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denotes that this is a ticketed session.Program: Oral and Poster Abstracts
Session: 321. Blood Coagulation and Fibrinolytic Factors: Poster I (61 abstracts)
Introduction
The acute traumatic coagulopathy (ATC) which develops within 30 min following severe trauma with tissue damage and shock is defined by an increased prothrombin time (PT) and international normalized ratio (INR). While reduced thrombin might be expected in conjunction with elevated PT, recent clinical studies reveal paradoxically elevated thrombin generation potential in patients with ATC. We therefore hypothesized that the quantity of thrombin and the timing of thrombin-fibrinogen interactions both have an impact on clot quality; the exuberant production of thrombin found in trauma results in improper clot formation.
Methods
In vitro studies were conducted in human blood products and simplified synthetic plasma (consisting of purified human coagulation factors in HEPES buffered saline). Turbidimetry was used to observe fibrin crosslinking, while thromboelastography (TEG) was used to quantify clot formation parameters. Quantitation of fibrin(ogen) degradation products (FDPs) was conducted with the STA-R Evolution coagulation analyzer and by ELISA. A fluorogenic substrate was used to observe thrombin generation.
Results
Increasing the amount of prothrombin or thrombin (0-1400nM) in prothrombin-immunodepleted citrated plasma resulted in reduced clot times. The same dose response was examined in a buffered mixture of fibrinogen (300 mg/dl), FXIII (31.25nM), Ca2+ (2mM), and FXa (170nM—only used with prothrombin samples). However, while increasing prothrombin increased clot strength in both FII-deficient plasma and in the synthetic plasma, direct addition of thrombin decreased clot strength and by 3-fold at 1000nM versus 100nM (Figure 1; * p<0.05; ** p<0.01; ***p<0.001 between groups at given concentration); fibrin density was similarly reduced in turbidimetric assays. In TEG, the thrombin dose response did not affect whole blood or platelet-rich plasma, but in platelet-poor plasma the same clot strength inhibition trend was observed. Thrombin generation from the combination of prothrombin (0-1000 nM), FXa (170 nM), and Ca2+ (2 mm) was found to be saturated above an initial prothrombin concentration of 500nM.
An examination of FDPs from plasmin-degraded fibrinogen ± thrombin ± FXIII showed that FDPs from both crosslinked and uncrosslinked fibrin had 4-fold higher amounts of fibrin monomer and D-dimer than those produced from plasmin digestion of pure fibrinogen. When FDPs from thrombin +fibrinogen ± FXIII were supplemented back into fresh fibrinogen (0-50% of the final mixture) and allowed to clot again, there was a concentration-dependent decrease in fibrin formation rate (control: 0.15 OD/min; 50% FDP: 0.07 OD/min; p<0.001) which was not observed in samples treated with non-thrombin exposed FDPs. The use of these FDPs allowed for simulations of trauma patient plasma to be constructed using concentrations of plasma proteins associated with both “normal” trauma and the hypocoagulable state which manifests in ATC. When combining relevant levels of thrombin, fibrinogen, FDPs, and antithrombin III as are found in trauma patients that trend toward either good or bad outcomes, differences in TEG tracings can be observed illustrating the validity of these in vitro systems (Figure 2).
Conclusions
The dose responses of prothrombin versus thrombin reveal the significance of the timing of these reactions on proper clot formation. The conversion of prothrombin to thrombin mediated through FXa and FVa results in strong clots under normal circumstances. These results were reflected in both turbidimetry and TEG, indicating that fibrin crosslinking is being hindered by the presence of excess thrombin even in the presence of the antithrombin III. Additionally, FDPs from crosslinked substrates reduce new clot-making efficiency. Excess thrombin and significant increases in FDPs (which can result from fibrinolytic feedback loops) both contribute to an in vitro phenotype that may represent an underlying factor in the development of ATC.
Disclosures: No relevant conflicts of interest to declare.
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