In vitro Influence of Fibrinogen and Factor XIII on Viscoelastic Characteristics of Clot Formation under Hypothermia and Acidosis

C l i n M e d International Library Citation: Ammon DA, Andruszkow H, Sieg L, Wilhelmi M, Weber CF, et al. (2015) In vitro Influence of Fibrinogen and Factor XIII on Viscoelastic Characteristics of Clot Formation under Hypothermia and Acidosis . Int J Blood Res Disord 2:019 Received: October 29, 2015: Accepted: November 25, 2015: Published: November 27, 2015 Copyright: © 2015 Ammon DA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Ammon et al. Int J Blood Res Disord 2015, 2:2 ISSN: 2469-5696


Introduction
Acute coagulopathy in trauma patients is generally associated with high mortality [1].Though decreasing during the last years, exsanguination is, beyond traumatic brain injury, the main cause of death in multiple trauma patients in the early onset [2,3].Hypothermia and acidosis occur in patients suffering from multiple injuries and independently induce coagulopathy [4].While acidosis alone severely impairs plasmatic coagulation, platelet function and coagulation time is more affected in presence of hypothermia [5].Beside a decreased enzymatic function of coagulant factors, another cause for coagulation impairment in different settings is loss of Tem International GmbH, Munich, Germany) have been described elsewhere [13], but will be repeated briefly in the following.Rotation thromboelastometry acts by using a pin which is dipped into a bloodfilled cup.The pin is rotating in a precisely defined angle.While the blood begins to clot fibrin polymers aggravate rotation which is charted on a diagram, which in this way enables the visualization of blood coagulation.All measurements were conducted due to manufacturer's instructions.We performed two ROTEM analyses: EXTEM and FIBTEM, both as extrinsically activated tests (activation by recombinant tissue factor).Additionally, FIBTEM contains Cytochalasin D which inhibits platelet function and displays fibrin polymerization only [14].Following EXTEM parameters were recorded: coagulation time (CT [s]) which describes the time from the beginning of the reaction until first clot formation; clot formation time (CFT [s]): time from first clot formation to a defined clot firmness (20 mm); clot firmness after 20 minutes (A20 [mm]) as a marker for the clot stability.Regarding FIBTEM A20 values were recorded.

Sample preparation
Samples were subdivided into four groups according to addition of different substrates: • Native (ISO: addition of 300 mcl 0.9% sodium chloride for compensation of dilutional effects) • Fibrinogen (FIB+: 0.25 mg fibrinogen in 300 mcl, equivalent to 4 g fibrinogen (Haemocompletan, CSL Behring, Hattersheim, Germany) in a 70 kg human) • FXIII (FXIII+: 0. For hypothermic measurements the ROTEM device was cooled down by the integrated soft-and hardware.The regular device integrated thermal control was used to reassure accurate temperature for the specific measurements.

Statistical analysis
The data were analyzed using the statistical package for the social sciences (SPSS; version 20; IBM Inc., Somers, NY, USA).Incidences are presented with counts or percentages while continuous values are presented as mean ± standard deviation (SD).Differences between the groups were evaluated with analysis of variance (ANOVA) for continuous data.The Tukey post-hoc test was used when appropriate to identify differences between the aforementioned classified groups.
A two sided p-value < 0.05 was considered to be significant.

Results
Blood samples were withdrawn from seven males and three females, ages 29 ± 6 years.ROTEM results are shown in figure 1, figure 2, figure 3 and figure 4.

FXIII+
As compared to ISO values FXIII+ did not affect any of the examined parameters significantly (CT, CFT, MCF, FIBTEM) at any measuring point.

FIB+
FIB+ on contrary significantly affected CT, CFT and MCF at all examination points as compared to ISO.Reconstitution of baseline values of CFT and MCF under hypothermia in EXTEM was not achieved at any pH-level.In FIBTEM however reconstitution of A20 in comparison to baseline was achieved at all pH-levels (p < 0.001).

Discussion
Coagulopathy is one main cause of mortality during major trauma in the early onset (46.0% with vs. 10.9% without coagulopathy) [1,4].
Two major aspects are responsible for development of coagulopathy, hypothermia and acidosis [15].Treatment aims to restore basic conditions for blood coagulation such as achieving and maintaining normothermia, normal pH, and normal values for calcium, platelets and coagulation factors which is recommended by the guidelines for treatment of patients with major trauma [16].While acidosis itself can be treated relatively easy and fast by application of sodium   [17].Although there are several options for rewarming hypothermic patients, it takes precious time.Ways of bridging this period of time and preventing coagulopathy are main subjects of interest.We could, referring to results we found in a preceding study, affirm that administration of fibrinogen improved all measured parameters: MCF (maximum clot firmness) and alpha-  angle (gradient angle of a ROTEM tracing) increased while CT and CFT shortened.Furthermore, we reconfirmed that hypothermia and acidosis synergistically lead to decreased clot firmness whereas both, clotting time and clot formation time, are prolonged while fibrin polymerization was not affected by either hypothermia or acidosis [10].
Fibrinogen is beside platelet function and FXIII the most important substrate for clot firmness [13].It takes part in primary as well as in secondary haemostasis [18].By binding platelets via the glycoprotein IIb/IIIa receptor on the platelet's surface [19] it is involved in the early phase of aggregation.After fibrinogen activation by thrombin, fibrin builds a network that in turn incorporates platelets and proteins in the late onset of coagulation [20].While the first compound of fibrin monomers is pretty loose it is cross-linked to covalent bounds by activated FXIII [9,21].FXIII is the zymogen of a transglutaminase that has many different functions of which not all are well understood yet.It exists in two different forms, a tissue form and one that is located in the plasma.The tissue form is presented on platelets, macrophages and the placenta and plays an important role in maintaining pregnancy and promotes wound healing [9].Patients suffering from FXIIIdeficiency show wound healing problems as well as prolonged and severe bleeding [22,23].In a prospective study on patients after intracranial surgery those with a decreased FXIII activity had a higher risk for postoperative hematoma [24].The plasma form is activated by thrombin which results in a proteolytic removal of an amino-terminal propeptide and a following, calcium dependent conformation change [25].Due to this change FXIIIa (activated FXIII) is able to build covalent cross-links of fibrin monomers to anneal clot firmness [9,25,26].Furthermore, by attaching alpha-2antiplasmin into the clot, FXIIIa also improves fibrinolysis-resistance and by engrafting fibronectin, collagen and other extracellular matrix proteins adhesion to the endothelium [8,9,[25][26][27][28].By inhibition of FXIII with antibodies in an in vitro study, Jámbor et al. verified a reduction of maximum clot firmness while clot formation time and clot lysis have been increased [29].Beside clot stabilization by crosslinking fibrinogen, glycoprotein IIb/IIIa, and cytoskeleton factors like actin and myosin [22,30], the underlying mechanisms are stimulating effects on angiogenesis and modeling of a matrix that ensures clotadhesion as well as facilitating migration of cells that take part in tissue repair such as monocytes and fibroblasts [31,32].
Main cause for our findings may be the concentration-depended platelet link-rate of fibrinogen which can be increased by raising the fibrinogen concentration [33], even in presence of anti-platelet drugs as for example clopidogrel [34] as well as in patients suffering from thrombocytopenia [13,35].There is evidence that high levels of fibrinogen may increase the risk for thrombosis, ischemic heart attack or stroke [36,37].Dependency of clot stability from fibrinogen levels in a dose dependent manner [38] as well as a strong correlation of postoperative thromboembolic events and elevated values of clot firmness have been described [39].In our study addition of FXIII showed no further benefit on clot formation under the given circumstances of hypothermia and acidosis -neither as single administration nor in combination with fibrinogen in comparison to the effect fibrinogen had exclusively.While our study participants showed normal FXIII values it could be possible that a ceiling effect of FXIII action can be responsible for our findings.In intensive care patients without hypothermia and acidosis but reduced FXIII levels a beneficial effect of FXIII on clot firmness has been described [40].Thus, we only can speculate if addition of FXIII in presence of reduced FXIII levels may increase clot stability under hypothermia or acidosis.Furthermore, speed of FXIII activation could possibly be limited due to unknown cofactors, since FXIII, its functions and influences are not fully explored yet.Further research is necessary.

Limitations
In this study all volunteers were healthy and had normal FXIII levels.There might be some point of saturation from which there is no benefit by adding extra FXIII.It requires further studies to detect a possible benefit of FXIII to trauma patients with a possible acquired FXIII-deficiency.However, even if the implemented tests connect close to physiological processes, an in vitro examination cannot consider all of the complex incidents taking place in the critically ill patient.Furthermore, the artificially simulated acidosis can just rudimentary be compared with the acidosis developed during trauma with its origin in inadequate oxygen supply and shock.Nevertheless the used in vitro model of acidosis and hypothermia represents a standard model.

Figure 1 :
Figure1: Results of rotation thrombelastometry coagulation time (CT) after extrinsic activation under hypothermia and acidosis.Groups are Native: control group with addition of isotonic saline solution for compensation of dilutional effects; Fib: addition of Fibrinogen; FXIII: addition of Factor XIII; Fib FXIII: addition of both substances.Asterisks ( * ) mark significant differences to ISO, # mark significant differences to FXIII.

Figure 2 :
Figure 2: Results of rotation thrombelastometry clot formation time (CFT) after extrinsic activation under hypothermia and acidosis.Groups are Native: control group with addition of isotonic saline solution for compensation of dilutional effects; Fib: addition of Fibrinogen; FXIII: addition of Factor XIII; Fib FXIII: addition of both substances.Asterisks ( * ) mark significant differences to ISO, # mark significant differences to FXIII.

Figure 3 :
Figure 3: Results of rotation thrombelastometry maximum clot firmness (MCF) after extrinsic activation under hypothermia and acidosis.Groups are Native:control group with addition of isotonic saline solution for compensation of dilutional effects; Fib: addition of Fibrinogen; FXIII: addition of Factor XIII; Fib FXIII: addition of both substances.Asterisks ( * ) mark significant differences to ISO, # mark significant differences to FXIII.

Figure 4 :
Figure4: Results of fibrin polymerization after extrinsic activation under hypothermia and acidosis.Groups are Native: control group with addition of isotonic saline solution for compensation of dilutional effects; Fib: addition of Fibrinogen; FXIII: addition of Factor XIII; Fib FXIII: addition of both substances.Asterisks ( * ) mark significant differences to ISO, # mark significant differences to FXIII.