The Pediatric Infectious Disease Journal, 2016,Sept:35(9):967-971.
Non-activated protein C in the treatment of neonatal sepsis: a retrospective analysis of outcome.
Ryszard Lauterbach, Barbara Wilk, Agata Bocheńska, Joanna Hurkała, Renata Radziszewska.
Department of Neonatology, Jagiellonian University Medical College, Kraków, Poland.
Objectives: Previously, we found that plasma protein C (PC) activity ≤ 10% significantly increased the risk of death during neonatal sepsis. The aim of present study was to elucidate the impact of PC zymogen supplementation in septic infants with plasma PC activity ≤10% on the treatment effects.
Methods: Plasma PC activity was determined in all infants when a presumptive diagnosis of sepsis was done. If the activity of plasma PC was ≤ 10%, either found at the beginning of sepsis or declined during the course of sepsis, we administered a non-activated PC zymogen, given as a single dose of 200 IU/kg.
Results: A significantly (≤10%) reduced activity of plasma PC was found in 18,5% of all infants with confirmed sepsis. It was 5 times more likely in infants with early-onset sepsis compared to infants with late-onset sepsis. Among 458 septic patients, death occurred in 19 infants (4.2%), exclusively with plasma PC activity ≤10%. The median birth weight and gestational age of treated infants were, respectively, 1010.0 g and 29 weeks.
Conclusions: There is an increased risk of death in septic neonates with significantly lowered plasma PC activity. Therefore we suggest either the necessity for its evaluation in the course of neonatal sepsis or supplementation when the plasma PC activity is below 10%.
PMID: 27254035; DOI: 10.1097/INF.0000000000001247
The inflammatory and pro-coagulant host responses to infection are closely related. Inflammatory cytokines activate coagulation cascade and acute thrombin formation, inhibit fibrinolysis, resulting in multi-organ failure and death [1, 2]. PC is a vitamin K-dependent natural anticoagulant, which exerts a crucial role in the modulation of coagulation, fibrinolysis and inflammation [3, 4]. Although the neonatal PC system seems well prepared to rectify a thrombin formation, lower levels of plasma PC, as antithrombotic defenses during sepsis, are more limited in neonates than in older children and adults .
Several studies have demonstrated that decreased activity of plasma PC in septic patients is associated with increased morbidity and mortality [6, 7]. However, in a large trial of pediatric sepsis, activated protein C given in septic children younger than two months, increased the incidence of intracranial hemorrhage . On the other hand, in several studies, either retrospective or prospective uncontrolled, the safety profile of protein C zymogen was considered acceptable, and potential beneficial effects were suggested . Normal values in neonates for plasma protein C activity depend on gestational age and range between 20% and 60% (in adults they range respectively between 65% and 150%) . Previously, we reported that evaluation of plasma protein C activity might have a prognostic value in the course of sepsis . We found that plasma protein C activity ≤ 10% significantly increased the probability of the occurrence of death during neonatal sepsis.
The biological rationale for PC zymogen supplementation is the anti-coagulant, anti-inflammatory, pro-fibrinolytic and anti-apoptotic action of protein C [12, 13]. Activation of protein C in the microcirculation may promote local anticoagulation and fibrinolysis without substantially increased systemic levels due to the short half time of the activated protein C within the circulation. Moreover, data obtained experimentally indicated that cytoprotection and modulation of inflammation, rather than anticoagulation, are the activities related to the survival advantage of activated protein C administration . A subsequent in vitro study has shown that the anti-inflammatory and cytoprotective properties are attributed to the zymogen rather than to the activated protein C . Also, an activation of protein C zymogen on endothelial cells was about five times more effective in protecting endothelial barrier properties than an equivalent concentration of exogenously administered activated protein C . Moreover, it was also suggested that the improvement in coagulation markers, observed after protein C zymogen administration, might result from the modulation of the inflammatory response elicited by the increased levels of protein C zymogen 
Replacement with protein C zymogen concentrate is an established treatment for congenital protein C deficiency, but its use in acquired deficiency state, such as exists during neonatal sepsis, and is not approved by governmental agencies. Moreover, the majority of the neonatal units do not evaluate protein C activity in septic neonates. It is important to underline that in our present study, the decision for starting the replacement therapy was undertaken only after septic infants had a measurement of plasma protein C activity. On the grounds of data obtained in our previous clinical study  we decided on a “cut-off” value of 10% or less. This would hence be appropriate to infuse non-active protein C zymogen concentrate. There was no large published study on preterm infants with confirmed sepsis, who received an infusion of human protein C zymogen concentrate. Moreover, no such study evaluated plasma protein C activity before and after treatment.
In our study, protein C zymogen concentrate was given as a single dose of 200 IU/kg. Previously we started to use a dose of 60-80 IU/kg body weights, which was generally recommended in cases of severe congenital protein C deficiency. However, an increase in protein C activity, measured 10-12 hours after protein C zymogen concentrate administration was not effective and there was an urgent need for repeated infusion to obtain protein C activity above 10%. Therefore, a dosage of protein C zymogen concentrate, given in septic neonates was increased to 200 IU/kg. We speculated that higher doses of concentrate could have advantages in preterm patients with sepsis in whom the biological half-life of protein C was markedly reduced. Whether this is an optimal dosage it should be assessed in blind controlled, multicenter studies. However, in our study, we observed a statistically significant increase in plasma protein C activity evaluated 10 – 12 hours after infusion and there were no adverse events associated with its administration. Also, there was no need for repeated infusions in the further course of sepsis in all infants treated with that dose of protein C zymogen concentrate.
The overall mortality rate in the relatively large (458 patients) group of infants with confirmed sepsis, mostly extremely immature (median of gestational age: 29 weeks) and with a very low birthweight (median of birthweight in group A: 1010g) was 4.2%. The most frequently presented mortality rate in this group of newborns oscillates around the value of 10-20% . Whether this percentage might have any association with the amount of septic neonates with plasma protein C activity ≤10%, which we found in our study (18.6%) it is difficult to prove because the majority of the neonatal units do not evaluate protein C activity in septic neonates. However, we speculate that dependence may exist.
The aim of our retrospective analysis was to evaluate how frequently a significantly reduced plasma protein C activity complicates sepsis developed in newborn infants and whether it influences mortality and clinical course of disease. We found that in the group of infants with confirmed sepsis, treated during the 6 year period of time, death occurred exclusively in several of those (22.4%), who presented a significantly decreased (median, 6.0%; range, 0.0% – 9%)) plasma protein C activity. Other authors, who also evaluated plasma protein C activity in term neonates with sepsis, found that all infants with plasma protein C activity below 12% died, and those whose respective values dropped below 6%, died with the symptoms of disseminated intravascular coagulation. It is necessary to remember, that none of the evaluated septic term infants in this study were treated with protein C zymogen concentration .
Finally, we conclude that evaluation of plasma protein C activity in premature septic patients, who otherwise have a high mortality, seems to be an important diagnostic tool with a sensitive prognostic value. We also realize that retrospective analysis in the absence of a control group of untreated patients such as ours is far from providing clinical efficiency in terms of mortality and morbidity. However, it cannot be ruled out that such beneficial effect on the clinical course of sepsis might have occurred due to PC zymogen administration.
Fig. 1. Full-term newborn with confirmed early-onset-sepsis; a superficial thrombosis on plantar surface developed on the third day of life – pathogen – Gram negative bacteria; plasma protein C activity (5%) was evaluated on the third day of life and protein C zymogen concentrate was given in a dose of 200 IU/kg.
Fig. 2. This patient on the 10th day of life
Fig. 3. This patient on the 14th day of life
- Amaral A, Opal SM, Vincent JL. Coagulation in sepsis. Intensive Care Med. 2004; 30: 1032-1040.
- Levi M, van der Poll T. Coagulation in patients with severe sepsis. Semin Thromb Hemost 2014, e-pub ahead of print September 2014 doi: 10.1055/S-004-28132.
- Esmon CT, Gu JM, Xu J et al. Regulation and functions of the protein C anticoagulant pathway. Haematologica. 1999, 84: 363-368.
- Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost. 2001; 86: 51-56.
- Cvirn G, Gallistl S, Muntean W. Effects of antithrombin and protein C on thrombin generation in newborn and adult plasma. Thromb Res. 1999; 93: 183-190.
- Fisher CJ, Yan SB. Protein C levels as prognostic indicator of outcome in sepsis and related diseases. Crit Care Med, 2000; 28; Suppl 9: S49-56.
- Macias WL, Nelson DR. Severe protein C deficiency predicts early death in severe sepsis. Crit Care Med , 2004; 32: 223-228.
- Nadel S, Goldstein B, William MD et al. for the Researching Severe Sepsis and Organ Dysfunction in Children: a Global Perspective (RESOLVE) Study Group: Drotrecogin alfa (activated) in children with severe sepsis: a multicenter phase III randomized controlled trial. Lancet 2007; 360; 836-843.
- Decembrino L, D’Angelo A, Manzato F et al. Protein C concentrate as adjuvant treatment in neonates with sepsis-induced coagulopathy: a pilot study. Shock, 2010; 34: 341-345.
- Andrew M, Paes B, Milner R, et al. Development of the human coagulation system in the healthy preterm infant. Blood, 1988; 72: 1651-1657.
- Lauterbach R, Pawlik D, Radziszewska R, et al. Plasma antithrombin III and protein C levels in early recognition of late-onset sepsis in newborns. Eur J Pediatr, 2006; 165: 585-589.
- Flutcher CA, Gardiner JE, Griffin JH, et al. Proteolytic inactivation of activated human factor VIII procoagulant protein by activated protein C and its analogy to factor V. Blood, 1984; 63: 486-489.
- Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. Chest. 2003; 124; (3, Suppl): 26S-32S.
- Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood. 2007, 109: 3161-3172.
- Bae JS, Yang L, Manithody C, et al. The ligand occupancy of endothelial protein C receptor switches the protease-activated receptor 1-dependent signaling specificity of thrombin from a permeability enhancing to a barrier-protective response in endothelial cells. Blood. 2007; 110: 3909-3916.
- Feistritzer C, Schuepbach RA, Mosnier LO, et al. Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells. J Biol Chem. 2006; 281: 2077-2084.
- Crivellari M, Della Valle P, Landoni G, et al. Human protein C zymogen concentrate in patients with severe sepsis and multiple organ failures after adult cardiac surgery. Intensive Care Med. 2009; 35: 1959-1963.
- Wynn JL, Wong HR. Pathophysiology and treatment of septic shock in neonates. Clin Perinatol. 2010; 37: 439-479.
- Beshlawy EA, Alaraby I, Hussein HA, et al. Study of protein C, protein S and antithrombin III in newborns with sepsis. Pediatr Crit Care Med, 2010; 11: 52-59