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From www.bloodjournal.org by guest on June 12, 2017. For personal use only. Formation of C8-CI-Inhibitor, Plasmin-a2-Plasmin-Inhibitor By Yanina Stimulation of clinical the classical are activated Using platelets complement. as for plasma nary was hours. pathways neutrophil was The these their complexes formation are cleared by assaying plasma blood of blood prolong the loss,’ and cell activation bleeding time, may lead circulation qualitative and quantitative ing thrombocytopenia,’3 ergic6 platelet and A2,8 aggregate and depletion formation,5 of a-granule granule enzyme, and human for during formed have neutrophils. and C5a’7’8 krein,’’6 and elastase,” vasculature that are enzymes degranulation. Under bypass aggregates in Three factor the poly- now XIIa,’3 neutrophil investigate seen the Thrombosis Sections, SchoolofMedicine, and the New Division Philadelphia; ofthe Submitted complement, the Ben Franklin ing. San Broad reprint Center, St, The publication payment. charge “advertisement” indicate © 1989 requests Society 1986. and the of Sur- 19. 1988. Grants No. and a grant from of Hematology published to Robert PA & Stratton, Health MD. Sciences article must with Meet- in abstract Inc. section 1734 that with of the pathways but of probably neutrophil activa- bypass. Inc. 3400 solely bypass, pathways are to produce activated neutrophil ago- AND METHODS components. purified dodecyl of 85 .tmol was insoluble urokinase. purified as described. wt) CI, was a generous prepared factor sulfate (mol weight was XII (SDS) 88 and by 85 kilodalton by Cl-inhibitor23 of of On a nonne- as a doublet (Kd) pNa activation gift of purified fragments.#{176}’’ gel it appears of D-Pro-Phe-Arg prepared activation with of a specific hydrolyzed/mm/mg. purified plasminogen and a2-plasmin-inhibitor24 by were Purified antibodies. IgG was purified from monospecific antisera raised in rabbits against Cl-inhibitor, a2-plasmin-inhibitor, and normal serum, respectively. F(ab’)2 fragments were prepared by digestion of rabbit anti-human IgG as described.25 Clinical cardiopulmonary bypass. In nine adults with normal function tests, formation samples from whom of enzyme obtained written inhibitor before, during, informed after and consent was was measured complexes in open-heart sur- for congenital (one patient), valvular (four patients), or ischemic heart disease (four patients). Blood samples (15 mL) were drawn from a radial arterial catheter: (a) before induction of anesthesia; (b) after anesthesia, before hepanin; (c) after heparin, before CCB; (d) five minutes after start of CCB; (e) 45 minutes after start of CCB; (f) just before CCB ended; (g) 60 minutes after CCB; and (h) 24 hours after CCB. Plasma was harvested from blood samples of all patients that had CCB with a bubble oxygenator, centrifugal pump, and arterial line filter as previously described.” gery Thrombosis Ctr, by sodium plasma N were defrayed in part by page therefore be hereby marked 18 U.S.C. evidence corn- activation. or fibrinolytic Kallikrein Plasmin liver 19140. costs ofthis This article in accordance W. Colman, and 1 nmol/L ± no cardiopulmonary plasma Bing. obtained, University 0006-497l/89/7302-0013$3.00/0 468 5-9. this fact. by Grune and 1986. Temple Philadelphia, Center Philadelphia. and HL-36579 indicate contact & Stratton, human David activity Fund. December 68:342a, York; Department of Health nmol/L. 2 was be associated clinical contact, prekallikrein’9 of Medicine, September Institute HL-24365, Partnership Francisco, Address accepted in part at the American in Blood Research 1988; by National HL-19055, Presented form 22, in part. Harrison ofPennsylvania, there results cardiopulmonary Purified University Research New 2 ± from Clinical cardiopulmonary bypass (CCB) circuits contained a centrifugal pump (Biomedicus, Eden Praire, MN), bubble oxygenaton (BOS-102; Bentley Labs, Irvine, CA), aterial line filter (model I 331) and cardiotomy reservoirs (Extracorporeal, Valley Forge, PA). Simulated extracorporeal circulation (SECC) perfusion circuits (surface area of 0.9 m2) included a spinal coil membrane oxygenator model 0800-2A (surface area of 0.8 m2) and a venous reservoir (model R-500-4), and were obtained from Sci-Med Life Systems, Minneapolis. Silicone tubing was obtained from Dow Chemical, Midland, MI. Beef lung hepanin was obtained from Upjohn, Kalamazoo, MI. Linbro flat-bottomed 96 well polystyrene E.I.A. microtitration plates were obtained from Flow Laboratories, Inc, Handen, CN. Urokinase was obtained from Abbott Diagnostics, Dr Hematology- Department Center, Surgery, University January Supported, the Temple 21 rose a from plasmin-a2-plasmin-inhibitor and may Over rose Chicago. the classical the Thrombosis Medical of Cardiothoracic Hospital and Medicine, Sections, Hospital-Cornell HL-l8828, Center of Hematology-Oncology York gery, Research Department These during circulation. complexes to However. or in vitro by Grune molecular From nmol/L complexes nmol/L. fibrinolysis, duced Oncology I ± complement e 1989 kalli- aggregation whether extracorporeal MATERIALS hydrolases,9”#{176} the the azurophilic form stimulate 2 formation. Colman nists. by surface activation of plasma been identified as in vitro agonists all plex during a2-adren- recirculation.’2 Coagulation We is with circuit. of thromboxane contents.3 specific peptides proteolytic there decreased lysosomal lactoferrin,” pulmonary we abnormalities.2 secretion neutrophils release granule constituent, conditions, 5 ± W. Cl 8-C1 -inhibitor -inhibitor and plasma protein increase postoperative pulmonary receptors,7 fibrinogen of in vivo tion has been associated with major alterations of platelets, includreduced sensitivity to aggregating Extracorporeal agents,3’4 to simulated hours. Bypass levels URING CARDIOPULMONARY BYPASS extensive contact between blood anticoagulated heparin and synthetic surfaces of the extracorporeal alterations 25 not in vivo. their baseline classical within D Consequences in concentration to baseline in vitro of two either cardiopulmo- increased reverted Jr. and Robert period to assays increase clinical Edmunds, kallikrein-C1 complexes a modest after complexes Since “sandwich” -inhibitor Cardiopulmonary during a agonists. and During L. Henry during or fibrinolytic of there completed. of enzyme-inhibitor measured that complex occurs whether kallikrein-C1 each C. Harpel, investigated sources found of bypass We immunosorbent we levels Peter neutrophils contact. potential Cl 8-C1 -and respectively. 24 and bypass. enzyme-linked specific Complexes T. Wachtfogel, cardiopulmonary Kallikrein-CI-Inhibitor, to Simulated assembled extracorporeal from standard Blood, circulation. Perfusion silicone rubber components, Vol 73, No 2 (February), circuits were polycarbonate 1989: pp 468-471 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. ENZYME-INHIBITOR COMPLEXES connectors, and described.4 Blood carbon dioxide tons were hundred coil spiral and IN CARDIAC membrane oxygenators gas compartments for I 5 minutes primed by applying milliliters of blood before were priming a vacuum were (3.3 mg/mL blood). system before barely occlusive lengths with drawn from The carbon temperature was blood the samples reservoir by of 2.4 times Plasma during was the donor recirculation as described.’ described, measuring C1,-Cf-inhibitor,26 complex were values indicate differences the mean as kallikrein-CI-inhibitor,27 between (ANOVA) groups.2’ was When either Cl-inhibitor can be detected sonbent assays. Similarly, with a2-plasmin-inhibitor. cific assays occurs to document during inhibitor nmol/L hours plasmin We used forms three 10 ± postbypass (Fig a stable sensitive were after nmol/L by 60 minutes 24 postbypass hours were after 45 postby(data not extracorporeal complex During formed rose SECC, significantly from a baseline of 2 ± 1 nmol/L to 1 1 ± 2 the first 30 minutes, with a steady increase to by 120 minutes (Fig 2). During SECC, levels (P < .00001) nmol/L within 21 ± 2 nmol/L of kallikrein-CT-inhibitor cantly (P < .02) from first circulation. of C1,-CT-inhibitor two minutes to I 5 by a2-plasmin-inhibitor increased from complex formed increased signifia baseline of 2 ± 1 nmol/L within the 120 1 .9 of 2.4 with (Fig a steady 2). rise to 25 Levels of ± plasmin- . complex formed were not significantly I 1 nmol/L before recirculation, with ± ± 4 nmol/L, ± minutes 0.7 nmol/L by 120 minutes (data a not shown). activation of these enzymes is performed Stimulation neutrophils occurs interaction. prevent the The consequences of application of prolonged as a direct result in approximately of platelets and of the blood-surface this cellular extracorporeal activation recircu- 2 C Levels of C 1,-CTfound to be 12 ± 2 nmol/L 45 minutes ofbypass, 24 ± 4 postbypass, and 1). The concentration 8 ± I nmol/L by 24 of kallikrein- , Fl I bypass annually. complex and spe- bypass. 1 nmol/L by 60 minutes immuno- circulation. formed complex prebypass, the enzyme reacts complex that enzyme-linked cardiopulmonary 1.05 ± 2 nmol/L by Cardiopulmonary 200,000 Americans antibody whether extracorporeal Clinical 0.5 ± postby- DISCUSSION kallikrein or CI, is formed, to form an enzyme-inhibitor by double by 24 hours complexes 7.39 ± 8 ± 2 by 60 In all cases RESULTS with 2.2 1 1 .4 the SEM. ± 2 nmol/L ± prebypass, 5 nmol/L shown). maximum and plasmin-a2-plasmin-inhibitor25 complexes. Statistical analysis. An analysis of variance used to test for significant and pass, 7 prebypass, nmol/L of bypass, 5 nmol/L forma- performed 1.8 ± and ± 1). Plasmin-a2-plasmin-inhibitor Simulated ventilated from postbypass, (Fig levels various a rate bath. directly 9.5 shimmed, at 37#{176}C by immersing water pass minutes from the for at Assays for measuring plasma enzyme-inhibitor tion. Enzyme-linked immunosorbent assays ± reservoir were mixture either at various directly into circuit blood) and glucose oxygenators maintained drawn (who I .0 L/min bag in a constant-temperature from donors the Cl-inhibitor complex was 8 ± 2 nmol/L nmol/L after 45 minutes of bypass, 14 minutes Five a precisely of dioxide Oxygena- were removed by at a rate 100% gas port. random to enter recirculation up to 120 minutes. Blood previously permitted pump oxygen-5% the reservoir harvested or from the roller of time was with the circuit. to the lower and any air bubbles starting a 95% L/min. Blood foaming, without as previously flushed had abstained from any medications for 2 weeks) venous reservoirs containing hepanin (5.0 U/mL gravity 469 BYPASS I 2 ‘S 0 C . E 0 L’- E ‘C 0 0) I-) . .0 2O 120 E 316 J l2 , uTj :: 0 J-T ‘C ll6 ; l1’i:t T ‘0 ANESTHESLA #{128}P5R1N #{216}YPSS$ POST 20 40 60 80 100 120 POST Fig 1 . C1,-C1-inhibitor and kallikrein-C1-inhibitor complex formation during CCB. Plasma levels of C1,-C1 -inhibitor and kallikrein-C1 -inhibitor complexes formed are expressed as nmol/L. Each column represents the mean ± SEM of nine separate patients. TIME (mm) Fig 2. C1,-C1-inhibitor and kallikrein-C1-inhibitor formation during SECC. Plasma levels of C1,-C1-inhibitor kallikrein-C1 -inhibitor complexes formed are expressed L. Each point represents the mean ± SEM of eight recirculations. complex and as nmol/ separate From www.bloodjournal.org by guest on June 12, 2017. For personal use only. 470 WACHTFOGEL lation to the addition, membrane treatment of anticoagulation alterations much of bypass. the In mechanisms activation morbidity this of pathways pulmonary with chose to activation. In and result in we complexes lation occur also measured Levels contact, and/or fibribypass would pro- the in a well-defined system,4 in the which absence allows the of hepatic of the activation chemotaxin, occurs C5a.’7”8 to both complement,29 Since the active of C3a to implicate mask classical component classical levels during of the plasma is has led path- alternative complement of pathway must circulation in both and 2). It is apparent that full extent of the activation components coagulation, ulate human the plasma kallikrein elevated CCB Levels and SECC in vivo clearance acts that has occurred. intrinsic kallikrein and (Figs we cannot exclude CT,-CT-inhibitor Levels of factor of extracorporeal stim- complex circulation (Figs that postbypass of 8 level However, to 20 levels 4 nmol/L ± 1 nmol/L ± of both a2plasmininhibitor showed Thus, through directly contact coagulation XIIa and kallikrein, the factor directly.2#{176} through the classical potential to activate Kallikrein-CT-inhibitor the C1,-C1-inhibitor complement neutrophils. complexes 1 complexes bly due to a combination order rate inhibitor constant complexes C1,-C1-inhibitor the rate Second, is 1.30 complexes ofactivation form (Fig of two for much 2). This factors. the formation x i0 mol/L’s’ is 0.44 x for prekallikrein is accelerated by high molecular of CT by activated factor XII (C5a), weight is not. has faster effect First, of iO the by factor kininogen Therefore, the than is probasecond kallikrein-CTwhile that mol/L’s’.37 60 to postbypass a (Fig and 1). plasminrise at in vitro complexes complement undergoing is known pathway.38 cardiopulmonary to activate Moreover, bypass the two of 48 in a prospec- tive study, presented with a sudden rise of airway pressure, acute pulmonary hypertension, and systemic hypotension bubble or membrane levels within of C3a32’33’4’ the lungs.42 evidence oxygenators and causes Although of alternative sharply plasma pathway activation,43 most investiga- of C433’” and thus by the alternative the first evidence that cardiopulmoboth the contact and classical comple- ment pathways. Presumably both pathways are initiated by activation of factor XII on the surface of the bypass circuit. Activation suggests, of both but does contact the enzyme previously of for specific pathways definitively system are wholly neutrophil inhibitors help to delineate nary bypass. during cardiopulmonary prove, that components observed of factor the role or partially responsible degranulation.” XIIa, of these kallikrein, pathways bypass of the or for The use CT, may in cardiopulmo- ACKNOWLEDGMENT XIIa while that kallikrein increases sequestration of leukocytes Collett et al reported direct pathway. complement factor XII system, both and indirectly pathway completely com- no significant This study provides nary bypass activates classical activates not urokiactiva- returned kallikrein-C1-inhibitor complexes factor XII pathway.36 the also and/or system but by 24 hours tors do not observe a consistent reduction conclude that complement is activated activate contact did possibility in CCB. rose significantly within and 2) indicating that plasma prekallikrein has been converted to the active enzyme. Kallikrein activates factor XII to factor XIIa and subsequently to factor XII fragments’9; fragments Surface In shortly after protamine injection,39 consistent with activation of the complement and/or kinin forming systems.#{176} Previous work has shown that cardiopulmonary bypass with either Addiand aer- of kallikrein-C1-inhibitor as a result the contact XIIa, degranulate.’3’6 chemotaxis34 CT. in turn complex of classical patients, 1 to mask pathway to aggregate and induces neutrophil obic glycolysis.35 are of neutnophils tionally, in SECC, rin-protamine be considered, since levels of C1,-C1-inhibitor complex, unique to the classical system, are elevated as a result of the extracorporeal plasmin-a2-plasmin-inhibitor of which any time postbypass (Fig 1). The mechanism of activation of the complement cascade is unknown, but formation of hepa- now Two of normal of CS CCB that C4 and prevent detection of elevated levels. Thus, while we found no evidence of activation of the fibrinolytic system to cell pathways than plexes circu- to the and activation the way.3#{176}33However, the alternative elevation investigators in response rapid However, in CCB the rate of clearance may the production of plasmin-a2-plasmin-inhibitor tion. blood-surface interaction or reticuloendothelial more products, such as tissue plasminogen activator nase, may explain the absence of fibrinolytic above extracorporeal probably in either CCB or SECC. This rules out fibrinolysis SECC. In this system the lack of endothelial cell minutes clearance. Neutrophil common formation simulated is C1 must then activate C2 and C3 and finally CS to form CSa. change during duce agonists capable of causing neutrophil degranulation. Due to the probable in vivo clearance of the enzyme-inhibitor complexes, formation addition, activate extracorporeal examine the possible We postulated that complement, cardiopulmonary during injury. degranulation, neutrophils associated study we neutrophil of the classical nolytic severe with heparin, of platelets and ET AL The authors wish to thank John Greenplate for expert technical assistance. REFERENCES 1. Hanker LA, Malpass TW, Branson HE, Hessel EA II, Slichter Si: Mechanism of abnormal bleeding in patients undergoing cardio- Rao AK, Addonizio VP ir, Stephenson LW, function during cardiac operation. i Thorac pulmonary 83:805, bypass: Acquired transient platelet dysfunction asso- release. Blood 56:824, 1980 2. Neville WE, Kontaxis A, Gavin T, Clowes GHA in: Postperfusion pulmonary vasculitis. Arch Sung 86:126, 1963 3. Edmunds LH in, Ellison N, Colman RW, Niewiarowski 5, ciated with selective a-granule 4. Edie RN: Cardiovasc Platelet Sung 1982 VL ir, Hicks RE, Niewiarowski 5, Edmunds LH Jr, RW: Function of human platelets during extracorponeal circulation. Am i Physiol 232:H622, 1977 5. Dutton RC, Edmunds LH in, Hutchinson JC, Roe BB: Platelet Colman Hennessy From www.bloodjournal.org by guest on June 12, 2017. For personal use only. ENZYME-INHIBITOR aggregate COMPLEXES emboli bypass produced IN CARDIAC in patients BYPASS during 471 cardiopulmonary membrane and bubble oxygenators and blood filters. J Thorac Cardiovasc Sung 67:258, 1974 6. Wachtfogel YT, Musial I, ienkin B, Niewiarowski 5, Edmunds LH Jr. Colman RW: Loss of platelet a2-adrenergic receptors during simulated extracorporeal circulation: Prevention with prostaglandin E,. J Lab Clin Med 105:601, 1985 7. Musial i, Niewiarowski 5, Hershock D, Morinelli TA, Colman RW, Edmunds LH ir: Loss of fibninogen receptors from the platelet surface during simulated extracorporeal circulation. 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EW: B2 levels 61 :808, 1980 Gnanadurai Lysosomal M, Salzman Sobel thromboxane utilizing a new assay for a2-macnoglobulin-kallikrein complexes. Biol Chem 260:4257, 1985 26. Hanpel PC, Cooper NR: Circulating C1-inactivator-CT,-CTr of plasma monary in 1982 plasma Jones HM, bypass Matthews N, Vaughan RS, Stark iM: Cardiopuland complement activation. Anaesthesia 37:629, From www.bloodjournal.org by guest on June 12, 2017. For personal use only. 1989 73: 468-471 Formation of C1s-C1-inhibitor, kallikrein-C1-inhibitor, and plasmin- alpha 2-plasmin-inhibitor complexes during cardiopulmonary bypass YT Wachtfogel, PC Harpel, LH Jr Edmunds and RW Colman Updated information and services can be found at: http://www.bloodjournal.org/content/73/2/468.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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