleanxt benefits are lacking. We report on a patient (L.P) with a two years history of Cl-INH infosion, totaling 250,ooO U/year (s&ndard dose for laryngeal edema in Cl-INH deficient patients l,OOO-2,000 U). Since his childhood, LP has soffercd from u&aria-like flares with cccasional respiratory distress. In 19% repeated measurements of C 1 -lNH, C4, Clq aod C3 were normal. Nevertheless, doe to poor response to haditionaI treatment the patient was seldom infused with Cl-INH concentrate, followed by immediate disappearance of symptoms. Since 1998 respiratory crises and m’ticaria flares increased in fizqoency and severity and Cl-W applreatly became the only effective trcatmcnt, despite the lack of serologic markers of hereditary or acquired deficiency and normal kinii and coagulation pathways. Aside from acute “respiratory crises”, controlled by Cl-D&l, the patient is well and tends to proselytize. RecentlyhehasconvertodtoCl-INHabosealadyaffectedby flooxetine-rekted an&e&ma and bmnchospasm. Frequent generous Cl-lNH (2,000 U/dose) infusions seem to cone01 her symptoms. We are tapering off the nemoleptic, hoping to dismiss infusions. A third patient was @z&d, on LP rocommcndation ( ! ), in the local hospital, for acute asphyxia of unclear origin. We are astonished in front of an “infective addiction to Cl-INH”. It could easily be discharged as a placebo effect in a psychiatric disorder, but, is, pcrbaps, its awkward (mis)use shedding some light on acute illnesses of obscure c&o&s and uncertain theraw?

166- Cl-INH BINDS TO ENDOTHELIAL CELLS AND PREVENTS CELL METABOLIC FAILURE AFTER COLD STORAGE - G. Gobbo, L. Bergamaschini, F. Macera, R. di Stefano, A. Agostoni. Department of Internal Medicine, University of Milan, Ospedale Maggiore, IRCCS, Milan, Department of Transplantation Surgery University of Pisa, Italy.

The vascular endothelial cells damage occurring during cold storage has an Important influence on the re-establishment of the organ function after transplantatron. Since complement is thought to play a major role in cold storage-reperfusron injury, we evaluated the Cl-INH effect on endothelial cells subjected to prolonged cold storage in vifro. Methods: human umbilical vein endothelial cells (HUVEC) at confluence were stored at 4°C in University of Wisconsin (VW) solution or in UW plus CllNH (125500 Pglml). After 24-72 hours we evaluated: a) HUVEC vrtality and metabolic activity by measuring the release of LDH and the abrlity to reduce MTT, respectively; b) deposition of Cl INH to HUVEC by a cell-surface ELISA: c) the activity of surface bound CIINH by the Inhibitron of exogenous Cls; d) the degree of apoptosis by microscopy and by cellular DNA fragmentation ELISA. In additional experiments after storage at 4°C. HUVEC were washed and further Incubated with normal human serum (10% rn M199) at 37°C for 2 h. Results: after 72 h at 4 C’ cell vitality was 6590% of basal value, both in HUVEC stored with UW or UW plus CIINH. The effect of CIINH plus UW on HUVEC metabolic achvrty progressed through 50% up to 75% of basal value in a dose- dependent manner. The protective effect was associated with the binding of CllNH to cell monolayer. CIINH reduced of about 60% the level of apoptosis Induced by cold storage. Cell bound Cl-INH retained the ability to Inhibit exogenous Cls. The addition of Cl-INH to the preservation solutron markedly reduced cell killing due to reperfusion with fresh human serum, as manrfested by 50% increase in cell survival as compared to cells stored rn VW alone. Conclusions: Cl-INH can bind to endothelial cells during cold storage preventing metabolic failure after prolonged cold storage Cells stored rn presence of Cl-INH were more resistant to cold induced apoptosis and to reperfusion injury.