Factor V has an anticoagulant cofactor activity that targets the early phase of coagulation

Abstract

Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa. Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV), which has been activated by FXa. Because full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that although FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ∼8-fold reduction in K_i compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FV^ΔIIa) that cannot be cleaved/activated by thrombin or FXa also enhanced TFPI-mediated inhibition of FXa ∼12-fold in the presence of protein S. In contrast, neither activated FV nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants, we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FV^ΔIIa (but not activated FV) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.

Keywords: FV; TFPI; anticoagulant; coagulation factor; enzyme inhibitor; inhibition mechanism; phospholipid; protein S; protein-protein interaction; tissue factor pathway inhibitor.

Figure 1.

Enhancement of TFPI-mediated inhibition of FXa by protein S and FV. A–D, TFPI (0–4 nm) alone (A) or in the presence of 100 nm protein S (B), 30 nm FV (C), or 30 nm FV and 100 nm protein S (D) was incubated with 200 μm substrate S-2765, 25 μm of phospholipids vesicles (DOPC/DOPS/DOPE, 60:20:20), and 5 mm CaCl₂. FXa (0.3 nm) was used to initiate the reaction, and FXa activity was measured through cleavage of the chromogenic substrate at 405 nm over 60 min. The results from a representative experiment (n = 5) are shown. PS, protein S.

Figure 2.

Comparison between recombinant and plasma-purified FV. Recombinantly produced in house FV (rFV) or ppFV were loaded on a 4–12% gel (4.95 μg/lane), blotted, and probed either with a polyclonal anti-human protein S antibody or a mixture of monoclonal antibodies directed against the K1, K2, and K3 domains and the C terminus of TFPI. As standard, 2 ng of recombinant TFPI and 69 ng of PS were used. For comparison, immunoblots of recombinant FV and ppFV (16.5 ng/lane) were probed either with a polyclonal antibody against FV (anti-FV pAb) or a monoclonal antibody directed against the light chain of FV (anti-FV LC mAb). Note that the monoclonal antibody has higher affinity for cleaved FV rather than full-length FV, as stated in the manufacturer's instructions.

Figure 3.

Enhancement of TFPI-mediated inhibition of FXa by protein S and FV is specific and phospholipid-dependent. A–D, FXa activity (0.3 nm) was followed in real time through cleavage of S-2765 (200 μm) at 405 nm in the presence of 25 μm phospholipids and the presence or absence of 0.25 nm TFPI, 100 nm protein S, and 30 nm FV (A). The same experiment was performed in the presence of anti-FV (300 nm, B), anti-protein S (1000 nm, C), and anti-FV (300 nm) + anti-protein S (1000 nm, D) antibodies. E and F, FXa activity (0.3 nm) was followed in real time through cleavage of S-2765 (200 μm) at 405 nm in the presence or absence of 1 nm TFPI, 100 nm protein S and 30 nm FV, and in the presence (E) or absence (F) of 25 μm phospholipids (DOPC/DOPS/DOPE, 60:20:20). PS, protein S. Control indicates reactions performed in the absence of TFPI, protein S, FV, and antibodies. Representative experiments are shown (n = 3).

Figure 4.

Effect of FV, FV^ΔIIa, and FVa on thrombin generation by FXa. Different FV forms (200 pm) were incubated with 1 μm prothrombin, 117 μm substrate S-2238, 25 μm of phospholipids vesicles (DOPC/DOPS/DOPE, 60:20:20), and 5 mm CaCl₂ before addition of FXa (0.6 pm). Thrombin activity was measured through cleavage of the chromogenic substrate at 405 nm over time.

Figure 5.

Enhancement of TFPI-mediated inhibition of thrombin generation by FXa in the presence or absence of FV and protein S. TFPI (0–16 nm) in the absence (●) or presence (○) of 100 nm protein S in the absence of FV (A) and in the presence of 200 pm FV (B), FV^ΔIIa (C), or FVa (D) was added to a prewarmed (10 min, 37 °C) solution containing 1 μm prothrombin, 117 μm substrate S-2238, 25 μm of phospholipids vesicles (DOPC/DOPS/DOPE, 60:20:20), and 5 mm CaCl₂. FXa (60 pm (A), 0.6 pm (B and C), or 0.06 pm (D)) was used to initiate the reaction, and thrombin activity was measured through cleavage of the chromogenic substrate at 405 nm over time. Thrombin formation was expressed as a percentage of the rate obtained in the absence of TFPI. The results are expressed as means ± S.E. (n = 4–7). Note the x axis scale change in B and C compared with A and D to better show the enhancement by FV and PS at lower TFPI concentrations. For each curve, only the linear range of curve was used for measuring thrombin activity as stated in the text and under “Experimental procedures.”