Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation

Abstract

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (< 20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.

Argatroban enhances fibrinolysis by differential inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor and factor XIII

“…With regard to inhibition of thrombin-mediated TAFI activation, it is likely that no important TAFI activation occurred in either argatroban or UFH anticoagulated samples, as the MRTG observed was less than 5 dynes/cm 2 per s, the critical speed of clot growth (and concordant thrombin generation/activity) that must be exceeded in this model to reliably activate TAFI [9]. These data [9] are consistent with the observation that TAFI activation is a threshold phenomenon, requiring an ambient thrombin concentration more than 120 nmol/l to inhibit fibrinolysis in similar, thrombelastographic-based models [10]. Finally, argatroban exposure did not affect MRL in TAFI-deficient plasma ( Table 3, Fig.…”

“…However, FXIIIa is more than 100-fold faster at crosslinking fibrin polymers than TAFIa is at cleaving Cterminal lysine residues. Thus, unless thrombomodulin activity is ideal, FXIIIa-mediated transglutaminase reactions will likely proceed at a more rapid pace than TAFIamediated events with similar thrombin generation, especially if near the 120 nmol/l threshold [10].…”

Thrombin generation and fibrinolysis in anti‐factor IX treated blood and plasma spiked with factor VIII inhibitor bypassing activity or recombinant factor VIIa

“…To evaluate the effects of FXa on LOT and LT, increasing concentrations of FXa (0.02–10 n m ) were used as a trigger. The results were compared to LOT and LT values obtained by increasing concentrations of rFVIIa (10, 60, 120, 360 n m ) and by thrombin (10 n m ; Recothrom ® ; ZymoGenetics, Seattle, WA, USA) as a trigger. Whole blood samples were incubated with tPA (0.15 μg mL −1 ) [16,17] and anti‐FIXa aptamer (48 μg mL −1 ) and used to study the effect of aPCC (0.4 U mL −1 ) and rFVIIa (60 n m ) on LOT and LT using thrombin (2 n m ) or tissue factor (TF; 2 μL of EXTEM ® , Pentapharm, Munich, Germany) as a trigger. Whole blood samples with aptamer and tPA or with tPA only served as controls.…”

“…Whole blood samples with aptamer and tPA or with tPA only served as controls. Normal coagulation time and maximal clotting firmness [16] in WB from healthy volunteers induced by 2 μL of EXTEM ® were defined as 189 ± 56 s and 61 ± 2 mm respectively and by thrombin 2 n m as 116 ± 24 s and 63 ± 2 mm respectively in a preliminary study. To evaluate the fibrinolytic potential of aPCC (0.4 U mL −1 ) and rFVIIa (60 n m ) on haemophilia B plasma, we performed ROTEM™ analyses in FIX‐deficient plasma in the presence of tPA (0.15 μg mL −1 ) [16,17] using thrombin (10 n m ) as a trigger. Plasma samples without aPCC or rFVIIa served as a control.…”

On the Mechanism of Action of Prolylcarboxypeptidase

“…FXI can be activated to activated FXI (FXIa) by FXIIa-mediated activation on the negatively charged surfaces or thrombinmediated activation on the platelet surface (Baglia et al, 2004), Figure 2. FXIa augments thrombin generation via interaction with a coagulation factor of the extrinsic pathway, factor IX (Baglia and Walsh, 1998;Taketomi et al, 2008). It appears that FXI along with activated platelet are a prerequisite for optimum thrombin generation in which the procoagulant activity of KKS is so just below the threshold point (Keularts et al, 2001).…”