Data from our in vitro study demonstrate that it is possible to restore coagulation properties by combining defined concentrations of coagulation factor concentrates in an albumin based colloid solution. The viscoelastic clot formation parameters A 10 and MCF observed in CRF were comparable not only to human plasma, but also to whole blood under presence of platelets. The surrogate parameter for thrombin generation, CT, was prolonged when compared to our internal control group, but reached normal levels for whole blood when analyzed in presence of platelets at 100 × 103/µl 14. Fluid substitutes like CRF could be an interesting treatment option, with clinical indications comparable to those of fresh frozen plasma, to avoid the development of multifactor coagulopathy under damage control resuscitation.
Massive bleeding, independent of its etiology (trauma, obstetrical or surgical), usually includes high ratio FFP transfusion guided by institutional MTPs, based on a large body of evidence favoring plasma against coagulation factor-free resuscitation fluids 11,18. This hemostatic resuscitation concept however is insufficient to avoid MT associated multifactor coagulopathies 19. Factor containing fluids for volume therapy, like CRF, that are characterized by optimized viscoelastic properties could be an appealing new treatment component. Additional POC monitoring would still allow for goal-directed top-up corrections for non-dilutional coagulopathy mechanisms like loss or consumption, but monitoring intensity could possibly be reduced. The immediate availability without thawing time and the universal applicability independent of blood group compatibility in a clinical emergency situation could be further attractive characteristics in favor of CRFs.
Human albumin 5% was chosen as carrier solution for the coagulation factor compound of our CRF for different reasons. First, colloids were favored against crystalloids, because accurately defined coagulation factor concentrations within a predefined volume of a resuscitation fluid would only make sense, if the underlying carrier liquid showed adequate and prolonged volume effects in the intravascular space. Second, the colloid should not interfere in a significant manner with the coagulation system. Although all colloid solutions show dilutional effects, albumin solutions, together with gelatins, seem to cause less colloid-specific, detrimental effects on platelet function or fibrin polymerization than other colloids, like dextrans, or starches 20,21. We preferred albumin against gelatins to optimize comparability to FFP in future trials. Experimental studies show that albumin based colloid solutions provide stabilizing effects on the endothelial barrier and show intravascular plasma expander effects of nearly 100% 22, 23. By contrast, no such effects on the endothelial barrier could be demonstrated for CFCs 24. The administration of well-balanced coagulation factors in carrier solutions with constant intravascular volume effects might be a safe way to treat bleeding associated coagulopathies, as “overshooted” peak plasma concentrations caused by the infusion of highly concentrated factor formulas (as under non-diluted CFC administration) would be avoided. The disruption of the glycocalyx integrity under special clinical conditions, however, seems to increase the endothelial permeability of albumin solutions and might negatively influence on the theoretical advantage of albumin against other colloids or crystalloids25.
Hemostasis is a result of a coordinated interaction between platelets and coagulation mechanisms 26. Coagulation mechanisms necessary for consolidation of platelet mediated primary hemostasis require a cascade of enzymatic reactions leading to the formation of fibrin 16, 27, 28. Results of the present study indicate that coagulation mechanisms can be reproduced using a restricted number of coagulation factors suspended in a neutral fluid. To our knowledge this is the first experimental study that has been able to demonstrate that the combination of commercially available CFCs in an initially coagulation factor- and blood-cell-free solution leads to the formation of a stable in vitro fibrin clot. The initiation of the coagulation in this fluid requires the use of EXTEM or FIBTEM reagents whose components (calcium, phospholipids and tissue factor) would trigger the activation of the coagulation factors VII, IX, X and II contained in commercial PCCs 6. These coagulation factors apparently lead to sufficient thrombin generation and warrant the basic activating mechanism of the coagulation system to sustain in vitro fibrin polymerization. FGN provides the structural clotting substrate supporting secondary hemostasis 29. The necessary concentration in CRF to reach normal TEM values (when combined with FXIII) were found in the range of physiological plasma concentrations, around 4 g/l for FGN and 0,5 − 1 IU/ml for FXIII. FXIII cross-links fibrin, completing blood coagulation and protecting the hemostatic plug from fibrinolytic activity at the clot formation site. In vitro studies demonstrated that supplementation with FXIII increases clot firmness assessed by TEM in perioperative patients with elevated FGN and reduced FXIII levels 30. However, in another in vitro model of MT in trauma, combination therapies with FGN and fresh frozen plasma, but not FXIII, improved both coagulation kinetics and fibrin-based clot strength 31, 32. Our present study indicates that increasing concentrations of FXIII enhance clot strength at fixed concentrations of PCC (1 IU/ml) and FGN (4 g/l). Consistently, there is further evidence that FXIII deficiency will impair FGN function and fibrin formation, suggesting an inverse link between low FXIII levels and enhanced thrombin generation, modifying the structure-function relationship of fibrin to support hemostasis 33. Data derived from clinical studies propose maintenance of 50–60% of FXIII activity to avoid bleeding tendency in the perioperative setting 34.
CRF compositions without FXIII, yielding comparable clot strength in TEM when compared to our final composition, are possible from a theoretical point of view. We decided to add FXIII to our final CRF composition despite the high potential of FGN on viscoelastic clot strength to maintain a close-to-physiological factor composition.
The safe upper limit of FGN treatment has not been precisely defined. It was initially suggested that levels of FGN should reach 1.5 to 2 g/l in bleeding patients 35. There is a clear tendency, as reported in different guidelines, to recommend elevating plasma FGN in some clinical situations 36–38. Taking into consideration the results of our TEM studies it may be difficult to maintain a well-balanced coagulation factor composition during the course of a long-lasting, high-dynamic bleeding event if supplements are only POC driven and punctual. In this context a fixed ratio of clotting factors in CRFs administered under volume therapy could provide more balanced stability within the complex multifactor system of blood coagulation than single factor substitutes as proposed in current algorithms.
CT in TEM is partially dependent on thrombin generation. Direct anticoagulants reducing thrombin generation definitively prolong CT 39. Platelets contribute to enhance thrombin generation, accelerate CT, and increase MCF. Additionally, platelet phospholipids dramatically contribute to the amplification of coagulation mechanism, thus potentiating thrombin generation and fibrin polymerization. Fibrin then interacts with activated platelets and plays a critical role for MCF. CT values of platelet-free CRF samples in our in-vitro experiments were significantly prolonged when compared to plasma CT levels of our internal control group. Several reasons may account for these findings:
First, our in vitro samples were completely free of any phospholipids or cell membrane fragments that could influence on factor activation. Consequently, the addition of platelets to CRF containing 1 IU/ml PCC, 4 g/l of FGN and 1 IU/ml FXIII leads to the normalization of CT and MCF. It could be assumed from our studies that, when combined with CRF, a platelet count around 100 × 103/µl should be required to fully reconstitute TEM parameters to levels observed in whole blood studies (see Fig. 2A-C). CT values above 80 s are considered to reflect pathological thrombin generation and are generally accepted as treatment threshold. CT values of CRF combined with platelets were significantly shorter than this generally recommended treatment thresholds 16 (Tables 1 and 5).
Second, the used PCC in our experiments contains heparin. Other study groups previously reported about CT sensitivity of extrinsically activated TEM tests 40. It is questionable if this phenomenon has any clinical relevance. The currently scientific rationale rather suggests that PCCs might possibly be associated to overshooted thrombin generation with potential to induce disseminated intravascular coagulation and that Antithrombin III (AT) supplements might mitigate this potentially dangerous adverse effect 9. The complete absence of AT in the final CRF composition is a major limitation of our experiments and the effects of PCC supplements in clinical situations with reduced AT levels will have to be analyzed in future trials.
A further limitation of our experimental studies is the complete absence of red blood cells. Red blood cells seem to exert a more important role in primary hemostasis, whereas their modulating effect on secondary hemostasis seems to be negligible 13. In fact, viscoelastic studies can be reliably performed in plasma samples 12, 13. Altogether, the transfer of our data into a clinical context must therefore be done very carefully. All factor components of CRFs have previously been safely administered in loose compositions for the management of bleeding associated coagulopathy 41. PCCs show a reliable safety profile and are now treatment of choice for the emergency reversal of Vitamin K antagonists 42, 43. Nevertheless, a careful assessment of the thrombogenic potential of fixed factor combinations for the treatment of a multiple factor deficit under massive bleeding will have to be performed in future studies.