and the Endogenous Thrombin Potential in Controls and Septic Patients
A. Siegemund, T. Siegemund, U. Scholz, S. Petros and L. Engelmann
Background
The physiologic effects of activated protein C (APC) are manifold. It inactivates the clotting factors Va and VIIIa, thereby inhibiting the prothrombinase and tenase activities. It also inhibits the proinflammatory consequences of local thrombin generation (platelet activation and secretion of vasoactive and proinflammatory substances) and activates endogenous fibrinolysis by binding to PAI-1.
In a randomized double-blind placebo-controlled study [1], administration of the activated protein C concentrate drotrecogin alfa (Xigris) has been found to result in an absolute mortality reduction of 6.1%. On the contrary, the positive effects of human inactive protein C concentrate have been described in small studies [2] and case reports [3] only. These are particularly patients with a heredi- tary protein C deficiency and with meningococcal sepsis, but also others with severe sepsis and consumption coagulopathy.
Considering these facts and the positive effects of the (inactive) human protein C concentrate (although not proven in a randomized clinical study), the present ex- vivo study was aimed to discuss the following questions:
1. Can the thrombin generation curve and the variables computed thereof be suitable to depict the complex processes involving protein C?
2. Are there differences between administration of activated and inactive human protein C regarding the thrombin generation curve?
3. Are there differences between healthy controls and septic patients in their reac- tion to the administration of protein C?
Patients and Methods
The study was conducted using blood samples from 10 healthy controls without thrombotic risk factors and patients with severe sepsis under treatment at the Medical ICU of the University of Leipzig. Sepsis was defined according to the ACP/SCCM criteria [4]. Blood was drawn via an antecubital venipuncture in con- trols and through an indwelling central vein catheter in patients after an informed consent. Patients did not receive any anticoagulant during the previous 12 hours before the blood draw.
I. Scharrer/W. Schramm (Ed.)
34thHemophilia Symposium Hamburg 2003
” Springer Medizin Verlag Heidelberg 2005
Thrombin generation was measured in platelet-rich plasma (PRP) as previously described [5]. The endogenous thrombin potential (ETP), thrombin peak (peak_h), time to peak (peak_t), start vs. end of the reaction as well as thrombin inhibition were computed. Protein C activity was measured with the Behring Coagulation
Thrombin peak, highest reaction rate (height of the peak, time to peak)
Endogenous thrombin potential (area under the curve)
End of reaction
Start of reaction Thrombin generation [FU/s]
Fig. 1a–c. Parameters of thrombin genertion curve
b
System (Dade Behring) applying the chromogenic method (Berichrom Protein C, Dade Behring Marburg GmbH). Measurements were carried out before and after addition of APC (drotrecogin-a, Xigris, Lilly Deutschland GmbH) and inactive pro- tein C (Ceprotin, Baxter Deutschland GmbH) ex vivo.
Data analysis was conducted using SPSS for windows version 10.0 (Chicago, Illinois). The Student t-test was applied for data comparison. Parametric data are given as mean ± SD, and a p <0.05 was considered statistically significant.
Results
Thrombin generation is dependent on platelet count in healthy controls as well as in patients. Therefore, inter-individual comparison is only possible with a defined platelet count (Fig. 2).
Drotrecogin-a reduced ETP and thrombin peak significantly (p<0.001); it also markedly delayed the start of thrombin formation (i.e. it prolonged the lag phase) (p<0.001). These reactions were concentration-dependent (Fig. 3). The inactive human protein C concentrate Ceprotin reduced the thrombin peak and prolonged the lag phase, but these effects were not statistically significant.
Another important physiological aspect is the thrombin inhibition, which is defined as the mean negative reaction velocity between maximum thrombin peak and the end of the reaction. It could be demonstrated that thrombin inhibition is also dependent on the platelet count and protein C activity (Fig. 4). At low platelet counts, small amount of APC is enough to prevent thrombin formation, while high-
Throm binpeak
Inhibition FU/[s-2]
time to end
End of Reaction Thrombin generation [FU/s]
Fig. 1c
c
er APC concentrations are needed with increasing platelet counts. The study on our healthy controls have shown that at a platelet count of 20 x 109/l only a quarter of the APC required at a platelet count of 200 x 109/l was enough for a comparable degree of thrombin inhibition.
In septic patients, it could be demonstrated that thrombin generation decreases with increasing APC concentrations in a similar manner to that observed in healthy controls (Fig. 5).
Discussion
Protein C plays a significant role as a physiological anticoagulant in the hemostatic system. After being activated via the thrombin-thrombomodulin mechanism, it inactivates the coagulation factors Va and VIIIa, thereby slowing the activities of the prothrombinase and tenase complexes, respectively. This process contributes to a marked reduction in thrombin generation. Protein C also activates endogenous fibrinolysis by binding to plasminogen activator inhibitor 1 (PAI-1). It also inhibits the proinflammatory consequences of local thrombin formation.
Platelets play a significant role in the process of thrombin generation, a role that has been underestimated in the past. Our data demonstrate that the amount of pro- tein C to be administered in a given subject should be dependent on the platelet count.
The endogenous thrombin potential represents all the factors involved in thrombin formation. Indirect measurements of thrombin generation, such as thrombin-antithrombin complex or the prothrombin fragments have also shown to be significantly reduced after administration of the activated protein C concentrate drotrecogin-a [1] or purified inactive human protein C [6]. In our study, the effect
Platelets [GPT/l]
Fig. 2. Thrombin peak as function of platelets
Thrombin generation [FU/s]
Thrombin generation [FU/s]
without 1.0 mg/l
without 2.0 mg/l time of measurement [s]
time of measurement [s]
Fig. 3. Influence of activated Protein C on Thrombin Generation (Platelets 100 Gpt/I)
of inactive human protein C on the thrombin generation curve was not significant.
The reason for this may be the lack of thrombomodulin or the endothelial throm- bin receptor in the test system, so that activation of protein C would be very slow.
The transition of protein C from its inactive zymogen to the active form is slowed in septic patients (7), so that administration of inactive protein C would not result in an optimal influence on the hemostatic system.
Concentration Xigris [µg/l]
Fig. 4. Inhibition of thrombin generation – Influence of activeted protein C and platelets
Protein C activity [% of normal]
Maximal Reaction Rate Thrombin Peak [FU/s]
maximal platelets
Fig. 5a. Influence of protein C activity in Sepsis – ETP (maximal reaction rate)
This is a preliminary study demonstrating that measurement of thrombin generati- on in platelet-rich plasma may be a valuable tool to describe the complex processes of coagulation in sepsis. Further investigations are necessary to elaborate on the role of active and inactive protein C.
References
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6. Conrad Conard J, Bauer KA, Gruber A, Griffin JH, Schwarz HP, Horellou MH, Samama MM, Rosenberg RD. Normalization of markers of coagulation activation with a purified Protein C concentrate in adults with homozygous protein C deficiency. Blood 1993; 82: 1159-64.
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29 (Suppl.): 69-74.
Endogenous thrombin potential [FU]
Protein C activity [% of normal]
maximal platelets
Fig. 5b. Influence of protein C activity in Sepsis – ETP (area under the curve)
H. Lenk (Leipzig)
