Use of Human Factor Vlla in the Treatment of Two

Hemophilia A Patients with High-Titer Inhibitors

ULLA HEDNERand WALTERKISIEL, Department for Coagulation Disorders,

University of Lund, Malmo, Sweden; Department of Biochemistry,

University of Washington, Seattle, Washington 98195

A B S T R A C T Two patients with hemophilia A com-plicated with high-titer alloantibodies have been treatedby repeated infusions of microgram quantities of purehuman Factor VIIa. Patient 1 was presented with agastrocnemius muscle bleeding that involved the kneejoint. Upon treatment with Factor VIIa the circum-ference of the muscle decreased and joint mobilityincreased substantially. Patient 2 was given FactorVIla concurrent with tranexamic acid in associationwith the extraction of two primary molars. No signif-icant gingival bleeding occurred after Factor VIla andtranexamic acid treatment. Furthermore, no delete-rious side effects or increase of the alloantibody levelwere observed in either patient throughout the FactorVITa infusion. These results, although limited and pre-liminary in nature, suggest that trace quantities ofFactor VIIa can act as a Factor VIII bypassing activityand restore hemostasis in these patients.

INTRODUCTIONPatients with severe hemophilia A complicated withhigh titers of antibodies against Factor VIII:CAg rep-resent a serious therapeutic problem. Recently, the useconcentrates of the unactivated and activated pro-thrombin-complex have met with limited success insome cases (1-5), while in others, evidence of dissem-inated intravascular coagulation or thromboemboliccomplications were observed (6-8). The putative he-mostatic agent(s) in these preparations has never beenidentified, although Factor VIIa has been suggestedas one possibility (9). Factor Vlla has an absolute re-quirement for tissue factor for expression of its pro-teolytic activity. In addition, Factor VIIa is not inhib-ited to any significant extent by antithrombin III inthe absence of heparin (10). Thus, it is possible thatupon administration, Factor VIIa may find its way

Received for publication 16 November 1982 and in re-vised form 15 February 1983.

unabated to the site of trauma, complex specificallywith tissue factor at the site, and rapidly initiate theformation of thrombin by the extrinsic pathway. Thismechanism would "by-pass" the intrinsic pathway,which requires normal levels of Factor VIII and FactorIX. With homogeneous preparations of Factor VIIa,our goal was to test this hypothesis in hemophilia Apatients complicated with alloantibodies. We reportherein the treatment of two patients with severe he-mophilia A with alloantibodies against Factor VIIILCAgby the inf usion of microgram quantities of homoge-neous preparation of human Factor VIIa.

METHODS

Coagulation assaysActivated partial thromboplastin time (General Diagnos-

tics Automated APTT, General Diagnostics, Div. of WarnerLambert Co., Morris Plains, NJ) was performed accordingto the manufacturer's specifications. Factor XII, Factor XI,Factor V, Owren's P + P (prothrombin plus Factor VII plusFactor X), fibrinogen, and fibrinogen/fibrin degradationproducts were assayed as previously described (11). Pro-thrombin time, thrombin time, and reptilase time were per-formed as described (11). Factor VIII coagulant activity(VIII:C) and Factor VIII-related antigen (VIII:RAg) wereassayed according to Nilsson (12) and Holmberg and Nilsson(13), respectively. Antithrombin III and a2-antiplasmin wereassayed by electroimmunoassay (14) as well as by amidolyticassay using S-2238 and S-2251, respectively. Factor X andprothrombin were assayed by one-stage assays according toBachmann et al. (15) and Hjort et al. (16), respectively. Fac-tor IX coagulant activity (IX:C) was assayed by a one-stageassay (12) and Factor IX antigen (IX:Ag) was assayed by theimmunoradiometric assay as described by Holmberg et al.(17). Factor VII was assayed by a one-stage assay using he-reditary Factor VII-deficient human plasma and humanthromboplastin (10). One unit of Factor VII activity is ar-bitrarily defined as that amount of activity present in 1 mlof normal, pooled human plasma. The inhibitory activity ofthe plasma was expressed in Bethesda units (BU)' (18).

1 Abbreviations used in this paper: BU, Bethesda units;BW, body weight.

1836 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. - 0021-9738/83/06/1836/06 $1.00Volume 71 June 1983 1836-184 1

Preparation of human Factor VIIaFactor VII was purified from human citrated fresh-frozen

plasma through the DEAE-Sepharose step essentially as de-scribed by Broze and Majerus (10). Fractions from theDEAE-Sepharose containing Factor VII activity were pooled,concentrated to -30 ml by ultrafiltration, and dialyzed over-night against 0.05 M Tris-HCl (pH 8.0)/0.15 M NaCl/10mMbenzamidine. The retentate was applied to a QAE-Se-phadex A-50 column (1.6 X 35 cm) previously equilibratedat 4°C with 0.05 MTris-HCl (pH 8.0)/0.15 MNaCl/l mMbenzamidine. Factor VII was eluted from the column witha linear gradient of CaC12 consisting of 150 ml of equili-brating buffer and 150 ml of equilibrating buffer containing10 mMCaCl2. The fractions containing Factor VII werecombined, made 10 mMin EDTA, and concentrated to 10ml by ultrafiltration. The concentrate was dialyzed overnightat 4°C against 25 mMTris-HCI (pH 8.0)/25 mMglycine/20 mMbenzamidine/5 mMEDTA, and subsequently sub-jected to preparative electrophoresis as described (19). Frac-tions were assayed immediately after elution from the pre-parative electrophoresis column, and those containing FactorVII activity pooled, and concentrated to -0.5 mg/ml byultrafiltration. Factor VII isolated by this procedure ap-peared as a single band by SDS-polyacrylamide gel electro-phoresis in the presence or absence of reducing agent. Themolecular weight noted by this technique was 50,000 andthe specific clotting activity was 2 U/jig. Single-chain FactorVII was converted to the two-chain Factor VIIa by limitedproteolysis using human Factor XIIa at an enzyme-to-sub-strate weight ratio of 1:100. Two-chain human a-Factor XIIawas generously provided by Dr. Kazuo Fujikawa, Universityof Washington. The Factor XII was isolated from humanplasma as described (20), and converted to two-chain FactorXIIa by human kallikrein in the presence of dextran sulfate(21). Before activation Factor VII was dialyzed at 4°Cagainst 20 liter of 0.01 MTris-HCl (pH 8.0)/0.15 MNaCl.The dialyzed Factor VII was then incubated at 37°C withthe appropriate amount of human Factor XIIa. Completeconversion of Factor VII to Factor VIla occurred within 2h under these conditions, and coincided with the formationof a two-chain Factor VIla molecule consisting of a heavychain (Mr = 34,000) and a light chain (M, = 24,000) heldtogether by a disulfide bond(s) (Fig. 1). The specific coag-ulant activity of Factor VII increased to a maximum of 50U/jig after activation by Factor XIIa. Factor VIla was thensterilized by Millipore filtration (Millex-HA 0.45, jm mem-brane, Millipore Corp., Bedford, MA) in the hospital phar-macy. Approximately 20-25% of the Factor VIIa activitywas lost during the sterilization-filtration process. Portionsof the sterilized preparations were analyzed for pyrogens(the National Bacterial Laboratory, Stockholm, Sweden) andfor HbsAg using the Ausria II assay (Abbott Laboratories,North Chicago, IL). The sterilized Factor VIla was storedat -80°C in 0.5-ml aliquots (- 10,000 U per vial) before use.Before infusion, each preparation was diluted with sterilesaline to the concentration desired.

Protocol for the infusion ofhuman Factor VIIaThese studies were conducted with the approval of the

Human Subjects Committee of the University of Lund,Malmo, Sweden, and followed guidelines established by theDeclaration of Helsinki. Informed consent was obtainedfrom each patient and their guardian before treatment. Be-fore infusion into patients, the thrombotic potential of the

--94L000. _ --67,000

q_pf.jw ~~--45,O000

mp --29,000

_I --20,000

- --14A400

1 2 3

FIGURE 1 SDSpolyacrylamide gel electrophoresis of humanFactor VIIa. Sample 1, 15 jg of unreduced Factor VIla;sample 2, 15 ,jg of reduced Factor VIIa; sample 3 containsa mixture of reduced standard proteins that include phos-phorylase b (94,000), bovine serum albumin (67,000), oval-bumin (45,000), carbonic anhydrase (29,000), soybean tryp-sin inhibitor (20,000), and a-lactalbumin (14,400). Electro-phoresis was carried out in 10% polyacrylamide gels aspreviously described (19).

Factor VIla preparations was examined in healthy dogs byessentially the same protocol described for the analysis ofprothrombin complex concentrates (11). No attempt wasmade to remove the trace amounts of Factor XIIa presentin the Factor VIIa preparations. Accordingly, these studiesexamined the thrombotic potential of Factor XIIa as wellas Factor VIla. Each preparation of Factor VIla was givento a mongrel (16-17 kg) in a dose of 50-100 U Factor VII/kg body wt (BW). The concentrate was given intravenouslyin 3 ml of sterile saline without anesthesia. The samplingwas performed by intermittent venous puncture before theinjection, and 15 min, 1, 4, and 24 h after injection. Eachtime, 30 ml of blood was withdrawn and anticoagulated with0.038% sodium citrate. After infusion of Factor VIla, ab-solutely no change in the coagulation or fibrinolytic profilewas observed in the dog plasma except the expected rise inplasma Factor VII (Table I). Furthermore, the dogs did notexhibit any untoward side effects during or after the injectionof Factor VIla.

Case reports

Case 1. A 13-yr-old male with severe hemophilia A wasadmitted to the hospital with bleeding in the left gastroc-nemius muscle. The circumference of the left leg just belowthe knee was 33.5 cm as compared with 30 cm for the right

Factor VIIa in Hemophiliacs with Inhibitors 1837

TABLE ICoagulation Analysis in Two Dogs Given 100 U VIIa/kg BWand 50 U VIIa/kg BW, Respectively

100 U/kg BW 50 U/kg BW

After After

Before 15 min I h 4 h 24 h Before 15 min l h 4 h 24 h

Platelets, 109/liter 320 270 330 311 404 315 348 316 320APTT, s 18 (18)° 18 (18) 17 (18) 17 (18) 17 (18) 18 (18) 18 (18) 18 (18) 17 (18) 18 (18)VIII:C, % 153 103 97 100 105 87 80 83 62 110IX:C, % 180 167 169 101 156 135 106 108 98 103P&P, % 116 128 172 180 152 136 175 152 162 150VII, % 83 500 430 235 102 90 235 242 235 109X, % 116 158 152 155 148 140 165 135 130 135V, % 100 110 95 105 90 100 95 110 90 110Fibrinogen, g/liter 2.8 1.5 2.4 2.3 2.9 1.7 2.5 2.5 1.9 1.8Ethanol gel test Neg Neg Neg Neg Neg Neg Neg Neg Neg Nega2-macroglobulin,

% 80 80 79 78 74 106 80 73 77 89

The numbers given within parentheses represent the APTT for normal dog plasma. Neg, negative.

leg. Secondary to the muscle bleeding the patient also ex-hibited an extension defect of 900 in the left knee. Labo-ratory records indicated that his antibody titer varied from0.3-3.0 BU/ml and increased 50-fold upon administrationof Factor VIII concentrates. The patient had repeated jointbleedings over the past 7 yr and was treated with a com-bination of high doses of Factor VIII and cyclophosphamideon eight different occasions. He has also been treated withactivated prothrombin complex concentrates (50 U/kg BWFEIBA [Immuno, Vienna] or 50 U/kg BWAutoplex [Hyland,Costa Mesa, CA) in association with minor bleedings, par-ticularly in the ankle joint. During most of these episodes,he experienced some improvement over a period of 24-28h after the infusion. In one such episode, he experiencedmild side effects including flushness in the face.

Factor VIIa dosage and hemostatic response. The pa-tient initially was given a dose of 50 U Factor VIIa/kg BWequivalent to 40-50 Mg of total protein (in 5 ml of sterilesaline). Following administration, the patient's plasma Fac-tor VII level increased from 90 to 135% (Table II). Thefollowing day his muscle was markedly softer and less tender(circumference of 33 cm) and the mobility in the left kneejoint was almost completely restored (extension defect of100). Another dose of Factor VIIa was administered (100 U/kg BW) upon which his Factor VII level rose from 90 to200% (Table II). The following day, the patient walked with-out difficulty (leg circumference 32 cm) and was then dis-charged from the hospital. After extensive physical activity,rebleeding occurred in the same muscle 36 h later and an-other dose of Factor VIIa (100 U/kg BW) was administered.4 h after this treatment, his muscle was considerably lesstender and clearly softer. The next day the swelling hadvirtually disappeared (circumference 31 cm), and the jointmobility was completely restored. Aside from the expectedrise in plasma Factor VII, no changes in the patient's co-agulation or fibrinolytic profile occurred throughout the Fac-tor VIla treatment (Table II).

Case 2. A 12-yr-old male with severe hemophilia A com-plicated with antibodies against Factor VIII:C (12 BU/ml)

was admitted to the hospital with bleeding from two primarymolars. The patient also had a moderate bleeding in his leftknee joint with marked discomfort in the patella region. Thispatient has been treated with high doses of Factor VIII com-bined with cyclophosphamide on four different occasions inassociation with various bleedings, particularly during in-traarticular injection of radioactive gold and teeth extrac-tions. Although a good hemostatic effect was achieved, amarked anamnestic rise in the antibody titer against FactorVIII occurred (up to 60 U/ml BU). In addition, activatedprothrombin complex concentrate, FEIBA (50 U/kg BWor100 U/kg BW), was given on two occasions in associationwith minor and moderate joint and muscle bleeding. A pos-itive effect of this treatment was dubious in this patient.Nonactivated Factor IX concentrate was also given on twooccasions in association with minor joint bleedings. No he-mostatic effect was observed with such a treatment, and thepatient refused further treatment with this concentrate. Thepatient has experienced extensive bleeding problems coin-cident with the loss of six primary teeth necessitating mul-tiple blood transfusions in spite of antifibrinolytic therapygiven intravenously every 6 h for periods of 2-3 wk. Oneach occasion he was unable to attend school for at least 2wk and was virtually unable to feed himself adequately.

Factor VIla dosage and hemostatic response. The pa-tient initially was given a dose of 50 U Factor VIIa/kg BW(40-50 jAg of protein in 1.3 ml of sterile saline). The fullyvisible bleeding from the teeth stopped promptly and ex-amination of the knee joint 1 h after the infusion revealeda complete absence of pain in the patella region. Anotherdose of Factor VIla (50 U/kg BW) was given 2 h after thefirst injection, followed by an equal dose 16 h later. No bleed-ing from the teeth occurred throughout the treatment pe-riod. The circumference of the left knee at two differentlevels was on admission 29.5 and 30.5 cm, respectively. 24h later the corresponding circumferences were 28.0 and 29.0cm. No changes occurred in the patient's coagulation or fi-brinolytic profile except for a slight rise in the plasma FactorVII level. The second treatment with Factor VIla concen-

1838 U. Hedner and W. Kisiel

TABLE IICoagulation Pattern in Patient 1 before and after the Treatments with Human Factor VIla

at Two Concentrations (50 U/kg and 100 U kg BW)

Day 1 Day 5Day 2 Day 6

Before Beforetreatment 1 h after VIla 24 h after Vlla 1 h after Vlla treatment 1 h after VIla 24 h after Vlla

50 U/kg 50 U/kg 100 U/kg 100 U/kg 100 U/kg

Platelet counts, 109/liter 295 290 275 260 315 305 290APTT, s 68 (31)° 61 (31) 72 (31) 78 (31) 77 (32)° 78 (32) 72 (32)VIII:C, % <2.5 <2.5 <2.5 <2.5 <2.5 <2.5 <2.5VIIIR:Ag, % 90 90 90 80 110 100 80IX:C, % 145 128 92 82 100 93 105XII, clotting assay, % 193 153 133 108 101 103 90

immunochemical, % 110 110 110 100 110 100 110Prothrombin time, s 15.2 13.2 15.0 13.9 15.2 13.2 16.6P & P, % 87 120 90 132 96 132 94VII, % 90 135 98 200 95 200 93V, % 104 102 91 98 110 96 100X, % 98 98 98 100 88 90 85Fibrinogen, glliter 5.5 NDt 5.0 NDI 5.3 NDt 4.7Ethanol gel test Neg Neg Neg Neg Neg Neg Neg

Neg, negative.e Numbers in parentheses represent the APTT for normal human plasma at the time of measurement.i ND, not determined.

trate occurred 1 wk later when the patient was presentedwith a huge fragile blood clot in the cavity formed by thespontaneous loss of a primary molar -12 h earlier. Due toits enormous size, the clot had to be removed. At this point,dental consults advised the extraction of two additional pri-mary molars. Accordingly, the patient was given anotherdose of Factor VIla (100 U/kg BW) and the two molars wereremoved under local anesthesia. The cavities created by theextracted teeth were filled with gauze soaked with tranex-amic acid. Another dose of Factor VIla (50 U/kg BW) wasadministered 4 h after the extraction. Tranexamic acid (10mg/kg BW) was administered intravenously at this time,and the same dose repeated every 6 h for 2 d. Thereafter,tranexamic acid was given orally in a dose of 60 mg/kg BWthree times daily for an additional 8 d. No further bleedingfrom the cavities was noted following extraction of the twomolars. In contrast to the large clot observed upon admission,no such clot ever formed in the cavities created by the ex-traction of the two molars in conjunction with Factor VIIaand tranexamic acid treatment. The clot observed in thesecavities was of a normal appearance, and by the second daythe tissue had retracted nicely.

RESULTS

Infusion of purified human Factor Vlla into two pa-tients with hemophilia A with antibodies against Fac-tor VIII:C produced no immediate adverse effects suchas chills, nausea, flush, or urticaria. Furthermore, noevidence of consumption coagulopathy, disseminatedintravascular coagulation, or increase in factor VIII:C

antibody titer were observed in either patient through-out the treatment. Table II contains the coagulationpattern of patient 1 before and after the administrationof Factor VIIa. The coagulation pattern of patient 2was virtually identical (data not shown). No hepatitishas occurred thus far in either patient (one patientfollowed for 1.5 yr and the other for 3 mo) followingthe last infusion of Factor Vlla.

The postinfusion recovery of Factor VIIa (measured15 min after infusion) ranged from 38 to 45%, assum-ing no extravascular equilibration and 40 ml blood/kgBW. Depending on the injection dose, the plasma Fac-tor VII activity of each patient returned to preinjectionlevels in -5-8 h. A shortening of the prothrombintime in the patient's plasma by an average of 2 s oc-curred invariably on each administration. The effectof Factor VIIa on the muscle bleeding (patient 1) andthe knee joint bleeding (patient 2) was evaluated bythe decrease in leg circumference 24 h after the ad-ministration. As for the muscle bleeding, a softeningof the tissue and a decreased tenderness were evidentwithin 2 h after the administration of Factor VIIa. Asubjective disappearance of the discomfort associatedwith the knee joint was also noted within a few hoursafter infusion of Factor VIIa.

Perhaps the most dramatic evidence of the hemo-static effectiveness of Factor Vlla was associated with

Factor VIla in Hemophiliacs with Inhibitors 1839

the mucocutaneous bleedings following the loss of aprimary tooth in patient 2. In this episode, the patienthad spontaneously lost a primary molar and developeda loose clot in the cavity. This clot was typical forpatients with hemophilia in that it was huge, of fragileconsistency, and hanging from the back of the throat.Signs of an ongoing diffuse bleeding around this clotwere also fully visible upon admission. Following ex-traction of two additional molars, and the administra-tion of Factor VIla and tranexamic acid, a small clotof a normal appearance could clearly be seen in thecavities and no diffuse bleeding ever occurred laterthan 30 min after the extraction.

DISCUSSION

A number of investigators have reported on the suc-cessful use of both ordinary prothrombin complex con-centrates (1, 2) and socalled activated prothrombincomplex concentrates (3-5) in the treatment of he-mophilia A patients complicated with inhibitors. How-ever, such concentrates also have been reported to bethrombogenic (6-8). Activated prothrombin complexconcentrates contain Factors IX, X, VII, and prothrom-bin in both activated and zymogen forms. Thus far,no consensus has been reached on which of these fac-tors possess Factor VIII bypassing activity, and whichfactor (or factors) is responsible for the thrombogenicproperties.

Factor VIla converts Factor X to Factor Xa in thepresence of tissue factor and calcium ions (22), thusestablishing a Factor VIII bypassing mechanism forthrombin formation. As pointed out by Seligsohn etal. (9), prothrombin complex concentrates appear tocontain a rather high ratio of Factor VIla/Factor VII.Even higher ratios were observed in activated pro-thrombin complex concentrates. Furthermore, theseinvestigators found an increase in plasma Factor VIIactivity in hemophilia B patients after infusion of pro-thrombin complex concentrates. On the other hand,they could not demonstrate any significant amountsof Factor IXa or Factor Xa in vivo in the same patients.These findings point to Factor VIIa as the active he-mostatic principle and as responsible for the FactorVIII bypassing activity.

Judging from our results in the two hemophilia Apatients with inhibitors against Factor VIII:C, purifiedFactor VIIa also seems to be effective in restoring he-mostasis in such patients. Patient 2 had extensivebleeding problems in the past coincident with the lossof five of six primary teeth, necessitating multipleblood transfusions on three of these occasions. It isnoteworthy to mention that patient 2 lost three teethduring Factor VIla therapy, two of which still had

significant roots and were extracted. In spite of this,the patient was back home after 4 d of hospitalizationwithout having experienced any measurable bleeding.The treatment was sustained by the administration oftranexamic acid according to the protocol recom-mended at tooth extractions in uncomplicated hemo-philia treated with Factor VIII/IX (23). This very pa-tient had been treated with tranexamic acid in exactlythe same way during all of his previous teeth bleedingepisodes. In spite of this treatment, bleeding episodesoccurred in all events before the administration ofFactor VIIa. Tranexamic acid most probably contrib-uted to the hemostatic effect observed with Factor VIIaby inhibiting local fibrinolysis as suggested by Bjorlinand Nilsson (23). Hanna et al. (5) noted a similar sus-taining effect of a fibrinolytic inhibitor, epsilon aminocaproic acid, on the hemostatic efficacy of activatedprothrombin complex concentrates at surgery in threesevere hemophilia A patients with antibodies againstFactor VIII. Treatment with Factor VIla most prob-ably would be augmented by concurrent administra-tion of a fibrinolytic inhibitor, since it is unlikely thatthe same degree of hemostasis can be achieved byFactor VIIa as that observed with Factor VIII in un-complicated hemophilia A patients.

Patient 1 in our study also experienced a rebleedingtwo days after hemostasis had been restored by FactorVIIa. A somewhat similar observation was made byKingdon and Hassell (24) in their dog model followingadministration of an activated prothrombin complexconcentrate (Autoplex, Hyland, Costa Mesa, CA). Theseinvestigators were able to overcome this rebleedingtendency by increasing the dose. In treating our secondpatient, we also administered Factor VIla for 3 d (twodoses a day). Thus, in order to achieve satisfactoryhemostasis with Factor VIla, it may be advisable togive several doses of at least 100 U/kg BWat intervalsof 6-10 h together with tranexamic acid.

No deleterious side effects were observed after ad-ministration of Factor VIIa in either patient. No in-crease in plasma Factor X activity was observed, in-dicating that Factor VIIa was not activating circulat-ing Factor X. Furthermore, no evidence of an activatedcoagulation system was found as shown by the absenceof fibrin monomers, as well as unchanged levels offibrinogen, antithrombin III, or a2-macroglobulin. Al-though we feel confident that the administered FactorVIIa played a significant role in the hemostasis of ourtwo patients, we also recognize that it is far too pre-mature to draw any sweeping conclusions regardingthe safety and efficacy of Factor VIla administrationin all hemophilia A patients complicated with alloan-tibodies. Wehope our findings will stimulate furtherresearch on Factor VIla as a potential Factor VIII by-

1840 U. Hedner and W. Kisiel

passing material for the management of a very diffi-cult clinical problem.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Tor-Erik Jonsson for his skill-ful help in the sterilization procedure. We also wish tothank Dr. Lars Wranne and Dr. Olle Berseus of the Hospitalof Orebro for their assistance in the treatment of patient 2.

This work was supported in part by grants from the Swed-ish Medical Research Council (19X-05447, 19X-00087),Greta and Johan Kocks Foundation and the Medical Faculty,University of Lund, and in part by research grant HL 16919from the National Institutes of Health. This work was per-formed in part during the tenure of a Established Investi-gatorship (Dr. Kisiel) of the American Heart Association anda Visiting Scientist Research Fellowship from the SwedishMedical Research Council (19V-6022).

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Factor VIlIa in Hemophiliacs with Inhibitors 1841