Inhibition of platelet-surface-bound proteins during coagulation under flow I: TFPI

Abstract

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Tissue factor pathway inhibitor (TFPI) is one such inhibitor, well known for its inhibitory action on the active enzyme complex comprising tissue factor (TF) and activated clotting factor VII. This complex forms when TF embedded in the blood vessel wall is exposed by injury and initiates coagulation. A different role for TFPI, independent of TF:VIIa, has recently been discovered whereby TFPI binds a partially cleaved form of clotting factor V (FV-h) and impedes thrombin generation on activated platelet surfaces. We hypothesized that this TF-independent inhibitory mechanism on platelet surfaces would be a more effective platform for TFPI than the TF-dependent one. We examined the effects of this mechanism on thrombin generation by including the relevant biochemical reactions into our previously validated mathematical model. Additionally, we included the ability of TFPI to bind directly to and inhibit platelet-bound FXa. The new model was sensitive to TFPI levels and, under some conditions, TFPI could completely shut down thrombin generation. This sensitivity was due entirely to the surface-mediated inhibitory reactions. The addition of the new TFPI reactions increased the threshold level of TF needed to elicit a strong thrombin response under flow, but the concentration of thrombin achieved, if there was a response, was unchanged. Interestingly, we found that direct binding of TFPI to platelet-bound FXa had a greater anticoagulant effect than did TFPI binding to FV-h alone, but that the greatest effects occurred if both reactions were at play. The model includes activated platelets' release of FV species, and we explored the impact of varying the FV/FV-h composition of the releasate. We found that reducing the zymogen FV fraction of this pool, and thus increasing the fraction that is FV-h, led to acceleration of thrombin generation.

Figure 1

Newly added reactions involving FV-h, FXa, and $\text{TFPI}\alpha$. (Left) Generation of FV-h through the activation of FV by FXa and secretion from platelet stores, binding/unbinding of coagulation factors to the platelet surface, and the assembly of prothrombinase. (Right) Binding of $\text{TFPI}\alpha$ to platelet-bound FXa, FV-h, and prothrombinase, and inhibition of prothrombinase assembly by $\text{TFPI}\alpha$. Line styles indicate different interactions: dense dashed lines, binding and unbinding of protein pairs; longer dashed lines, catalytic reaction steps; solid double arrow, binding and unbinding of proteins with platelet surface; arrow with faded tail, secretion of protein from platelet. Light shade with black font indicates TFPI-bound and inhibited cofactors, and heavier shade with white font indicates $\text{TFPI}\alpha$-bound and inhibited enzymes. FV is also secreted from platelet stores and can be activated by thrombin, but this is not shown because they were included in an earlier model. Circled numbers indicate reaction number listed in Table 1. To see this figure in color, go online.

Figure 2

Effects of the TF level and shear rate on the lag time and the thrombin concentration threshold behavior. (A and C) Lag times and (B and D) thrombin concentration after 10 min for a range of TF densities and three different shear rates. Black curves represent the results from the new model, which includes the additional $\text{TFPI}\alpha$-mediated inhibition reactions, and gray curves represent the results from the old model, where $\text{TFPI}\alpha$ could only bind to fluid-phase FXa and then to TF:VIIa. All simulations were run with $\left[\text{TFPI}\alpha \right]$ = 0.5 nM. Vertical dashed lines indicate TF densities of interest. The flat horizontal lines in (A) and (C) at lag time >30 indicate that the thrombin concentration did not reach 1 nM within 30 min for the corresponding TF density.

Figure 3

Thrombin concentration versus time for the indicated $\text{TFPI}\alpha$ levels for shear rate 100/s and with (A) TF = 2.5 fmol/cm² and (B) TF = 6 fmol/cm². Note the different timescales. To see this figure in color, go online.

Figure 4

Lag times as functions of TF and $\text{TFPI}\alpha$ levels with the specified $\text{TFPI}\alpha$ binding reactions. Lag times for cases in which (A) $\text{TFPI}\alpha$ binding with both FV-h and FXa are turned off, (B) $\text{TFPI}\alpha$ binding with FV-h is turned on and $\text{TFPI}\alpha$ binding with FXa is turned off, (C) $\text{TFPI}\alpha$ binding with FXa is turned on and $\text{TFPI}\alpha$ binding with FV-h is turned off, and (D) $\text{TFPI}\alpha$ binding to both FV-h and FXa are turned on. For parameter values in the region without color, the thrombin concentration did not reach 1 nM thrombin within 40 min. Lag time versus TF density for $\text{TFPI}\alpha$ = 0.5 nM with and without $\text{TFPI}\alpha$ binding FV-h or/and FXa for (E) $\text{TFPI}\alpha$ = 0.5 nM and (F) $\text{TFPI}\alpha$ = 4.0 nM. These curves correspond to the slices in the heatmap indicated by the horizontal lines in (A)–(D). Shear rate is fixed to 100/s. To see this figure in color, go online.

Figure 5

Thrombin generation for $h_5=0,0.5,1.0$ for shear rate 100/s. (A) The $\text{TFPI}\alpha$ concentration is 0.5 nM and the TF density was either 2.5 or 6 fmol/cm². (B) The $\text{TFPI}\alpha$ concentration is 2.5 nM and the TF density was either 4 or 10 fmol/cm².