MINERVA ANESTESIOL 2005;71:373-8

Use of Protein C concentrate in pediatric patients with sepsis

M I CO NE R PY V A RI M GH E D T
Aim. Protein C (PC) is a plasma glycoprotein implicated in modulating coagulation and inflammation. Its levels decrease in sepsis and related diseases, where it has also proved to be a prognostic indicator of outcome. Infusion of exogenous PC, although not able to decrease mortality in severe sepsis and septic shock, can safely resolve the coagulation imbalances related to these pathological states. Methods. A retrospective study was performed about utilisation of PC in severe sepsis and septic shock patients in three italian PICUs during a one-year period. Data from 29 patients were analysed. Age, PIM 2, mortality and length of stay were compared between treated and non treated patients. Treated patients were also analysed for PC dosage received, length of treatment, and modification of hemocoagulation parameters, before PC infusion and every 24 hours. Results. In treated patients, the activity of PC, PT and PTT activity and fibrinogen improved significantly from basal to day 5 (p<0.05). Diminution of d-dimer was not quite significant (p=0.0514). Rise in platelets count and antithrombin III activity was not significant. No adverse reactions related to Protein C concentrate were observed. No difference in mortality was observed between the two groups. Conclusions. Although PC is included in guidelines for management of severe sepsis and septic shock, only 38 %, of observed patients received PC treatment. Even in the treated group, patients received a lower dosage of PC, and for a shorter period, than recommended.

P. SILVANI, A. CAMPORESI, E. LICARI, A. WOLFLER

Intensive Care Unit, V. Buzzi Childrens Hospital Milan, Italy

In accordance to previous studies, we did not observe differences in mortality between treated and untreated patients. Our results showed a significant increase in plasma PC activity, following infusion of PC concentrate. This increase in PC appeared sufficient to restore some, but not all, of the abnormalities in the coagulation system. A large randomized, phase 3, placebo-controlled trial in children with severe sepsis and septic shock is advisable to establish effective role of therapy with PC in reducing mortality of these patients. Key words: Severe sepsis - Septic shock - Protein C.

Address reprint requests to: Silvani P, Terapia Intensiva Pediatrica, Ospedale dei Bambini V. Buzzi, via Castelvetro 32, Milano, Italy. E-mail: paolo.silvani@tiscali.it

rotein C (PC) is a glycoproteic, vitamin K dependent, plasma zymogen. After activation, operated by thrombin thrombomodulin complex on vascular endothelium surfaces, activated PC (APC) plays different roles in modulating coagulation, fibrin degradation and inflammation.1 Coagulation abnormalities are commonly observed during sepsis. In this disease, activation of the extrinsic pathway combined with depression of the inhibitory mechanisms of coagulation and fibrinolytic system result in a procoagulant state that may lead to microvascular thrombosis and organ dysfunction. Indeed, PC levels decreases during

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TABLE I.Reports concerning the use of PC in pediatric septic patients.

1999 2000

Ettingshausen CE et al.7 White B et al.8

8 patients (2 months 18 yeras) 36 patients (3 months 76 years)

M I CO NE R PY V A RI M GH E D T
2004 Pettenazzo A et al.9 8 patients (1 month 7 yeras)

sepsis and PC level is a prognostic indicator of outcome in sepsis and related diseases.2, 3 Otherwise, some clinical and histopathologic features of congenital homozygous PC deficit are very similar to clinical aspects of menigococcal sepsis and purpura fulminans. This observation led to consider therapeutics use of PC infusion in sepsis. Since 1993,4 several authors reported studies concerning the use of PC in sepsis (Table I). Recently, de Kleijn 10 published a randomized, double-blind, placebo controlled, dose-finding study of PC concentrate in children with severe meningococcal sepsis and purpura fulminans. The study was designed to assess the efficacy of the activation process of PC and investigate the optimal dosing regimen and safety of infused PC concentrate. Conversion of PC to APC was observed in almost all patients who received PC concentrate but higher-than-normal levels were achieved in patients who received either the cumulative dose of 400 IU/kg/day or 600 IU/kg/day, respectively. No serious adverse reactions related to PC concentrate were observed in the study. Although no difference in mortality was reported among treated and not treated patients (as would expected from a phase II trial that was not powered to detect mortality and morbidity differences), treated patients showed a resolution of coagulation imbalances. PC infusion (either as PC concentrate or as recombinant human activated PC) is included in Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock.11, 12 Although efficacy and safety of recombinant human APC for severe sep-

Materials and methods

We performed a retrospective analysis on utilization of PC concentrate in the treatment of severe sepsis and septic shock 14 in 3 Italian PICUs (Ospedale dei Bambini V. Buzzi, Milano; Clinica de Marchi, Milano; Ospedale S.Orsola-Malpighi, Bologna). Clinical documentation, relative to one year of activity (year 2004) has been reviewed. Twenty-nine children with severe sepsis and septic shock were admitted to the three Units. We considered the following parameters: age, pediatric index of mortality (PIM 2), 15 mortality and length of stay (LOS) in treated and non-treated patients. Treated patients were also analysed for PC dosage received, length of treatment, and modification of hemocoagulation parameters, before PC infusion (basal) and every 24 hours (day 1-5). Statistical alanysis

Statistical analyses were performed with the InStat software (GraphPad Software, Inc). Data are presented as mean (95% confidence interval [CI]) for continuous variables and percentages [95% CI]) for discrete variables. Statistical analyses of demographic data, PIM 2, LOS were performed with Students t test. Mortality between the two groups was analysed with Fishers exact test. PC activity, platelets number, PT and PTT activity, fib-

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sis in adults is established,13 no randomised studies using recombinant human activated PC have been performed in children.

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1993 1995 1997

Gerson WT et al.4 Rivard GE et al.5 Smith OP et al.6

1 patient (13 years) 4 patients (3 months - 15 years) 12 patients (3 months 27 years)

Amputation of three toes No deaths 2 amputations No deaths 2 amputations Reduction in mortality (observed vs expected) 2 deaths 1 amputation 3 deaths 4 amputations Reduction in mortality (observed vs expected), reduction in need for amputation (observed vs. expected) 2 deaths

Year

Authors

Population

Outcome

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TABLE II.Principal parameters of population analysed. Ns= not significative.

TABLE III.Sepsis related diagnosis, diagnosis on admission and isolated microorganism of population analysed.

M I CO NE R PY V A RI M GH E D T
Treated (N= 11)

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Age (months) PIM 2 Observed mortality (%) LOS (days)

22.2 25.7 16.79 15.2 27.7 11.5 8.9

41 50 10.77 15.6 22.2 8.5 8.7

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Non treated (N=18)

Treated (N=11)

Non treated (=18)

ns ns ns ns

Sepsis related diagnosis

Diagnosis on admission

Microrganisms

Severe sepsis = 3 Septic shock = 8 Meningitis = 5 Central venous cannulation related infection = 1 Peritonitis = 1 Blood stream infection = 3 Foodborne bacterial infections = 1 Neisseria meningitidis = 4 Escherichia coli = 2 Xanthomonas sp. = 1 Staphylococcus aureus = 1 Streptococcus pneumoniae = 1 Not determined = 2

Severe sepsis = 15 Septic shock = 3

Acute rensal failure = 1 Respiratory failure = 12 Meningitis = 3 Blood stream infection = 2 Neisseria meningitidis = 4 RSV = 5 Streptococcus pneumoniae = 2 Ebstein Barr virs = 1 Streptococcus haemolyticus = 1 Mycoplasma = 1 Not determined = 4

rinogen, d-dimer were analysed by using analysis of variance (ANOVA) for repeated measures. P<0.05 was considered to be significant. Variations among days of observation (basal vs day 1-5) were analysed by TukeyKramer comparison test. Results

count and antithrombin III activity was not significant. No adverse reactions related to PC concentrate were observed. Variations of PC activity, plateletsnumber, PT INR, PTT ratio, antithrombin III activity, ddimer and fibrinogen levels are plotted (as median, min, max) in Figures 1-7. Discussion

PC concentrate was utilized in eleven children. Principal parameters of population analysed are summarized in Table II. Sepsis related diagnosis, diagnosis on admission and isolated microorganism are summarized in Table III. Treatment with PC was carried on for 59 hours (range 24-120), average dosage was 324 UI/kg/day (range 66-400). In patients treated with PC, the activity of PC, PT and PTT activity and fibrinogen improved significantly from basal to day 5 (p<0.05). Diminution of d-dimer was not quite significant (p=0.0514). Rise in plateles

This study presents some limitations (retrospective design, lack of control group for variation of hemocoagulation parameters, low number of patients observed). Nevertheless, several considerations can be made. Although PC is included in guidelines for management of severe sepsis and septic shock,11, 12 only 38%, of observed patients received PC treatment. The majority of patients classified as septic shock received PC treatment, whereas only the minority of patients classified as severe sepsis did. The reasons of this low rate of treatment are not clear. Moreover, even in the treat-

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250 PC activity (%) 200 150 100 50 0 Basal 1 2 Days 3 4 5 d-dimer (gr/ml)

150

100 50 0

Basal

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1 2

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3 4 5 Days Basal

**

Figure 1.PC activity. *=p<0.05, **=p<0.01.

M I CO NE R PY V A RI M GH E D T
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 600 PT (INR)

Figure 5.d-dimer (g/mL fibrinogen). P>0.05 not significative.

**

PLT (x 1000)

500 400 300

**

***

200 0

100

Basal

1 2 Days 3 4

***

***

Days

Figure 2.PT INR. **=p<0.01, ***=p<0.001.

Figure 6.Platelets number (1000). P>0.05 not significative.

3.5

3.0

PTT (ratio)

2.5

**

1.5

***

***

***
4

**
5

1.0

0.5

Basal

Days

AT III (%)

2.0

140 120 100 80 60 40 20 0

Figure 3.PTT ratio. **=p<0.01, ***=p<0.001.

Basal

2 Days

Figure 7.Antithombin III (%). P>0.05 not significative.

1400 1200 1000 800 600 400 200 0

Fibrinogen (mg(dl)

Basal

Days

Figure 4.Fibrinogen (mg/dL). p>0.05 not significative.

ed group, patients received a lower dosage of PC, and for a shorter period, than recommended.10 Probably the high cost of treatment hampers its adequate and full application.

In accordance with the study of de Kleijn,10 we did not observe differences in mortality between treated and untreated patients. Observed mortality exceeded predicted in both, treated and not treated patients. Castellanos-Ortega 16 demonstrated that PIM failed to classify correctly children affected by meningococcal septic shock, underestimating significantly the mortality rate in the intermediate and high-risk strata. Furthermore, in severe sepsis and septic shock, mortality is strictly related to the rapidity and quality of conventional treatment (fluid resuscitation, vasopressors/inotropes, antibiotics).17

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M I CO NE R PY V A RI M GH E D T
Conclusions Treatment with PC concentrate is safe in children with septic shock and severe sepsis and leads to increases of plasmatic PC activity and normalization of hemocoagulation parameters. A large randomized, phase 3, placebo-controlled trial in children with severe sepsis and septic shock is advisable to establish effective role of therapy with PC in reducing mortality of these patients.
Acknoledgments.Leoncino S, Terapia Intensiva Pediatrica Clinica de Marchi, Milano and Iannella E, Terapia Intensiva Pediatrica Ospedale S. Orsola Malpighi. Bologna.

Our results showed a significant increase in plasma PC activity, following infusion of PC concentrate. However, PC activity level on days 4-5 decreased, loosing statistic significancy if compared to basal. The increase in PC appeared sufficient to restore some, but not all, of the abnormalities in the coagulation system. While PT, PTT and fibrinogen normalized following infusion of PC concentrate, decrease of d-dimer was not quite significant. On the contrary, de Kleijn 10 demonstrated normalized levels of d-dimer, occurring earlier in patients treated with higher doses of PC concentrate. These observations might be related to low dosages utilized and shortness of treatment.10

i dati ottenuti da 29 pazienti. Let, il PIM 2, la mortalit e la durata del ricovero sono stati confrontati tra i pazienti trattati e quelli non trattati. I pazienti trattati sono anche stati valutati a seconda della dose di proteina C somministrata, della durata del trattamento e delle modificazioni dei parametri emocoagulativi, prima dellinfusione di proteina C ed ogni 24 ore. Risultati. Nei pazienti trattati lattivit della proteina C, del tempo di protrombina (PT), del tempo di tromboplastina parziale (PTT) e del fibrinogeno migliorata significativamente dai valori basali al giorno 5 (p<0,05). La diminuzione del d-dimero non stata veramente significativa (p<0,0514). Laumento della conta piastrinica e dellattivit dellantitrombina III non stato significativo. Non sono state osservati effetti collaterali relativi al concentrato di Proteina C. Circa la mortalit, nei due gruppi non si osservata alcuna differenza. Conclusioni. Sebbene la proteina C sia inclusa nelle linee guida per la gestione della sepsi grave e dello shock settico, solo il 38% dei pazienti studiati stato sottoposto ad trattamento con essa. Anzi, nel gruppo dei pazienti trattati il dosaggio della proteina C era inferiore a quello raccomandato, cos come la durata del trattamento. In accordo con studi precedenti non abbiamo osservato differenze circa la mortalit tra i pazienti trattati e quelli non trattati. I nostri risultati hanno evidenziato un aumento significativo dellattivit della proteina C nel plasma dopo infusione di concentrato di proteina C. Questo aumento dei valori della proteina C sembra essere sufficiente a rimediare ad alcune, ma non a tutte, le anormalit del sistema coagulativo. Per stabilire il ruolo effettivo della terapia con proteina C sulla mortalit auspicabile che venga eseguito uno studio clinico di fase 3, controllato, versus placebo, su bambini con sepsi grave e shock settico. Parole chiave: Sepsi - Shock settico - Proteina C.

Riassunto

Impiego del concentrato di Proteina C nei pazienti pediatrici con sepsi Obiettivo. La proteina C una glicoproteina plasmatica implicata nella modulazione della coagulazione e dellinfiammazione. I suoi livelli diminuiscono nella sepsi e in corso di patologie correlate, dove si anche dimostrata essere un indicatore prognostico del decorso. Linfusione di proteina C esogena, sebbene non sia in grado di diminuire la mortalit della sepsi grave e dello shock settico, pu risolvere in sicurezza lo sbilancio coagulativo correlato a questi stati patologici. Metodi. In 3 centri italiani di terapia intensiva pediatrica stato eseguito uno studio retrospettivo, che ha preso in considerazione un periodo di tempo pari ad 1 anno, sullutilizzazione della proteina C nella sepsi grave e nello shock settico. Sono stati analizzati

1. Amaral A, Opal SM, Vincent JL. Coagulation in sepsis. Intensive Care Med 2004;30:1032-40. 2. Fisher CJ Jr, Yan SB. Protein C levels as a prognostic indicator of outcome in sepsis and related diseases. Crit Care Med 2000;28 Suppl 9:S49-56. 3. Macias WL, Nelson DR. Severe protein C deficiency predicts early death in severe sepsis. Crit Care Med 2004;32 Suppl 5:S223-8. 4. Gerson WT, Dickerman JD, Bovill EG. Severe acquired protein C deficiency in purpura fulminans associated with disseminated intravascular coagulation: treatment with protein C concentrate. Pediatrics 1993;91:418-22. 5. Rivard GE, David M, Farrel C. Treatment of purpura fulminans in meningococciemia with protein C concentrate. J Pediatr 1995;126:646-52. 6. Smith OP, White B, Vaughan D. Use of protein-C concentrate, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans. Lancet 1997;350:1590-3. 7. Ettingshausen CE, Veldmann A, Beeg T. Replacement

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