Genetic and Clinical Determinants of Chronic Thromboembolic Pulmonary Hypertension: The Role of PAI-1 Polymorphism

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The effort of the authors to retrospectively analyze such a large number of patients (204) with pulmonary thromboembolism is commendable.

However, the manuscript proposed for publication does not bring a consistent scientific and practical additional information:

1. The role of congenital hypercoagulability (thrombophilia) and acquired hypercoagulability (comorbidities, medication, predisposing conditions, risk factors) in the occurrence of pulmonary thromboembolism is well known.
2. In practice, there are several risk scores (Padera Prediction Score, Caprini) for PE, clinical probability scores for PE (Wells score, Revised Geneva Score) that ensure comfort in clinical practice for the earliest possible diagnosis of patients with pulmonary embolism.
3. Recognized thrombophilia as the predictors of PE are: factor V Leiden, thrombin mutation G202101, antithrombin deficiency, protein C and S deficiency, antiphospholipid antibodies. The PAI-1 4G/5G polymorphism is not a recognized predictor of pulmonary embolism by the current guidelines (ASH, ACCP, ISTH). Moreover, a series of large studies suggest that PAI-1 4G/5G, where present, has a modest and not independent role in predicting PE.

Author Response

However, the manuscript proposed for publication does not bring a consistent scientific and practical additional information:

1. The role of congenital hypercoagulability (thrombophilia) and acquired hypercoagulability (comorbidities, medication, predisposing conditions, risk factors) in the occurrence of pulmonary thromboembolism is well known.

-Response:

We thank the reviewer for this important comment and fully agree that the role of congenital and acquired hypercoagulability in the occurrence of acute pulmonary thromboembolism (PTE) is well established.

However, we would like to respectfully emphasize that the primary focus of our study is not the risk of acute PTE, but rather the determinants of progression from acute PTE to chronic thromboembolic pulmonary disease (CTEPD), which represents a distinct and less well-characterized clinical entity.

While classical thrombophilic conditions are known contributors to venous thromboembolism, their role in impaired thrombus resolution and chronic vascular obstruction remains controversial. Importantly, our study demonstrates that most traditional thrombophilic mutations (Factor V Leiden, Factor II, MTHFR variants) were not associated with CTEPD development, whereas the PAI-1 4G/5G polymorphism, a key regulator of fibrinolysis rather than coagulation, showed a strong association.

This finding suggests that genetic determinants influencing fibrinolytic capacity, rather than procoagulant tendency alone, may play a more relevant role in the pathogenesis of chronic thromboembolic disease. By integrating genetic data with clinical, echocardiographic, and biomarker profiles, our study provides novel insight into why some patients fail to achieve complete thrombus resolution despite adequate anticoagulation, thereby progressing to CTEPD.

To clarify this distinction, we have revised the Introduction sections to more explicitly differentiate between risk factors for acute PTE and predictors of chronic thromboembolic sequelae, and to highlight the potential mechanistic role of impaired fibrinolysis in CTEPD development.

Manuscript revision: Although thrombophilic factors are well recognized in the occurrence of acute pulmonary thromboembolism, their contribution to thrombus persistence and the development of chronic thromboembolic disease remains incompletely understood.

 

2. In practice, there are several risk scores (Padera Prediction Score, Caprini) for PE, clinical probability scores for PE (Wells score, Revised Geneva Score) that ensure comfort in clinical practice for the earliest possible diagnosis of patients with pulmonary embolism.

Response:

We thank the reviewer for this valuable comment and fully agree that several well-validated risk assessment and clinical probability scores, such as the Caprini, Wells, and Revised Geneva scores, play an essential role in the early identification and diagnosis of acute pulmonary embolism in clinical practice.

However, we would like to emphasize that these tools are specifically designed to assess the risk or probability of acute PE occurrence, rather than to predict long-term outcomes or the development of chronic thromboembolic sequelae after an established PE diagnosis.

To date, there is a lack of validated clinical or genetic risk stratification models that can reliably identify patients at increased risk of progressing from acute PE to chronic thromboembolic pulmonary disease (CTEPD). Our study addresses this unmet clinical need by focusing on post-PE disease evolution, integrating clinical variables, echocardiographic findings, biomarkers, and genetic predisposition.

Importantly, we demonstrate that genetic factors related to fibrinolytic impairment, particularly the PAI-1 4G/5G polymorphism, together with comorbidities and early right ventricular involvement, are associated with an increased risk of CTEPD—an outcome not captured by existing PE risk or probability scores.

3. Recognized thrombophilia as the predictors of PE are: factor V Leiden, thrombin mutation G202101, antithrombin deficiency, protein C and S deficiency, antiphospholipid antibodies. The PAI-1 4G/5G polymorphism is not a recognized predictor of pulmonary embolism by the current guidelines (ASH, ACCP, ISTH). Moreover, a series of large studies suggest that PAI-1 4G/5G, where present, has a modest and not independent role in predicting PE.

Response:

We thank the reviewer for this important and accurate clarification. We fully agree that the PAI-1 4G/5G polymorphism is not recognized as an established predictor of acute pulmonary embolism, and it is not included among classical thrombophilic conditions in current guidelines (ASH, ACCP, ISTH).

Importantly, our study does not propose PAI-1 4G/5G as a risk factor for the occurrence of acute PE. Instead, our focus is on its potential role in the post-embolic disease course, particularly in impaired thrombus resolution and the development of chronic thromboembolic pulmonary disease (CTEPD).

Unlike classical thrombophilic mutations that predominantly affect coagulation pathways, PAI-1 is a key regulator of fibrinolysis. The 4G allele has been associated with increased PAI-1 expression and reduced plasminogen activation, which may contribute to persistent organized thrombi despite adequate anticoagulation, a hallmark of chronic thromboembolic disease.

Consistent with the reviewer’s comment, previous studies have demonstrated that the association between PAI-1 4G/5G and acute PE is modest and often not independent. However, the relevance of this polymorphism may be more pronounced in the context of thrombus persistence rather than thrombus formation. Our findings support this distinction, as classical thrombophilic mutations were not associated with CTEPD, whereas the PAI-1 polymorphism showed a significant association.

To avoid misinterpretation, we have revised the Discussion sections to explicitly state that PAI-1 4G/5G is not a predictor of acute PE, but may represent a modifier of thrombus resolution and chronic disease progression.

Manuscript revision: Although the PAI-1 4G/5G polymorphism is not recognized as a risk factor for the occurrence of acute pulmonary embolism, one of the most notable findings of our study was its association with the development of CTEPD.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did a good job in writing this manuscript on "Genetic and Clinical Determinants of CTEPH: The Role of PAI-1 polymorphism".  The authors did a single center retrospective cohort study and included 204 patients diagnosed with acute PTE between December 2023 - December 2024.  The authors collected the baseline demographic , clinical, laboratory and echocardiographic data.  Genetic analysis assessment of Factor II, Factor V Leiden, MTHFR C677T, MTHFR A128C, Factor XIII V34L, and PAI-1 4G/5G polymorphism was performed.  Patients were followed for at least 12 months for the development of CTEPH.  Among the genetic variants only the PAI-1 4G/5G polymorphism was significantly associated with CTEPD (p=0.001).  The authors mention several limitations.  The authors conclude that advanced age, comorbid diseases, elevated cardiac biomarkers and genetic predisposition are significant predictors of CTEPD after acute PTE emphasizing the multifactorial pathogenesis of CTEPD highlighting the need for close monitoring in genetically predisposed patients.  However, please note the following points:

1. How did you determine the power of the study.  Did you do any statistical analysis to determine the relevant number of patient recruitment for statistically valid interpretation of results.  
2. Given "the relatively small number of CTEPD events may have reduced the statistical power to detect associations with less prevalent genetic variants", please mention how you could have overcome this.  Discuss extending the study for a longer time and multicenter design could have helped in getting robust data.    
3. The authors hypothesize "that specific genetic polymorphisms, particularly those influencing fibrinolysis may increase susceptibility to CTEPD beyond the contribution of traditional clinical risk factors", please mention in the Discussion why Thrombin Activatable Fibrinolytic Inhibitor (TAFI) gene polymorphisms were not assessed.  The authors may consider at least mentioning about it citing references in the literature to improve Bibliography.  
4. Please consider to improve the Bibliography by including some recently published (2025) paper "Genetic Analysis of Patients with CTEPH: A  single-Center Observational study and discuss the findings of this study in the Discussion section.  (Genes (Basel) 2025 Nov 6; 16(11):1336.  The author studied the Tier 1 and 2 genes related to coagulation, fibrinolysis and platelet disorders - as recommended by ISTH.
5. The authors mention in the Conclusions in the Abstract "Our study demonstrates that advanced age, comorbid diseases, elevated cardiac biomarkers and particularly the presence of the PAI-1 4G/5G polymorphism are significant predictors of CTEPD after acute PTE".  However, in the text  the authors write "Although, the majority of patients who developed CTEPD carried the PAI-1 mutation, a considerable number of patients without CTEPD also had this genetic variant.  Therefore, the predictive value of the PAI- 4G/5G polymorphism should be interpreted with caution".  Please discuss the true predictive value of PAI-1 4G/5G in the light of limited power of the study.  
6. As mentioned in the Limitations, the invasive right heart catheterization was not available that precluded the definitive confirmation of CTEPH, which questions the validity of the results obtained.  
7. The authors mention in the Limitations "more comprehensive genetic profiling could provide additional insight into the genetic predisposition to chronic thromboembolic disease", please mention what could be included in that type of comprehensive genetic profiling which was not included here.  The authors may wish to discuss the 2025 paper mentioned in comment number 4 above.

Author Response

1. How did you determine the power of the study. Did you do any statistical analysis to determine the relevant number of patient recruitment for statistically valid interpretation of results. 

Response:

We thank the reviewer for this important methodological question. We performed an a priori sample size estimation using G*Power (v3.1.9.7) based on the between-group difference observed in a key continuous parameter (CTEPD+ vs CTEPD−; mean 51.4 vs 36.9; SD 16.0 vs 14.9). Assuming α=0.05, power=0.95, and the required minimum sample was 34 (effect size d=0.938). The final cohort (n=204) exceeded this threshold.

We acknowledge that although this calculation supports adequacy for detecting the specified between-group difference in the continuous outcome, the relatively small number of CTEPD events limits the precision of multivariable and genetic subgroup analyses; this has been addressed in the limitations.

Manuscript Revision: An a priori sample size estimation was performed using G*Power software (version 3.1.9.7). Based on an expected between-group difference in a key continuous parameter, the calculated effect size was d=0.938. With α=0.05, power=0.95, and the minimum required sample size was 34.   

2. Given "the relatively small number of CTEPD events may have reduced the statistical power to detect associations with less prevalent genetic variants", please mention how you could have overcome this.  Discuss extending the study for a longer time and multicenter design could have helped in getting robust data. 

Response:

We thank the reviewer for this constructive suggestion and fully agree that the limited number of CTEPD events may have reduced the statistical power to detect associations with less prevalent genetic variants.

This limitation could have been addressed by extending the study duration, which would allow the inclusion of a larger number of patients and capture additional CTEPD events, given the relatively low incidence and delayed manifestation of chronic thromboembolic disease.

Furthermore, a multicenter study design would substantially increase sample size, enhance event accrual, and improve the representativeness and generalizability of the findings.

In addition, a multicenter approach would enable more comprehensive evaluation of rare genetic variants and facilitate subgroup analyses that are not feasible in single-center cohorts. Prospective multicenter registries with standardized follow-up protocols and harmonized genetic analyses may therefore provide more robust and definitive evidence regarding the role of genetic predisposition in CTEPD development.

These considerations have been explicitly addressed in the Discussion section as directions for future research.

Manuscript revision: The relatively small number of CTEPD events may have limited the statistical power to detect associations with less prevalent genetic variants. Extending the study period would likely increase event accrual, while a multicenter design could provide a larger and more diverse patient population, allowing more robust analyses of genetic subgroups. Future prospective multicenter studies with longer follow-up and standardized genetic assessment are warranted to validate and extend our findings.

3. The authors hypothesize "that specific genetic polymorphisms, particularly those influencing fibrinolysis may increase susceptibility to CTEPD beyond the contribution of traditional clinical risk factors", please mention in the Discussion why Thrombin Activatable Fibrinolytic Inhibitor (TAFI) gene polymorphisms were not assessed.  The authors may consider at least mentioning about it citing references in the literature to improve Bibliography.  

Response:

We thank the reviewer for this insightful comment and agree that thrombin-activatable fibrinolytic inhibitor (TAFI) represents an important regulator of fibrinolysis and may be biologically relevant to chronic thromboembolic disease.

TAFI gene polymorphisms were not assessed in the present study primarily due to methodological and practical constraints, including the predefined genetic panel available at our institution during the study period and the retrospective design. Our genetic analysis was therefore limited to the most commonly used and clinically accessible thrombophilia-related polymorphisms in routine practice.

We acknowledge that TAFI, through its inhibitory effect on fibrin-bound plasminogen activation, may contribute to impaired thrombus resolution and chronic vascular obstruction. To address this important point, we have added a dedicated discussion of TAFI and its potential relevance to CTEPD pathophysiology, supported by references from the existing literature. We also highlight TAFI as a promising target for future studies employing broader genomic or targeted fibrinolysis-related gene panels.

Manuscript revision: In addition to PAI-1, thrombin-activatable fibrinolytic inhibitor (TAFI) has been identified as another key modulator of fibrinolysis. TAFI attenuates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin, thereby reducing plasminogen binding and plasmin generation. Several studies have suggested that TAFI gene polymorphisms and elevated TAFI activity may be associated with venous thromboembolism and impaired thrombus resolution. However, TAFI polymorphisms were not assessed in the present study due to the retrospective design and limitations of the predefined genetic testing panel. Future studies incorporating a broader spectrum of fibrinolysis-related genes, including TAFI, may provide a more comprehensive understanding of the genetic determinants of chronic thromboembolic pulmonary disease (20,21).

4. Please consider to improve the Bibliography by including some recently published (2025) paper "Genetic Analysis of Patients with CTEPH: A  single-Center Observational study and discuss the findings of this study in the Discussion section.  (Genes (Basel) 2025 Nov 6; 16(11):1336.  The author studied the Tier 1 and 2 genes related to coagulation, fibrinolysis and platelet disorders - as recommended by ISTH.

Response:

We thank the reviewer for this valuable and timely suggestion. We agree that the recently published 2025 single-center observational study in Genes provides important insights into the genetic background of chronic thromboembolic disease and is highly relevant to our work.

Accordingly, we have included this study in the revised Bibliography and expanded the Discussion section to incorporate its key findings. In that study, Bereczky et al. performed next-generation sequencing of ISTH-recommended Tier 1 and Tier 2 genes related to coagulation, fibrinolysis, and platelet disorders, as well as selected vascular genes, in patients with CTEPH compared with pulmonary embolism controls without CTEPH. The authors did not identify a single recurrent causative mutation; instead, they reported a heterogeneous distribution of rare, non-synonymous variants across multiple biological pathways, supporting a polygenic and multifactorial genetic architecture.

We have discussed how these findings are consistent with our results, which similarly suggest that genetic predisposition contributes to the development of chronic thromboembolic pulmonary disease within a broader multifactorial framework rather than through a single deterministic variant. In particular, the absence of exclusive pathogenic variants in fibrinolysis-related genes such as CPB2 (encoding TAFI) in the Genes study underscores the complexity of fibrinolytic regulation and supports our cautious interpretation of the statistically significant association observed for the PAI-1 4G/5G polymorphism.

The Discussion has been revised to explicitly integrate this recent genomic evidence and to highlight the need for larger, multicenter, and harmonized genetic studies to further elucidate the polygenic determinants of CTEPD and CTEPH.

Manuscript Revision: A recent single-center observational study by Bereczky et al. (22) used next-generation sequencing to interrogate ISTH-recommended Tier 1 and Tier 2 genes related to coagulation, fibrinolysis, and platelet disorders, along with vascular genes, in patients with CTEPH compared with PE controls without CTEPH. The authors reported no single recurrent causative variant, but rather a heterogeneous distribution of rare, non-synonymous variants across multiple pathways, including coagulation, altered fibrinolysis, and impaired angiogenesis, supporting a polygenic disease architecture. Notably, although CPB2 (encoding TAFI) has been proposed as a candidate gene in chronic thromboembolic disease, no exclusive variants in CPB2 were identified among CTEPH cases, underscoring the complexity of fibrinolysis-related genetic contributions and the need for larger, harmonized cohorts.These findings are consistent with our results, which also suggest that genetic factors contribute to CTEPD development within a broader multifactorial framework. In our cohort, the observed association between genetic predisposition and CTEPD supports the concept that chronic thromboembolic disease arises from the interaction of multiple genetic and clinical determinants rather than a single causative variant, highlighting the importance of multifactorial mechanisms in CTEPD pathogenesis.

 

5. The authors mention in the Conclusions in the Abstract "Our study demonstrates that advanced age, comorbid diseases, elevated cardiac biomarkers and particularly the presence of the PAI-1 4G/5G polymorphism are significant predictors of CTEPD after acute PTE".  However, in the text  the authors write "Although, the majority of patients who developed CTEPD carried the PAI-1 mutation, a considerable number of patients without CTEPD also had this genetic variant.  Therefore, the predictive value of the PAI- 4G/5G polymorphism should be interpreted with caution".  Please discuss the true predictive value of PAI-1 4G/5G in the light of limited power of the study. 

 

Response:

We agree that the predictive value of the PAI-1 4G/5G polymorphism should be interpreted with caution in light of the limited number of CTEPD events. However, it is important to emphasize that the association between PAI-1 4G/5G and CTEPD was statistically significant in our cohort, indicating a non-random relationship.

Although a considerable proportion of patients without CTEPD also carried this polymorphism, the significantly higher prevalence among patients who developed CTEPD suggests that PAI-1 4G/5G may contribute to disease susceptibility. The limited event number may have reduced the power to fully delineate its independent predictive value and discriminative performance at the individual level.

Therefore, we interpret the PAI-1 4G/5G polymorphism as a statistically significant but contributory genetic factor, acting within a multifactorial framework rather than as a standalone predictor. The Abstract have been revised accordingly to reflect both the statistical significance of the association and the methodological limitations related to power.

Manuscript revision: Our study demonstrates that advanced age, comorbid diseases, elevated cardiac biomarkers, and genetic predisposition are significant predictors of CTEPD after acute PTE. These findings emphasize the multifactorial pathogenesis of CTEPD, highlighting the need for close monitoring in genetically predisposed patients. associated with the development of CTEPD after acute PTE, while the PAI-1 4G/5G polymorphism may contribute to CTEPD susceptibility within a multifactorial context.

 

6. As mentioned in the Limitations, the invasive right heart catheterization was not available that precluded the definitive confirmation of CTEPH, which questions the validity of the results obtained.

Response:

We thank the reviewer for this important comment. We fully agree that invasive right heart catheterization is required for the definitive diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH). However, we would like to clarify that the primary outcome of our study was chronic thromboembolic pulmonary disease (CTEPD) rather than hemodynamically confirmed CTEPH.

In line with current ESC/ERS recommendations, CTEPD represents a clinically relevant entity characterized by persistent thromboembolic lesions and functional impairment, which may be identified using non-invasive imaging and echocardiographic findings in the absence of systematic right heart catheterization. Our study was therefore deliberately designed to evaluate predictors of post-embolic chronic thromboembolic disease within this broader clinical spectrum.

Accordingly, the absence of routine right heart catheterization does not invalidate our results but defines the scope and applicability of our conclusions, which are limited to CTEPD rather than CTEPH. We have emphasized this distinction throughout the manuscript and have explicitly acknowledged the lack of invasive hemodynamic confirmation as a limitation, while avoiding extrapolation to hemodynamically confirmed CTEPH.

7. The authors mention in the Limitations "more comprehensive genetic profiling could provide additional insight into the genetic predisposition to chronic thromboembolic disease", please mention what could be included in that type of comprehensive genetic profiling which was not included here.  The authors may wish to discuss the 2025 paper mentioned in comment number 4 above.

Response:

We thank the reviewer for this important clarification request. By “more comprehensive genetic profiling,” we refer to a broader genomic approach that extends beyond targeted thrombophilia panels and includes next-generation sequencing of genes involved in coagulation, fibrinolysis, platelet function, and vascular remodeling, as recommended by the International Society on Thrombosis and Haemostasis (ISTH).

Specifically, such profiling could incorporate ISTH-recommended Tier 1 and Tier 2 genes, including but not limited to genes regulating fibrinolysis (e.g., PAI-1, CPB2/TAFI), coagulation factors, platelet signaling pathways, endothelial function, and angiogenesis-related pathways. In addition, rare and low-frequency variants, copy number variations, and non-synonymous mutations not captured by conventional thrombophilia panels may be identified using this approach.

In this context, we have expanded the Discussion to incorporate the recent 2025 single-center observational study published in Genes, which applied next-generation sequencing to ISTH Tier 1 and 2 genes in patients with CTEPH. That study demonstrated a heterogeneous distribution of rare variants across multiple biological pathways without identifying a single recurrent causative mutation, supporting a polygenic and multifactorial genetic architecture. We discuss how these findings align with our results and further emphasize the need for larger, harmonized genomic studies to clarify the genetic determinants of chronic thromboembolic disease.

Manuscript revision: Finally, only selected thrombophilic mutations were analyzed; future studies incorporating broader genomic approaches, including next-generation sequencing of genes involved in coagulation, fibrinolysis, platelet function, and vascular remodeling, may provide additional insights into the genetic predisposition to chronic thromboembolic disease (22).

 

 

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for incorporating reviewer's suggestions.  The manuscript reads well now.

Congratulations