Eur J Pediatr (1996) 155 : 20-25 �9 Springer-Verlag 1996

F.-M. Miiller W. Ehrenthal G. Hafner D. Sehranz

Purpura fulminans in severe congenital protein C deficiency: monitoring of treatment with protein C concentrate

Received: 16 May 1994 Accepted: 4 April 1995

F.-M. Mt~ller �9 D. Schranz University Children's Hospital, Langenbeckstrasse 1, D-55131 Mainz, Germany

W. Ehrenthal �9 G. Hafner Institute of Clinical Chemistry, University Hospital, Langenbeckstrasse 1, D-55131 Mainz, Germany

F.-M. Mtiller (YTI) University Children's Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany Fax: 0049-241-8888423

A b s t r a c t This report describes the successful use of protein C concen- trate to treat severe purpura fulmi- nans in a homozygous protein C-de- ficient infant for 8 months until oral anticoagulation was initiated. While fresh frozen plasma was previously used in such cases to replace protein C in the acute phase, the availability of a monoclonal antibody purified protein C concentrate now allows specific replacement of protein C, avoiding problems of fluid overload. An occlusive-hydrocolloid bandage proved to be effective in local treat- ment o f skin lesions. D-dimer, fibrin monomer, thrombin-anti thrombin complex and prothrombin fragment 1+2 were useful markers in monitor- ing and optimizing protein C re- placement therapy.

Key w o r d s Purpura fulminans �9 Severe congenital protein C deficiency �9 Protein C concentrate �9 Coagulation marker

Abbrev ia t ions aPTT activated partial thromboplastin time �9 DIC disseminated intravascular coagulation �9 F1 +2 prothrombin fragment 1+2 �9 F M fibrin monomer - t-PA tissue plasminogen activator �9 TAT thrombin-anti thrombin complex

Introduction

Protein C is a vitamin K-dependent plasma protein which is synthesized in the liver and activated on the endothe- lium of the cell wall by a complex of thrombin and the en- dothelial thrombin receptor thrombomodulin. Activated protein C (APC) exerts an anticoagulant effect by the in- activation of the activated forms of factors V and VIII, which leads to a decrease in thrombin formation [5, 13]. APC has also been shown to have profibrinolytic effects [6]. Severe congenital protein C deficiency can lead to life-threatening neonatal thrombosis and purpura fulmi- nans, both of which are frequently associated with dis- seminated intravascular coagulation (DIC). We have treated an infant with homozygous protein C deficiency

and severe purpura fulminans with a monoclonal antibody purified protein C concentrate for 8 months and investi- gated the usefulness of laboratory parameters o f coagula- tion in monitoring and optimizing the treatment o f severe protein C deficiency.

Materials and methods

Protein C concentrate (Human), Vapor-Heated, Immuno(Vienna, Austria) is an investigational new drug which has been used to treat patients with congenital and acquired protein C deficiency [e.g., 10, 11, 16, 41]. The protein C is isolated from a virus-inacti- vated prothrombin complex concentrate (Bebulin VH, Immuno AG) which is obtained from cryosupernatant by DEAE-Sephadex chromatography and is licensed for the treatment of congenital and acquired deficiencies of factor IX in the United States of America, Canada and Europe. All plasma units used are tested for the ab-

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sence of hepatitis B surface antigen,absence of antibody to HIV-1 and -2, absence of antibody to hepatitis C virus and for alanine aminotransferase levels not exceeding twice the upper limit of nor- mal. To further reduce the risk of virus transmission, the pro- thrombin complex concentrate undergoes a two-step procedure consisting of vapour heating at an excess pressure of 190 mbar at 60~ for 10 h, and at 375 mbar at 80~ for 1 h. To separate pro- tein C, the prothrombin complex bulk powder is subjected to cal- cium-dependent monoclonal immuno-affinity chromatography. The monoclonal antibody is an epitope-affinity-purified IgG1 pro- duced in a serum-free tissue culture. The eluted protein C under- goes several ultrafiltration and diafiltration steps and pasteurized therapeutic human albumin is added. The preparation undergoes an additional vapour heat treatment as well as further ion exchange chromatography steps to eliminate murine immune globulins.

The protein C content is measured in international units (IU), defined as the protein C activity in 1 ml of a normal human plasma pool, based on a snake venom (Protac) activity assay calibrated against the first international standard of protein C [19]. Reconsti- tution of the freeze-dried concentrate yields a solution containing 8-12 mg/ml total protein, 7-11 mg/ml NaC1, and 3-5 mg/ml Na3cit- rate �9 2 H20. The protein C activity of the solution is 100-150 IU/ml. The factor II activity is below 0.14 IU/ml, factor IX below 0.11 IU/ml, factor VII below 0.21 IU/ml, and factor X = 0.05 IU/ml.

For coagulation analyses, 1.8 ml of venous blood was drawn into a 2 ml test tube containing 0.2 ml sodium citrate (0.11 mol/1). After thorough mixing, the samples were kept at room temperature and centrifuged within 1 h at 3400 g at 4~ for 10 min. Aliquots for later analysis were frozen at - 70 ~ C. Protein C and antithrom- bin III were determined with chromogenic substrate tests on a Hi- tachi 717 analyser (Boehringer-Mannheim, Germany). Hetero- genic enzyme immunoassays were used to determine D dimer and soluble fibrin (fibrin monomer) (Boehringer-Mannheim) as well as thrombin-anti thrombin complex and prothrombin fragment 1+2 (Behringwerke, Marburg, Germany) [9, 35, 36, 43]. Activated par- tial thromboplastin time (aPTT; Pathromtin, Behringwerke) and fibrinogen (Fibrinogen A, Boehringer Mannheim) were deter- mined using standard techniques. Platelet count in EDTA blood was determined using a Coulter counter STKS.

Case report

The patient was born in the 38th week after an uneventful preg- nancy and an uncomplicated delivery. Within the first few hours of life she developed a slightly crusted haematoma measuring 0.5 cm on her left thigh at the site of an intramuscular vitamin K injection. On both legs haemorrhagic areas appeared which subsequently evolved to necrosis (Fig. 1). A diagnosis of venous thrombosis was proposed and intensive therapy with heparin was initiated at a dose of 500 U/kg daily. Intensive replacement therapy with fresh frozen plasma was initiated, but was discontinued when the symptoms be- gan to recede. On the 13th day of life, she again developed fulmi- nant symptoms of venous and arterial thrombosis and was trans- ferred to the intensive care unit of the Children 's Hospital in Mainz under therapy with heparin, corticosteroids, antibiotics and xantinol nicotinate. Laboratory values on admission to each hospi- tal are given in Table 1. A diagnosis of protein C deficiency was proposed. At this t ime the child had extensive haemorrhagic- necrotic lesions on both legs, with severe blistering on her right leg (Fig. 2). In addition, she had a retinal haemorrhage of the left eye with central retinal detachment. Treatment with a dose of 500 U/kg daily of heparin was continued until a final diagnosis could be es- tablished. Fresh-frozen plasma was infused in a dose of 5 ml/h, 150-250 IU of anti thrombin III twice daily and tissue plasminogen activator (t-PA) once a day as a bolus injection of 0.2 mg/kg and then an infusion of 0.8 mg/kg over 2 h. With the aim of inhibiting platelet aggregation, prostacyclin was administered in a dose of 10 ng/kg per minute.

Tab le 1 Laboratory values for a neonate with severe congenital protein C deficiency immediately after appearance of initial symp- toms of purpura fulminans at 24 h of life and on admission to spe- cialized clinic on day 13 of life

24 h Day 13

Platelets/nl 140 259 PTT (sec) 30 89 Fibrinogen (mg/dl) 132 122 Anti thrombin III (%) 66 62 Fibrin monomer - positive D-dimer (ng/ml) a - 2771 TAT (ng/ml) b - 10.1

a Normal value < 400 ng/ml b Normal value < 5 ng/ml

Since protein C activity levels determined at this time in the con- sanguineous parents were 48% in the mother and 61% in the fa- ther, both were judged to have heterozygous protein C deficiency. The patient 's protein C activity level was below 10% even after re- placement therapy with fresh-frozen plasma and there was good agreement between the chromogenic assay and clotting and im- munological assays (data not shown). A diagnosis of type I homo- zygous protein C deficiency was therefore made on the patient 's 16th day of life.

After obtaining informed consent from the parents, replacement therapy with protein C concentrate was initiated on the 20th day of life with a dose of 130 IU three times daily. Levels of protein C ac- tivity and coagulation markers were determined 1 h before and 1 h after administration of protein C concentrate. In addition, the co- agulation markers D-dimer, fibrin monomer (FM), thrombin-an- t i thrombin complex (TAT), and prothrombin fragment 1 +2 (F 1 +2) were used to monitor treatment and adjust dosage of the protein C concentrate. As shown in Fig. 3, a number of adjustments were made in the dosage levels to achieve protein C levels in the normal range. After stabilization, the maintenance dose was 500 IU once daily. Under protein C treatment and with the use of an occlusive- hydrocolloid bandage locally, the skin lesions healed astonishingly well in a relatively short period of time (Fig. 4). However, the reti- nal haemorrhage with central retinal detachment which had devel- oped in the left eye during the first days of life was irreversible.

The maintenance dose was temporarily split into two daily ad- ministrations to allow the implantation of a Broviak catheter. Pro- tein C concentrate therapy was then continued on an outpatient ba- sis with once daily administration at the paediatrician's office for a total of 8 months without adverse effects. When she was 8 months old, the patient developed a catheter-related infection which led to withdrawal of the catheter. After the infection had been treated with antibiotics, oral anticoagulation was successfully initiated un- der protective replacement therapy with the protein C concentrate.

Results

D u r i n g the in i t i a l p h a s e o f t r e a t m e n t w i t h c o n t i n u o u s in-

f u s i o n o f h e p a r i n a n d f r e s h - f r o z e n p l a s m a , the D - d i m e r

l eve l b e g a n to d e c r e a s e a n d f i b r i n o g e n l e v e l s n o r m a l i z e d . T h e p l a t e l e t c o u n t i n c r e a s e d in i t ia l ly , b u t d e c r e a s e d a f t e r d i s c o n t i n u a t i o n o f f r e s h f r o z e n p l a s m a . A f t e r r e i n s t a t e -

m e n t o f f r e s h f r o z e n p l a s m a a n d s i m u l t a n e o u s t h e r a p y

w i t h p r o s t a c y c l i n a n d t -PA, f i b r i n o g e n i n c r e a s e d i m m e d i - a t e ly a n d p l a t e l e t c o u n t a l so b e g a n to r i se a g a i n (Fig. 5).

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60 ] I before substitution 150

lh after substitution 140 1 3 0 F F P 5 m l / h 2 5 0 U 4 x / d 2 5 0 U 3 x / d 2 5 0 U 2 x / d 5 0 0 U /d 2 5 0 U/d

'1 [ - - 1 II I I I [ - - I 120 110 100 90

O 80 ' ~ 70

~_ a0

0 ti I IIIr 40

20 ~ ] ,o

0 I I I I I I I I I I I I I I I I I I

Fig. 1 Purpura futminans in a neonate with severe congenital pro- tein C deficiency: haemorrbagic lesions

Fig. 2 Puq~ura fulminans in a neonate with severe congenital pro- tein C deficiency: necrotic areas with severe blistering

Fig.3 Protein C activity levels in a neonate with severe congeni- tal protein C deficiency before and after infusion of protein C con- centrate

Fig.4 Healing of skin lesions 1 month after the start of protein C replacement therapy in a neonate with severe congenital protein C deficiency

infusion schedule, protein C activity ranged between 20% and 70% 1 h prior to administration of the protein C con- centrate and between 80% and 150% 1 h after administra- tion (Fig. 3). Table 2 shows the results (mean value, stan- dard deviation, n) during the maintenance period (500 IU/day) for the four different coagulation markers ordered in relation to protein C levels.

Discussion

D-dimer levels normalized after initiation of therapy with the protein C concentrate. Levels of D-dimer remained normal during both the perioperative and postoperative period when the catheter was inserted and throughout the period of protein C therapy.

Of the 51 coagulation analyses performed, 6 could not be evaluated due to coagulation activation when blood samples were drawn (TAT > 60 gg/1). Depending on the

In newborn infants with low levels of protein C it is diffi- cult to distinguish hereditary from acquired protein C de- ficiency. Acquired protein C deficiency has been de- scribed in paediatric and adult patients in association with a variety of conditions such as DIC, sepsis, liver disease and 1-asparaginase therapy [21, 24]. Although in some in- dividuals prothrombin and protein C levels may be in the adult range by the end of the 1 st year of life, mean values

23

Platelets/nl Fibrinogen (mg/dl) 500

400 ~ / ' ""',,, .-

2~176 f t ' /'l::l'2tl~l~t. " ~ / Fibrinogen ,ooiv . . . . . . . . .

6 I i I i i i I 1 i i i i i i p i ~ i

1

500

400

300

200

lOO

o 2 3 4 5 6 7 8 9 1011121314151617181920

Day

Fig. 5 Course of platelets and fibrinogen in a neonate with severe congenital protein C deficiency. Heparin was given continuously and frozen plasma intermittently from days 1 to 19, prostacyclin and t-PA were given from days 13 to 19

remain significantly lower than in the adult until after 3 years of age, with a lower limit of normal of 40% until the age of 11 [1, 24, 31,40]. When homozygous protein C de- ficiency is suspected, protein C levels should be measured not only in the propositus, but also in the parents and sib- lings so that the hereditary nature of the defect can be con- firmed [20, 25-28, 42]. For diagnosis of protein C defi- ciency, protein C activity tests may be considered superior to antigen determination [3, 29, 30, 33]. Direct analysis of the protein C gene is now also possible [38, 45].

Previously, continuous infusions of fresh-frozen plasma or prothrombin complex concentrates were administered to replace protein C in the acute phase of purpura fulmi- nans until oral anticoagulation could be initiated [27, 28]. However, administration of fresh-frozen plasma can lead to hyperproteinaemia, fluid overload and hypertension and carries increased risk of infection [45]. The availabil- ity of a monoclonal antibody purified protein C concen- trate now allows specific replacement without these disad- vantages. Intravenous administration of heparin has been

shown to have no positive effect on purpura fulminans [15, 44].

During the acute phase of purpura fulminans in our pa- tient, the laboratory results were consistent with DIC [27, 28], including decreased platelet count, decreased fibrino- gen, increased fibrin split products, and prolonged aPTT. After initiation of treatment with fresh-frozen plasma fib- rinogen levels normalized, D-dimer levels decreased and a noticeable reduction in the haemorrhagic areas was ob- served. The irreversible detachment of the retina in the left eye may have been related to a decrease in fibrinogen levels and thrombocytopenia. In severe congenital protein C deficiency thrombo-embolic episodes in the eye and central nervous system develop at least partially in utero [45]. In a similar case of homozygous protein C defi- ciency with purpura fulminans reported by Dreyfus et al. [11] fresh-frozen plasma replacement therapy increased fibrinogen levels, but platelet count normalized only with protein C replacement therapy. It is interesting to note that prostacyclin and t-PA administration in addition to fresh frozen plasma might have helped to prevent a further de- crease in the platelet count in our patient. However, prostaglandins have not been shown to have a positive ef- fect on consumptive coagulopathy and purpura fuhninans [27, 28,451.

Conard et al. [8] have shown that F l+2 and fibrino- peptide A decrease after administration of protein C con- centrate, demonstrating that protein C is converted to ac- tivated protein C in vivo. In our patient, a dose of 250 IU (70 IU/kg) of the protein C Concentrate four times daily achieved protein C activity levels over 100% 1 h after in- fusion during the acute phase. This resulted in a rapid ef- fect on Fl+2, TAT, FM and D-dimer levels. TAT and F1+2, with relatively short half-lives, showed the quickest and most pronounced reaction, while the effect on D- dimer and on FM was somewhat delayed (data not shown). As can be seen from Table 2, all markers were in the pathological range when protein C activity was below 30%.

In agreement with other reports, protein C concentrate therapy was well tolerated by our patient during an 8- month period and there were no side-effects [2, 11, 12, 23, 41]. Our goal was to postpone the initiation of oral anti-

Table 2 Coagulation markers used in monitoring protein C replacement therapy ordered in relation to level of protein C activity: F1+2, TAT, D-dimer and FM. Mean values are given for each level of protein C activity + SD

Protein C (%) F 1+2 (ng/ml) TAT (ng/ml) D-dimer (ng/ml) FM (~l.g/ml) Number of measurements

6.5 + 1.5 1.22 _+ 0.28 8.28 + 1.21 1629 + 521 > 42 - 5 15.3+ 2.4 1.18+0.53 9.02+2.29 1193+854 >42 - 6 31.1+ 5.0 0.88+0.20 5.11+2.29 774+774 21.3+11 8 47.8 + 3.7 0.56-+ 0.12 3.45 + 1.23 717 + 717 15.5 + 7.1 6 83.8 + 8.9 0.64 _+ 0.21 3.96 + 0.98 440 + 165 13.7 +_ 5.6 5

117 _+ 16 0.77+0.35 4.44_+2.53 544+203 13.5+ 4.3 11

24

coagula t ion until the infant was at least 6 months or o lder to prevent potent ia l adverse effects on bone growth [14]. Oral ant icoagula t ion can resul t in coumar in - induced skin necrosis in he terozygous protein C def ic ient individuals , s ince differences in metabol ic c learance rates be tween protein C and the p rocoagulan t v i tamin K-dependen t fac- tors cause a transient hypercoagulab le state [7, 12, 34]. However , the balance of the coagula t ion sys tem in homo- zygous protein C-def ic ient individuals is a l ready shifted towards coagula t ion activation, so that oral ant icoagula- t ion represents no further r isk for these pat ients [15, 17, 27, 28, 37, 39, 45].

In conclusion, a diagnosis of homozygous prote in C or protein S [18, 21, 22, 32] def ic iency should a lways be cons idered when a newborn infant begins to deve lop signs of purpura fulminans. Ear ly diagnosis and adequate re- placement therapy may be life-saving. Initial therapy should

consist of continuous administrat ion of fresh frozen plasma, and replacement therapy with protein C concentrate should be ini t iated as soon as it can be made avai lable . The coag- ulat ion markers D-dimer , FM, TAT and F l + 2 are useful for moni tor ing therapy. Oral ant icoagula t ion is current ly the t reatment o f choice for long- term therapy, but should not be ini t iated at too ear ly an age, due to the potent ia l for adverse effects on bone growth. A successful l iver trans- plant has only been repor ted in one case [4]. Long- te rm therapy with the prote in C concentrate is now a possibi l - ity, while a combina t ion of oral ant icoagulat ion and inter- mit tent p rophylac t ic protein C therapy may represent the most v iable opt ion for the immedia te future.

Acknowledgements We are grateful to Dr. Hans Peter Schwarz and Immuno AG, Vienna, Austria for supplying the protein C con- centrate. We also thank Kathryn Nelson for valuable suggestions and linguistic expertise.

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