Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

Abstract

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.coagulation | coagulation factor V | coagulation factor XI | feedback activation T he mechanism behind the activation of the intrinsic pathway of coagulation in vivo has been subject to discussion since the introduction of the model of coagulation (1, 2). The importance of the intrinsic pathway for physiological hemostasis is apparent from the bleeding phenotypes that are seen in factor VIII (FVIII) deficiency, factor IX (FIX) deficiency and, to a lesser extent, in factor XI (FXI) deficiency. In vitro, activation of the intrinsic pathway can be initiated by factor XII (FXII) on negatively charged surfaces, which results in clotting. However, the role of FXII-dependent coagulation in physiological hemostasis is controversial. FXII deficiency, for instance, protects against thrombosis in murine models (3), which conflicts with the protective role of high FXII antigen levels in epidemiological studies on thrombosis in humans (4), as well as with the absence of bleeding symptoms in FXII-deficient individuals. Additionally, FXII hyperactivity leads to angioedema, rather than to thrombosis (5), which corresponds with evidence that activation of the kallikrein-kinin system by FXII can take place independent of coagulation (6).As FXI deficiency, unlike FXII deficiency, is associated with mild bleeding phenomena, a FXI activator other than FXII is likely. Thrombin, the final and central enzyme of the coagulation cascade, can directly activate FXI, which leads to downstream formation of more thrombin (7,8). This feedback activation mechanism offered an alternative explanation for the existence of the intrinsic pathway: The amplification of minute amounts of thrombin, ...

Polyphosphate is a cofactor for the activation of factor XI by thrombin

“…Although multiple studies have reported the activation of FXI by thrombin using purified proteins, [8][9][10][11][12] some have questioned whether this reaction can proceed to any significant extent in plasma. [13][14][15] Figure 2.…”

“…A solution to this conundrum was proposed in 1991 by Naito and Fujikawa 8 and by Gailani and Broze,9 who reported that thrombin up-regulates its own generation by feeding back to activate FXI, leading to a "revised model of coagulation." [9][10][11] More recently, Matafonov et al identified that ␤-thrombin and ␥-thrombin, proteolyzed derivatives of ␣-thrombin, also can activate FXI in plasma. 12 The proposal that FXI activation by thrombin plays a significant role in blood clotting in vivo is somewhat controversial.…”

Activation of factor XI by products of prothrombin activation

“…21 A recent report suggests that FV modestly enhances FXI activation by ␣-IIa. 27 We tested both FV and FVa in the chromogenic substrate assay but saw no evidence for an effect on FXI activation with any type of thrombin.…”

“…21 A recent report suggests that FV modestly enhances FXI activation by ␣-IIa. 27 We tested both FV and FVa in the chromogenic substrate assay but saw no evidence for an effect on FXI activation with any type of thrombin.Previously, we described an FXII-deficient plasma system in which thrombin generation from endogenous prothrombin requires FXI, Ca 2ϩ , and a trigger, such as a small amount of ␣-IIa. 19 Results supported the conclusion that ␣-IIa activates FXI in plasma.…”

“…Values were converted to nanomolar FXIa using a standard curve. Effects of FV or FVa on FXI activation by thrombin were determined by the method of Maas et al 27 Briefly, FXI (30nM) was incubated with thrombin (10nM) with or without FV or FVa (30nM, a gift from Dr Paul Bock) in Tyrode buffer with 3mM CaCl 2 at 37°C for 10 minutes. Argatroban was added to 5M, and FXIa generation determined by monitoring cleavage of S-2366.…”