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J Thorac Cardiovasc Surg 1998;115:1179-1188
© 1998 Mosby, Inc.

CARDIOPULMONARY SUPPORT AND PHYSIOLOGY

Heparinless cardiopulmonary bypass with active-site blocked factorIXa: A preliminary study on the dog

Talia B. Spanier, MDa, Mehmet C. Oz, MDa, Oktavijan P. Minanov, MDa, Ronit Simantov, MDe, Walter Kisiel, PhDd, David M. Stern, MDa,b, Eric A. Rose, MDa, Ann Marie Schmidt, MDa,c

From the Departments of Surgery,a Physiology,band Medicine,c Columbia University College of Physicians andSurgeons, New York, N.Y., the Department of Pathology,d Universityof New Mexico, Albuquerque, N.M., and the Division of Hematology/Oncology,eCornell University Medical College, New York, N.Y.

Received for publication July 30, 1997. Revisions requested Sept. 11, 1997. Revisions received Dec. 18, 1997. Accepted for publication Dec. 19, 1997. Address for reprints: Talia Spanier, MD/Ann Marie Schmidt, MD,Columbia University College of Physicians and Surgeons, 630 West 168 St., P&S17-501, New York, NY 10032.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Appendix: Preparation and...
 References
 
Objective: Cardiopulmonary bypass is apotent stimulus for activation of procoagulant pathways. Heparin, thetraditional antithrombotic agent, however, is often associated with increasedperioperative blood loss because of its multiple sites of action in thecoagulation cascade and its antiplatelet and profibrinolytic effects.Furthermore, heparin-mediated immunologic reactions (that is, heparin-inducedthrombocytopenia) may contraindicate its use. Cardiopulmonary bypass with aselective factor IXa inhibitor was tested to see whether it could effectivelylimit bypass circuit/intravascular space thrombosis while decreasingextravascular bleeding, thereby providing an alternative anticoagulant strategywhen heparin may not be safely administered.
Methods:Active site-blocked factor IXa, a competitive inhibitor of the assembly offactor IXa into the factor X activation complex, was prepared by modification ofthe enzyme's active site by the use of dansyl glutamicacid-glycine-arginine-chlormethylketone. Twenty mongrel dogs (five were givenstandard heparin/protamine; 15 were given activated site-blocked factor IXadoses ranging from 300 to 600 µg/kg) underwent 1 hour of hypothermiccardiopulmonary bypass, and blood loss was monitored for 3 hours after theprocedure.
Results: Use of activatedsite-blocked factor IXa as an anticoagulant in cardiopulmonary bypass limitedfibrin deposition within the extracorporeal circuit as assessed by scanningelectron microscopy, comparable with the antithrombotic effect seen withheparin. In contrast to heparin, effective antithrombotic doses of activatedsite-blocked factor IXa significantly diminished blood loss in the thoraciccavity and in an abdominal incisional bleeding model.
Conclusion:These initial studies on the dog suggest that administration of activatedsite-blocked factor IXa may be an effective alternative anticoagulant strategyin cardiopulmonary bypass when heparin is contraindicated, affording inhibitionof intravascular/extracorporeal circuit thrombosis with enhanced hemostasis inthe surgical wound.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Appendix: Preparation and...
 References
 
Cardiopulmonary bypass (CPB) presents a substantial prothromboticstimulus in cardiac operations; thus its successful performance requires potentanti-coagulation.Go 1 Heparin,the traditional antithrombotic agent, however, is not without untoward sideeffects. Its multiple sites of action in the coagulation cascade and itsprofibrinolytic and antiplatelet effects contribute to an adverse bleedingdiathesis and potential morbidity and death in many patients undergoing CPB.Go Go 1-4Furthermore, complications associated with the use of heparin, includingheparin-induced thrombocytopenia with potential for pathologic thrombosis, alsorender its use contraindicated in a subset of patients. The need forpharmacologic reversal of heparin effect with protamine may itself contribute toadditional perioperative morbidity with the possibility of life-threateningprotamine allergy and subsequent hemodynamic collapse.Go Go 5,6

Although multiple strategies to perform CPB in the absence of heparinhave been suggested (low-molecular weight heparins, thrombin inhibitors,iloprost, ancrod, orgaran, or dermatan sulfate), none are routinely used in theclinics in the United States, likely because their use has been associated withsuch complications as potential for sensitization, cross-reactivity withheparin, profound hemodynamic instability, or excessive bleeding.Go Go 7-13Other strategies, such as addition of antifibrinolytic agents, although reducingblood loss, nevertheless do not obviate the use of heparin/protamine.Go Go 14-16

In situations where the use of heparin is relatively contraindicated, wehave sought an alternative anticoagulant that would selectively inhibitintravascular coagulation, resulting from the contact of circulating blood withextracorporeal membranes, filters, and tubing, while enhancing extravascularhemostasis with decreased blood loss in the surgical wound. These considerationsled us to focus on the role and unique location of factor IX/IXa in thecoagulation cascade.

Factor IXa expresses its procoagulant activity after incorporation intothe intrinsic factor X activation complex, consisting of factors IXa, VIII(a)assembled on an appropriate cellular surface in the presence of calcium ions.This complex then activates factor X, and the newly formed factor Xa thenbecomes integrated into the prothrombinase complex, thereby generating thrombin.In the presence of relatively low levels of tissue factor, such as that presentin the intravascular space/extracorporeal circuitry during the initial phase ofCPB, inhibition of the participation of factor IX/IXa would prevent activationof coagulation mediated by both the tissue factor–factor VIIa and contact(intrinsic) pathways. However, in the presence of high levels of tissue factor,such as that present on the cut surface of the sternum and in the pericardium,the tissue factor–factor VIIa pathway would directly activate factor X,thus resulting in the generation of thrombin and clot formation in the surgicalwound.Go 17

Because selective reversible inhibitors of factor IXa have not beendeveloped, we prepared active site-blocked factor IXa (IXai), in which theactive site residues serine 376 and histidine 221 were acetylated and alkylated,respectively, with dansyl-Glu-Gly-Arg chloromethylketone, and used this agent asa competitive inhibitor of the assembly of factor IXa into the intrinsic factorX activation complex.Go Go 18,19 Indeed, IXai has already beentested as a selective anticoagulant in an electric-current canine coronarythrombosis model.Go Go 20,21 In those studies, comparedwith dogs receiving active site-blocked Xa, dogs receiving IXai manifestedinhibition of intravascular thrombosis with preservation of extravascularhemostasis (as demonstrated in an incisional abdominal wound model).

In this report, we demonstrate that administration of IXai in canine CPBresults in the inhibition of intravascular thrombosis within the extracorporealcircuit, with diminished bleeding tendency within the surgical wound. These datasuggest that use of IXai may be a potential alternative anticoagulant strategyin CPB, especially in situations where the use of heparin is relativelycontraindicated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Appendix: Preparation and...
 References
 
Preparation and purification of IXa/IXai
Factor IXai was prepared and characterized as described in the appendix.

CPB in dogs
Twenty mongrel dogs (27 to 35 kg) underwent CPB for 1 hour afterinduction of anesthesia with isoflurane and appropriate endotrachealventilation. The heart was suspended in a pericardial cradle after mediansternotomy. Anticoagulant (either factor IXai [range 300 to 600 µg/kg)] orheparin 300 IU/kg) was infused intraatrially; CPB was established withcannulation of the right atrial appendage and ascending aorta (70 ml/kg/min, 32°C, mean arterial pressure 55 to 70 mm Hg).

CPB with a roller head pump (Stockert Instrumente, Munich, Germany)primed with lactated Ringer's solution (1 liter) and a Cobe membrane lungoxygenator (Cobe Cardiovascular, Inc., Arvada, Colo.) with sterile surgicalgrade Tygon tubing/filters (Norton Performance Plastics, Akron, Ohio) wasmaintained for 1 hour. The aorta was crossclamped, and the heart was arrestedwith cold crystalloid cardioplegic solution. An aortotomy was performed tosimulate an aortic valve repair. Throughout the procedure a left atrial ventcatheter was attached to a cardiotomy sucker. The pericardium was continuouslyevacuated through an additional cardiotomy sucker so as not to allow pericardialpooling of blood. The aortotomy was subsequently repaired after appropriatedeairing; the heart was rewarmed, and the aortic crossclamp was released. Dogswere then weaned from CPB and decannulated.

In dogs treated with heparin, protamine (2 mg/kg) was administeredintravenously at the termination of CPB. After cessation of CPB, animals wereobserved for up to 3 hours to measure the extent of blood loss. At hourlyintervals after bypass, blood that pooled in the pericardial and pleural spaceswas suctioned, and the amount obtained was carefully quantitated and recorded.Continuous hemodynamic measurements were made and recorded before, during, andup to 3 hours after institution of CPB. One surgeon performed all of theexperiments; no modifications of the operating technique were made throughoutthe study.

The experimental animals were divided into five groups. Group 1 (n = 5), the control group, received standard dosesof heparin/protamine (300 IU/kg and 2 mg/kg, respectively). Animals treated withIXai received a single dose of IXai (group 2 [n =3], 300 µg/kg; group 3 [n = 3], 360µg/kg; group 4 [n = 6], 460 µg/kg;and group 5 [n = 3], 600 µg/kg). Nopharmacologic reversal was administered to animals treated with IXai.

All animals received humane care in compliance with the "Principlesof Laboratory Animal Care" formulated by the National Society for MedicalResearch and the "Guide for the Care and Use of Laboratory Animals"prepared by the National Academy of Sciences and published by the NationalInstitute of Health (NIH publication 86-23, revised 1985). This study wasapproved by the Columbia University Institutional Animal Review Committee andwas conducted according to Columbia University policy.

Assessment of fibrin deposition in the CPB tubing and filters
At termination of CPB, the tubing and filters were removed and subjectedto analysis by scanning electron microscopy as previously described.Go 22

Analysis of blood samples and tissue
Routine hematologic analysis was performed before the initiation of CPB,every 15 minutes during CPB, and then at hourly intervals after CPB to determinehemoglobin and hematocrit values, levels of platelets and fibrinogen, whiteblood cell count, prothrombin time, activated partial thromboplastin time, andactivated clotting time (ACT; International Technidyne, Edison, N.J.). Celite(Celite Corporation, Quincy, Mass.) ACT was evaluated on a Hemachron model 801(International Technidyne, Edison, N.J.). After the dogs were killed, necropsywas performed; the heart, lungs, liver, and kidney were removed, fixed informalin (10%), and examined by histochemistry with hematoxylin and eosinfor evidence of clot/fibrin deposition and the presence of microemboli orbleeding.

Platelet aggregation studies
Platelet-rich plasma was isolated from blood collected from a normalvolunteer (no aspirin intake for at least >7 days), and platelet aggregationwas measured in response to epinephrine (100 µm) or collagen (190 µg/ml)on a PAP 4A aggregometer (Bio/Data, Horsham, Pa.) as previously describedGo 23 in the presence of variousconcentrations of IXai, heparin, or a combination of IXai/heparin.

Assessment of extravascular bleeding tendency
The bleeding tendency at extravascular sites was assessed with a modifiedincisional bleeding time as previously described.Go 20 Briefly, a uniform abdominal wallincision (1 cm deep, 5 cm long) was made, and a preweighed 4 x 4 inchgauze was inserted for 5 minutes. The gauze was then removed and reweighed, andthe weight of blood loss quantitated. Measurements were taken at baseline,immediately after institution of CPB, and at one-half hour intervals until theanimal was killed.

The modified cephalin clotting time (MCCT)
To detect levels of IXai in plasma as a means of monitoring its use inCPB, we developed a rapid, reproducible test using factor IX deficient plasma,Celite diatomaceous earth, barbital buffer, calcium chloride, cephalin, andstandard dog plasma (Sigma Chemical Co., St. Louis, Mo.). Factor IX-deficientplasma (0.1 ml) was mixed with Celite (0.024 mol/L) in barbital buffer (0.05mol/L) in silicone-coated glass tubes for 2 minutes in a shaking water bath at37° C (Fig. 1, A). To this mixture was added an optimizedconcentration of cephalin (1:32) in barbital buffer (0.05 mol/L) (Fig. 1,BGo), control or test-dogplasma (0.05 ml), and lastly, calcium chloride (0.001 mol/L). Time to clotformation was then visually measured for each point. The limit of detection ofIXai in this assay was 0.06 µg/ml (Fig. 1, CGo).



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  		Fig. 1. MCCT.A, Assay procedure. Factor IX-deficient plasmawas incubated with Celite (0.024 mol/L in barbital buffer [0.05 mol/L]) insilicone-coated glass tubes for 2 minutes in a shaking water bath at 37° C.An optimized concentration of cephalin (1:32; B)in barbital buffer (0.05 mol/L) was added, along with control or test-dog plasma(0.05 ml) and calcium chloride (0.001 mol/L). Time to clot formation was thenvisually determined. C, Determination of thelimit of detection of the MCCT was performed with different concentrations ofIXai and normal dog plasma. Data were plotted as log MCCT versus log [IXai] ofeach value. The limit of detection in this assay is 0.06 µg/ml.

 
Statistical analysis
Data are expressed as the mean ± standard error. Serial datafrom each group were evaluated by the repeated measures analysis of variance andthen by paired t test with the use of InStat2.01 statistical analysis program (Graphpad Software, San Diego, Calif.) on aMacintosh computer (Apple Computer, Cupertino, Calif.).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Appendix: Preparation and...
 References
 
IXai was prepared and purified from a mixture of the human vitamin-K–dependentcoagulation factors (factors II, VII, IX, and X; Proplex; BaxterPharmaceuticals, Duarte, Calif.). Factor IX was first purified from thismaterial and migrated as a single band on sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) in the absence or presence of mercaptoethanol (10%)with an apparent relative molecular mass of approximately 68 kDa (Fig. 2,lanes 1 and 2,respectively). On activation of factor IX with XIa,purified factor IXa migrated as a single band on nonreduced SDS-PAGE gels, witha relative molecular mass of approximately 45 kDa, and as two bands,corresponding to the heavy and light chains of factor IXaß on reduced gels(Fig. 2Go, lanes 3 and5, respectively). After inactivation withdansyl Glu-Gly-Arg chloromethylketone and extensive dialysis, the resultingfactor IXai, devoid of procoagulant activity, migrated identically to IXa onSDS-PAGE (Fig. 2Go, lanes 4[nonreduced] and 6 [reduced]). Subsequent to removal of detectable levels ofendotoxin, IXai was then tested in canine CPB.



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  		Fig. 2. Purification of factorIXai from Proplex. Factor IX was purified from Proplex and activated with factorXIa as described. IXa was then reacted withdansyl-Glu-Gly-Arg-chloromethylketone to yield IXai. Factors IX, IXa, and IXaiwere run on SDS-PAGE under nonreducing or reducing conditions as indicated: Lane1 (10 µg); lane 2 (10 µg); lane 3 (10 µg); lane 4 (15 µg);lane 5 (10 µg); and lane 6 (15 µg).

 
CPB was performed in 20 dogs for 1 hour at 32° C with standardheparin/protamine or varying doses of IXai as an anticoagulant. Compared withdogs receiving heparin (n = 5), dogsreceiving IXai (460 µg/kg [n = 6]and 600 µg/kg [n = 3]) demonstratedsimilar hemodynamic profiles (70 ml/kg/min flow, 32° C, 55 to 70 mm Hg meanarterial pressure), with no evidence of increased pressures in the CPB tubing orcircuitry at any time during the procedure. In addition, no visible clot wasnoted in the circuity. However, in dogs treated with the lowest dose of IXai(300 µg/kg [n = 3]), a visible clotformed in the bypass tubing along with the development of excess line pressures,thereby necessitating premature termination of CPB. In dogs treated with IXai(360 µg/kg [n = 3]), although avisible clot formed in the CPB circuit/tubing, CPB was successfully maintainedfor 1 hour with no increased line pressures. In a limited number of animalstreated with the optimal antithrombotic dose of IXai (460 µg/kg; 6 dogs),CPB was extended to 1.5 hours to demonstrate the feasibility of the use of IXaiover this time period. Similar results were found when compared to 1 hour of CPB(data not shown).

To detect fibrin or platelet deposition within the bypass circuit, thetubing and filters were removed immediately on the termination of CPB andanalyzed by scanning electron microscopy. CPB performed with heparin or IXai(460 µg/kg or 600 µg/kg) resulted in similar levels of fibrindeposition (Fig. 3, left panel). However, at 360 µg/kg IXai,significantly increased fibrin and platelet deposition was noted in the arterialfilter (Fig. 3Go, right panel).



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  		Fig. 3. Scanning electronmicroscopy of the CPB filters. Dogs were treated with IXai (460 µg/kg) orheparin (300 IU/kg/protamine 2 mg/kg) and underwent CPB for 1 hour. At the endof CPB, the filters were removed and subjected to scanning electron microscopyat the indicated magnifications. CPB performed with IXai (460 µg/kg) wasassociated with similar amounts of clinically inapparent fibrin deposition,compared with heparin (left panel). At 360 µg/kg IXai, visible fibrin andplatelet deposition was observed (right panel).

 
At an effective antithrombotic dose of IXai (460 µg/kg), no adverseeffects were noted in the visceral organs. Three hours after termination of CPB,necropsy revealed no gross or microscopic thrombosis or bleeding in the heart,lungs, liver, or kidney in animals treated with IXai compared with thosereceiving heparin (data not shown).

We had hypothesized that an important distinction between IXai andheparin in CPB would be the divergent effects of these agents on extravascularhemostasis. Consistent with this hypothesis, in contrast with heparin-treatedanimals, a hemostatic clot was observed along the cut surface of the sternum andin surgical tissue planes throughout the procedure in IXai-treated animals.Furthermore, blood loss in the thoracic cavity was diminished in dogs treatedwith IXai (in dogs treated with IXai, 360 µg/kg, 520 ± 53 ml;in dogs treated with IXai, 460 µg/kg, 520 ± 60 ml) comparedwith dogs receiving standard heparin/protamine (1275 ± 115 ml;p < 0.05). However, at 600 µg/kgIXai, an increased bleeding tendency was observed that was similar to that foundin animals treated with heparin (950 ± 300 ml; p =0.2) (Fig. 4), suggesting a dose-dependent effect.



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  		Fig. 4. Thoracic-cavity bloodloss in dogs undergoing CPB. Dogs underwent CPB with either IXai (300 to 600µg/kg) or heparin/protamine for 1 hour; dogs were then observed for 3hours after discontinuation of CPB. At the end of the observation period, dogsreceiving heparin accumulated 1275 ± 115 ml blood in the thoraciccavity; those dogs receiving either 360 µg/kg or 460 µg/kg IXaiaccumulated significantly less blood in the thoracic cavity (530  ±55 ml and 520 ± 30 ml, respectively). At 600 µ/kg IXai,increased bleeding was observed in the thoracic cavity (950 ± 300ml). *Indicates p < 0.05 in dogstreated with IXai compared with heparin by analysis of variance.

 
Further assessment of the extravascular bleeding tendency in dogs treatedwith IXai versus heparin was demonstrated in an abdominal incision model,essentially a modified bleeding time. On administration of IXai (460 µg/kg),significantly decreased bleeding was observed compared with animals treated withstandard doses of heparin (Fig. 5). In contrast, treatment with 600 µg/kgof IXai resulted in bleeding similar to that observed with heparin (Fig. 5Go). Taken together, these data suggest that compared withheparin, extravascular bleeding tendency is diminished in dogs treated witheffective antithrombotic doses of IXai.



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  		Fig. 5. Incisional wound model.To establish the effects of IXai on bleeding outside the operative field, astandardized incisional model in the abdomen was used as described earlier. Atan effective antithrombotic dose of IXai (460 µg/kg), there was noevidence of enhanced bleeding at the time points assessed compared to baseline.In contrast, significantly increased bleeding was observed after infusion ofheparin during CPB, which returned to baseline after heparin reversal withprotamine. At a dose of IXai of 600 µg/kg, significantly increasedbleeding was observed during CPB, comparable to that seen with heparintreatment. Data are reported as means ± standard error. *Indicatesp < 0.05 in dogs treated with IXaicompared with heparin by analysis of variance.

 
Use of IXai as an anticoagulant in CPB did not adversely affect routinelymeasured hematologic values. Compared with heparin, in dogs receiving effectiveantithrombotic doses of IXai (460 µg/kg), no untoward effects wereobserved on either white blood cell count or hematocrit values (Fig. 6,A and B). Levels of platelets were notsignificantly different in dogs treated with IXai or heparin, although a trendwas observed toward less variability in dogs treated with IXai (Fig. 6,C).



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  		Fig. 6. Hematologic analysis.Dogs underwent CPB with either IXai (460 µg/kg) or heparin/protamine. Atthe indicated times, blood was withdrawn for analysis of white blood cell count(A), hematocrit value (B),and platelets(C). No significant differences were observedbetween groups. Each group represents mean values observed in four dogs.

 


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  		Fig. 6, cont'd. D, Plateletaggregation studies as described were performed on platelet-rich plasma fromnormal volunteers in response to epinephrine (100 µm) in the presence ofvarious concentrations of IXai: lane 1, 5.0 µg/ml; lane 2, 1.0 µg/ml;lane 3, 0.8 µg/ml; lane 4, 0.4 µg/ml; lane 5, 0.2 µg/ml; andlane 6, 0.1 µg/ml; or in the presence of IXai/heparin: lane 7, IXai 0.4µg/ml/heparin 9 U/ml; or heparin alone: lane 8, 13.5 U/ml; and lane 9, 9U/ml. Arrow indicates the point at which the agonist (epinephrine) was added. Inall cases, primary and secondary waves of aggregation were normal.

 
Platelet aggregation studies performed in the presence of increasingconcentrations of IXai (0.1 to 5.0 µg/ml) or IXai (0.4 µg/ml) incombination with heparin (9 U/ml) revealed no abnormalities in primary orsecondary wave of aggregation in response to either epinephrine (Fig. 6,D) or collagen (datanot shown). In addition, no spontaneous aggregation was observed in the presenceof IXai (0.1 to 5.0 µg/ml) or heparin/IXai (9 U/ml and 0.4 µg/ml,respectively; data not shown).

Coagulation assays revealed that compared to dogs treated with heparin,dogs treated with IXai (460 µg/kg) demonstrated no significant increase inprothrombin time (Fig. 7, A), activated partialthromboplastin time (Fig. 7, B), or ACT (Fig.7, C). These findings are most likely explainedby the lack of sensitivity of these assays for assessment of effectiveantithrombotic doses of IXai. However, to realistically use IXai in CPB, itwould be important to be able to rapidly and reproducibly detect theantithrombotic activity of IXai. We therefore developed the MCCT as describedearlier. Compared with preoperative values (MCCT of 21  ± 1.2seconds), after a single dose of IXai immediately before CPB (460 µg/kg),MCCT rose to approximately 80 seconds through at least 1 hour of CPB andreturned to baseline by 3 hours after CPB (Fig. 8). MCCT for heparin-treated dogs was morethan 100 seconds throughout bypass, returning to baseline after protamineadministration (Fig. 8Go). Similar results were found indogs treated with IXai that did not undergo any surgical procedure, with afourfold increase in MCCT after drug administration, which returned to baselineby 3 hours after administration (data not shown).



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  		Fig. 7. Analysis of(A) prothrombin time (PT),(B) activated partial thromboplastin time(PTT), and (C)ACTin dogs undergoing CPB. Blood was withdrawn at the indicated times, andcitrated plasma was prepared. PT remained control in dogs treated with IXai (460µg/kg) and activated PTT was mildly elevated. In contrast, dogs treatedwith heparin/protamine demonstrated significant elevations of PT andactivated PTT. ACT values (117 ± 32.0 seconds at baseline in alldogs), which returned to baseline after the administration of protamine, rose to>480 seconds during CPB in the dogs treated with heparin. In contrast, indogs treated with IXai (460 µg/kg), ACT did not rise significantlyabove baseline (133 ± 23.0 seconds). *Indicatesp < 0.05 in dogs treated with IXaicompared with heparin by analysis of variance.

 


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  		Fig. 8. Determination of theMCCT in CPB. Dogs were placed on CPB with an effective antithrombotic dose ofIXai (460 µg/kg), and MCCT was measured throughout the procedure. Baseline(preoperative) MCCT was 21 ± 1.2 seconds, which rose to 80 secondsafter administration of IXai. Levels returned to baseline by 3 hours after CPB.Similarly, dogs were placed on bypass with heparin and reversed after bypasswith protamine.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Appendix: Preparation and...
 References
 
Multiple stimuli converge in CPB to present a potent prothromboticstimulus.Go 1 The use of heparinanticoagulation successfully prevents intravascular/extracorporeal circuitthrombosis at the expense, however, of impaired hemostasis in the surgicalwound. In these studies, we demonstrated in a canine model of CPB thatprevention of the assembly of factor IXa into the factor IXa-VIIIa-X complex(factor X activation complex) on an appropriate cell surface effectively permitsflow of blood over the extracorporeal circuits without increased pressures orthrombus formation within the intravascular space/extracorporeal circulation. Incontrast to heparin, however, these data suggest that targeted inhibition of theparticipation of factor IXa in the procoagulant pathways enhances hemostasis inthe surgical wound, with diminished blood loss in the thoracic cavity and in anincisional abdominal wound model.

Despite the complex set of intrinsic (contact)/extrinsic (tissue factor)–mediatedpathway interactions that together underlie the coagulopathy of CPB, the uniqueposition of IX/IXa in the clotting cascade may render it an ideal target forselective antithrombotic intervention in CPB. Indeed, multiple studiesGo Go 24-26have indicated an important role for the contact system in CPB, as would beexpected on the intimate contact of circulating blood with the extracorporealcircuitry: (1) reduced levels of factor XII indicate early activation of theclotting system in CPB; (2) levels of factor XIIa and factor XIIa activity areelevated in the plasma of patients undergoing CPB; and (3) factor XII/XIIabecome localized on tubing in the bypass circuit. An important role for theextrinsic pathway in the coagulation abnormalities observed in CPB has also beensuggested: low levels of tissue factor expressed by monocytes that have adheredto the bypass circuitry (after 2 hours) may result in selective activation of IXby the tissue factor-VIIa pathway in that setting, likely to be effectivelyinhibited by IXai.Go 27Furthermore, given the progressively increased expression of endothelial tissuefactor from the luminal to the adventitial side of the blood vessel, high levelsof tissue factor in the surgical wound have critically limited access to theintravascular space/extracorporeal circulation, thus precluding intravascularthrombosis, while effecting tissue factor-VIIa-mediated activation of theextrinsic pathway and extravascular hemostasis, a process not affected by IXai.Go Go 28,29Indeed, recently it has been suggested that a major procoagulant perturbation incardiac surgery/CPB is the wound itself, with high levels of tissue factor notedin the extravascular space.Go 30

In conclusion, although we performed this procedure in a limited numberof dogs, these initial data suggest that active site-blocked factor IXa mightrepresent an alternative antithrombotic strategy in CPB, in which decreasedbleeding tendency in the surgical wound results in the absence of apparentintravascular/extracorporeal circuit thrombosis. Studies in baboons are inprogress to delineate site- and time-dependent activation of coagulationpathways in CPB performed with IXai. In this nonhuman primate model, the use ofsensitive and specific human-based reagents, which will delineate the extent ofactivation of coagulation and fibrinolytic pathways, will be invaluable indetermining the potential efficacy of IXai as a clinical anticoagulant strategy.


   Appendix: Preparation and purification of IXa/IXai
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix: Preparation and...
References
 
Factor IXai was prepared by applying Proplex (a mixture of the human vitamin-K dependent coagulation factors [factors II, VII, IX and X] supplied by Dr. Roger Lundblad, Baxter Pharmaceuticals, Duarte, Calif.), reconstituted in TRIS-buffered saline solution (TBS; pH 7.5) containing CaCl2 to a column of calcium-dependent anti-human factor IX monoclonal antibody (CaFIX-1) coupled to Affi-Gel 10 (BioRad Laboratories, Hercules, Calif.) equilibrated at 4° C with TBS containing CaCl2 (0.01 mol/L). After sample application, the column was washed extensively with TBS containing CaCl2 (0.01 mol/L) and NaCl (0.5 mol/L), and factor IX was subsequently eluted in TRIS-HCl (0.1 mol/L; pH 8.0) containing EDTA (0.03 mol/L). Minimal residual contaminants were then removed with Q-Sepharose Fast Flow chromatography (Pharmacia Biotech, Inc., Piscataway, N.J.). Purified factor IX (Fig. 2Go) was then activated at 37° C by incubation with purified human factor XIa (1:1000 enzyme:substrate ratio)Go 31 in TRIS-HCl (0.05 mol/L; pH 8.0) containing CaCl2 (0.005 mol/L) for 1 hour. Factor IX (Fig. 2Go) was then activated in the presence of factor XIa, and the IXa thus formed was reacted with a 100-fold molar excess of dansyl-Glu-Gly-Arg chloromethylketone for 3 hours at 37° C. Dansyl-Glu-Gly-Arg chloromethylketone was prepared in HCl (0.01 mol/L) at a concentration of approximately 5 mg/ml and incubated with factor IXa in TBS (final pH of the solution; 7.5). After incubation, the mixture was dialyzed overnight at 4° C versus 20,000 volumes of phosphate-buffered saline solution. The final product, IXai, devoid of procoagulant activity, migrated identically to IXa on SDS-PAGE (Fig. 2Go) and was used for experiments after filtration (0.2 µm) and chromatography on DeToxi-gel columns (Pierce, Rockford, Ill.). These preparations had no detectable lipopolysaccharide at a protein concentration of 1 to 2 mg/ml, with the Limulus amebocyte assay (Sigma, St. Louis, Mo.).


   Acknowledgments
 
We thank Mary Lynn Gaddis at Baxter Healthcare Systems(Irvine, Calif.) and Dr. Steven McCormick (New York Eye and Ear Hospital, NewYork, N.Y.) for performing scanning electron microscopy on the arterial filtersprocessed after canine CPB.


   Footnotes
 
Mehmet Oz, MD, is an Irving Fellow of Columbia University.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix: Preparation and...
 References
 

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