![Proposed mechanisms of TFPI inhibition of factor X activation
In 1998, Baugh et al. [9] proposed a scheme with two reaction pathways for TFPI inhibition of factor X activation: direct and indirect. In our work, we consider a modification of Baugh’s scheme by restricting it to elementary reactions only and removing the multi-step reaction 5. (A) Representative structures for coagulation factors and associated labels. (B) Biochemical reactions involved in the direct inhibition of factor X activation. (C) Biochemical reactions involved in the indirect inhibition of factor X activation. (D) Schematic for the reactions involved in the activation of factor X by TF:VIIa and both the direct and indirect pathways of inhibition by TFPI.](https://www.researchgate.net/publication/385859019/figure/fig1/AS:11431281290580782@1731698818589/Proposed-mechanisms-of-TFPI-inhibition-of-factor-X-activation-In-1998-Baugh-et-al-9_Q320.jpg)
![Experimental measurements and uncertainty in model predictions of factor X activation
(A) Factor X (170 nM) activated by TF:VIIa (0.032 to 1.024 nM) in the presence of TFPI (2.4 nM). (B) Factor X (170 nM) activated by TF:VIIa (0.128 nM) in the presence of TFPI (2.4 nM), preincubated with factor Xa (0.00 to 1.00 nM). Data extracted from Figure 3B of [9]. The curves show model predictions using median and literature values presented in Table 1, and the uncertainty in model predictions using posterior estimates (see S1 and S2 Figs): 70%, 90%, and 99% credible intervals about the median model prediction. Note that there is a replicate experimental condition between both experiments. The Xa = 0 nM pre-incubation condition in Experiment Two (B) matches the TFPI 0.128 nM curve in Experiment One (A).](https://www.researchgate.net/publication/385859019/figure/fig2/AS:11431281290636929@1731698818867/Experimental-measurements-and-uncertainty-in-model-predictions-of-factor-X-activation-A_Q320.jpg)
![Functional enzyme at steady state over flow rate
For each model, the steady state concentration of functional enzyme ([E] and [E:S]) is presented for flow rates from 10⁻³ (Low Flow) to 10³ sec⁻¹ (High Flow).](https://www.researchgate.net/publication/385859019/figure/fig5/AS:11431281290646051@1731698819793/Functional-enzyme-at-steady-state-over-flow-rate-For-each-model-the-steady-state_Q320.jpg)
November 2024
·
16 Reads
Blood coagulation is a vital physiological process involving a complex network of biochemical reactions, which converge to form a blood clot that repairs vascular injury. This process unfolds in three phases: initiation, amplification, and propagation, ultimately leading to thrombin formation. Coagulation begins when tissue factor (TF) is exposed on an injured vessel’s wall. The first step is when activated factor VII (VIIa) in the plasma binds to TF, forming complex TF:VIIa, which activates factor X. Activated factor X (Xa) is necessary for coagulation, so the regulation of its activation is crucial. Tissue Factor Pathway Inhibitor (TFPI) is a critical regulator of the initiation phase as it inhibits the activation of factor X. While previous studies have proposed two pathways—direct and indirect binding—for TFPI’s inhibitory role, the specific biochemical reactions and their rates remain ambiguous. Many existing mathematical models only assume an indirect pathway, which may be less effective under physiological flow conditions. In this study, we revisit datasets from two experiments focused on activated factor X formation in the presence of TFPI. We employ an adaptive Metropolis method for parameter estimation to reinvestigate a previously proposed biochemical scheme and corresponding rates for both inhibition pathways. Our findings show that both pathways are essential to replicate the static experimental results. Previous studies have suggested that flow itself makes a significant contribution to the inhibition of factor X activation. We added flow to this model with our estimated parameters to determine the contribution of the two inhibition pathways under these conditions. We found that direct binding of TFPI is necessary for inhibition under flow. The indirect pathway has a weaker inhibitory effect due to removal of solution phase inhibitory complexes by flow.
![FIGURE 2 The [PSI + ] Weak variant persists after mating to the dominant [PSI + ] Strong variant. (A) Wildtype (WT) [psi − ], [PSI + ] Weak , and [PSI + ] Strong haploids were mated to a WT [PSI + ] Strong haploid without treatment (untreated, dark gray), in the presence of 3 mM GdnHCl (treated, light gray), and in the presence of 3 mM GdnHCl following growth of the [PSI + ] Strong haploid (1N) in the presence of GdnHCl for 12 h (titrated, white). The number of [PSI + ] Strong propagons from the resulting zygotes and a [PSI + ] Strong haploid was quantified and presented as box-whisker plots. Horizontal lines indicate 25th, 50th, and 75th percentiles; whiskers indicate 10th and 90th percentiles; diamonds indicate means; outliers are presented as dots. The number of zygotes analyzed is presented in parentheses below each box plot. The equality of means across all groups was tested using Welch's ANOVA test (p* ANOVA ); p-values were determined using pairwise unequal variance t-tests and are presented above each box plot for comparisons within a cross. The absence of a p-value for comparisons within a cross indicates a lack of significant difference. Full statistical comparisons are available in Supplementary Table S3. (B) The number of [PSI + ] Weak propagons from the zygotes presented in (A) was quantified and presented as box-whisker plots as in (A). The equality of means across all groups was tested using Welch's ANOVA test (p* ANOVA ); p-values were determined using pairwise unequal variance t-tests and are presented above each box plot for comparisons within a cross. The absence of a p-value for comparisons within a cross indicates a lack of significant difference. Full statistical comparisons are available in Supplementary Table S4. (C) The persistence of the [PSI + ] Weak variant was observed by microscopy in crosses between [PSI + ] Strong and [PSI + ] Weak haploids expressing Sup35(G20D)-GFP under the control of the P tetO7 promoter. GFP foci were detected in cells at the periphery of the resulting microcolony, indicated by white arrows in the inset (200-fold magnification).](https://www.researchgate.net/publication/383115477/figure/fig1/AS:11431281274782874@1725062629280/The-PSI-Weak-variant-persists-after-mating-to-the-dominant-PSI-Strong-variant_Q320.jpg)
![FIGURE 4 The dominant [PSI + ] Strong variant competes for soluble Sup35 more efficiently than the recessive [PSI + ] Weak variant. (A) Wildtype [PSI + ] Strong (blue) and [PSI + ] Weak haploids (salmon) were mated to a [PSI + ] Weak haploid expressing GFP-tagged Sup35 under the control of the P MFA1 promoter. Fluorescence intensity was quantified in the mother lobe of a singly budded zygote following repeated photobleaching of the daughter (left), and the rate of fluorescence loss was calculated and presented as box-whisker plots (right, n ≥ 11). Horizontal lines indicate 25th, 50th, and 75th percentiles; whiskers indicate 10th and 90th percentiles; diamonds indicate means. An unequal variance t-test was used to determine p values (B) A [PSI + ] Weak haploid expressing a GFP-tagged Sup35 mutant (G20D) under the control of the P MFA1 promoter was mated to wildtype [PSI + ] Strong (blue) and [PSI + ] Weak (salmon) haploids and analyzed as in (A, n ≥ 13).](https://www.researchgate.net/publication/383115477/figure/fig2/AS:11431281274792558@1725062629474/The-dominant-PSI-Strong-variant-competes-for-soluble-Sup35-more-efficiently-than-the_Q320.jpg)
