Association between hemostasis and acute mesenteric ischemia: a Mendelian randomization analysis

doi:10.21203/rs.3.rs-4356518/v1

Background: Hemostatic abnormality has been observed in patients with acute mesenteric ischemia (AMI) but causal effects of hemostatic factors on AMI are still not clear.

Methods: We performed a two-sample Mendelian randomization(MR) analyses using summary-level genome-wide association study (GWAS) data to explore the potential causal effects of 23 hemostatic factors on acute mesenteric ischemia.

Results: Statistically significant association between genetically determined plasminogen levels and acute mesenteric ischemia were observed. A genetically predicted one SD increase of plasma plasminogen was associated with an odds ratio of 0.41(95% confidence interval [CI] 0.24 to 0.68, P = 0.0007). Sensitivity analysis of weighted median method also supported the result of main analysis. Other hemostatic factors in this study were not shown to have significant associations with AMI.

Conclusion: Genetically decreased blood level of plasminogen was causally associated with genetically predicted risk of AMI. Our result suggested potential causal roles hemostatic factors in the susceptibility and pathogenesis of AMI.

hemostasis

acute mesenteric ischemia

Mendelian randomization analysis

Acute mesenteric ischemia (AMI) is a severe vascular emergency caused by insufficient blood supply to the intestine. AMI can be occlusive or non-occlusive and has diverse forms. Common causes include mesenteric arterial embolus and thrombosis, mesenteric venous thrombosis and other conditions (e.g. shock, hypovolemia) that impair mesenteric perfusion. AMI is associated with 17.7% of emergent laparotomies for acute abdomen and can result in bowel ischemia, necrosis and death. Bowel resections are common in patients with AMI. Clinical outcomes of AMI remain poor.Timely diagnosis and intervention are often delayed and mortality rates of AMI range from 49–80% [1–3]. Accurate diagnosis and prompt treatment of patients with AMI are critical to improve the overall outcomes. It is also crucial to identify those who are at high risk of developing AMI.

Both inherited and acquired coagulation abnormalities are observed in acute mesenteric ischemia [4, 5]. A recent pathological study on ischemic bowels of patients with COVID-19 infections revealed thrombi in small intestinal vessels and hypercoagulable states [6]. D-dimer is a marker of activation of coagulation and fibrinolysis and is investigated as a biomarker of AMI. But its diagnostic accuracy is still controversial and there is currently a lack of definite biomarkers for accurate diagnosis of AMI [7]. Anticoagulation and thrombolysis treatments are used in several subtypes of AMI patients but their effects have not been evaluated or confirmed by high-quality trials [1–3]. Although various associations were observed, the roles of coagulation and fibrinolysis in AMI are still not clear. To the best of our knowledge, no studies have evaluated causal effects of hemostatic factors on AMI.

Mendelian randomization (MR) studies use genetic variations to assess causal relationship between the exposure and outcome. Genetic variants are allocated randomly at conception and confounding bias can be reduced by MR compared with conventional observational analyses. Single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWASs) were utilized as instrumental variables (IVs) [8]. If a hemostatic factor has causal effect on pathogenesis of AMI, its genetically predicted circulating levels should have impact on the risk or progression of the disease.

The aim of this study was to evaluate potential causal effects of genetically predicted measures of hemostasis on acute mesenteric ischemia using Mendelian randomization. We analyzed associations between 23 hemostatic factors including platelet traits, coagulation and fibrinolysis factors and AMI by leveraging GWAS data.

Study Design and data sources

This study is reported according to the Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization (STROBE-MR) (additional checklist) [9,10]. Figure 1 illustrates the overall study design. We conducted two-sample MR analyses using summary-level GWAS data. All data in this study was available publicly thus ethical approval was not required. Three core assumptions for instrumental variables were made. SNPs must be associated with the exposure, are are independent of the confounding factors and only affect the outcome through their effects on the exposure. Data from 23 GWASs for exposures and one GWAS for the outcome were included in the study.

We performed systematic search and retrieved data on GWASs of participants of only European ancestry (until March,2024) in the GWAS Catalog, UK Biobank and FinnGen. The exposures are genetically-predicted hemostatic factors in plasma and the outcome is acute mesenteric ischemia. The largest GWAS summary statistics were included. After identification and evaluation, a total of 23 hemostatic parameters were included as instrumental variants were retrieved from GWAS catalog: vWF level (n=5,356) [11], TM (thrombomodulin) level (n=21,758) [12], platelet count (n=408,112) [13], plateletcrit (n=408,112) [13], mean platelet volume (n=408,112) [13], platelet glycoprotein (n=3,301) [14], prothrombin (n=10,708) [15], PT/INR(n=34,919) [16], fibrinogen level (n=10,708) [15], coagulation factors (V,VII,VIII,IX,Xa) (n=10,708) [15], thrombin level (n=10,708) [15], tissue factor level (n=21,758) [12], antithrombin level(n=10,708) [15], activated protein C level (n=3,301) [14], D-dimer level (n=10,708) [15], plasminogen level (n=10,708) [15], tissue-type plasminogen activator level (n=21,758) [12], plasminogen activator inhibitor level (n=341,448) [17], alpha-2-antiplasmin level (n=3,301) [14], plasmin level (n=3,301) [14], tissue factor pathway inhibitor level (n=3,301) [14], protein C level (n=3,301) [14] and protein S level (n=21,758) [12]. Summary-level data for AMI was obtained from GWAS catalog(143 cases, 456,205 controls) [18]. Diagnosis of AMI was made based on International Classification of Diseases codes (ICD-9,Acute vascular insufficiency of intestine). Full summary statistics for the exposures and outcome are available for download from the GWAS catalog website (https://www.ebi.ac.uk/gwas/downloads/summary-statistics). Overview of GWAS used was shown in Supplement table 1.

Mendelian Randomization

Independent genome-wide significant SNPs of each hemostatic factor were used as genetic instruments. Included instrumental variables should be strongly associated with the exposures (p ≤5×10⁻⁸) and SNPs with an F statistic <10 were excluded [19]. Linkage disequilibrium (LD) clumping was performed with r²< 0.001 and window size of 10,000 kb [20]. The strength of each genetic instrument was assessed through the F statistic, calculated as F = R²(N − 2)/(1 − R²), where R² denotes the proportion of variance explained by the genetic instrument and N represents the effective sample size of the GWAS for the SNP-micronutrient association [19]. The R² value was determined using the formula 2 × MAF (1 − MAF) beta2, where beta signifies the effect estimate of the genetic variant on the exposure, measured in standard deviation (SD) units, and MAF indicates the minor allele frequency [19-20]. Then, we removed SNPs associated with diseases or risk factors potentially associated with AMI (http://www.phenoscanner.medschl.cam.ac.uk/). The remaining SNPs were used in the MR analysis. The inverse variance-weighted (IVW) method was the main method for MR analysis and and the weighted median and MR-Egger methods were conducted to improve the IVW model-based estimation [21]. P < 0.05 was considered nominally significant. Wald ratios for all SNPs were calculated. MR analyses estimated odds ratio (OR) of per SD change of the genetically predicted circulating levels of hemostatic factors. All MR analyses were performed using the TwoSampleMR package (version ) in R (version ) [22].

Pleiotropy and and Sensitivity Analysis

We performed MR-Egger regression to examine the potential presence of pleiotropic effects of the SNPs. The intercept term in MR-Egger regression serves as a valuable indicator to determine if directional horizontal pleiotropy is influencing the results of the MR analysis. We employed both the IVW method and MR-Egger regression to identify heterogeneity. The degree of heterogeneity was measured using the Cochran Q statistic, and a P value of < 0.05 was considered significant heterogeneity. Moreover, to identify any potentially influential SNPs, we conducted leave-one-out test, whereby the MR was re-run while excluding each SNP in turn.

The detailed randomization analysis results of exposures associated with hemostatic factors in plasma, including numbers of SNPs, ORs(95%CI) and p values, are presented in Supplement table 2. Forest plot results of all IVW are shown in Figure 2. Through multiple Mendelian randomization analyses, we observed statistically significant association between genetically determined decrease of plasminogen levels and acute mesenteric ischemia. A genetically predicted one SD increase of plasma plasminogen was associated with an OR of 0.41(95% confidence interval [CI] 0.24 to 0.68, P = 0.0007) by IVW analysis. Weighted median analysis also supported the result of main analysis. Other hemostatic factors in this study were not shown to have significant associations with AMI. Information of SNPs after clumping process for plasminogen was show in Supplement table 3. The Results of pleiotropy and sensitivity analyses were shown in Table 1. The p-values of MR-Egger intercept analysis and Cochran’s Q test were both above 0.05. The leave one out analysis and funnel plot were presented in Supplement Figure 1 and 2.

In this two-sample Mendelian randomization study, we found genetically decreased blood levels of plasminogen was causally associated with genetically predicted risk of acute mesenteric ischemia. No causal effects of other factors involved in this study were found. Our study provided evidence on the potential role of coagulation and thrombosis in the pathogenesis of AMI.

Plasminogen is a single-chain zymogen and its activation results in plasmin which plays the central role in fibrin degradation and removal of blood clot. Plasminogen is one of the most abundant circulating hemostatic proteins in plasma but its level is rarely tested as a diagnostic marker in routine clinical practice [23]. Our study revealed a negative association of plasminogen level and AMI, which indicated that blood plasminogen measurement might help with risk stratification and early diagnosis of AMI. It is also suggested that deficient fibrinolysis by low blood plasminogen levels might contribute to thrombosis or embolism in AMI.

Interestingly, in COVID-19 patients with acute respiratory distress syndrome, plasminogen inhalation improved clinical outcomes possibly due to its fibrin-degrading effects on pulmonary microcoagulopathy [24, 25]. Supplementation of plasminogen to increase the fibrinolytic activity could be a potentially innovative therapy to reduce intestinal injury caused by mesenteric ischemia and also a possible preventive strategy for high-risk population of AMI. Plasminogen also played a role in inflammation by interacting with immune cells. Future investigations could be performed not only on restoration of mesenteric perfusion for AMI but also on targeting plasminogen in thromboinflammation of AMI [26, 27].

A meta-analysis evaluating the diagnostic accuracy of D-dimer in transmural mesenteric ischemia and showed a sensitivity of 87.6% and specificity of 83.6%[4]. D-dimer is a fibrin degradation product and has been widely used clinically as a marker of fibrinolysis. This MR study did not reveal significant association of genetically determined levels of D-dimer and AMI, which suggested that elevations of D-dimer in AMI could be secondary change of thrombosis.

There are limitations in out study. Only participants of European ancestry were involved in the study and evaluation of other racial groups will be needed. All hemostatic measures in this study were from general population. Diagnostic accuracy of these factors should be evaluated in high-risk populations of developing AMI.

In this systemic MR study, we revealed genetically decreased blood levels of plasminogen was causally associated with genetically predicted risk of AMI. Our result suggested potential causal roles of plasminogen in the susceptibility of AMI and involvement of hemostatic factors in the pathogenesis of AMI. Further research on coagulation and fibrinolysis in ischemic diseases of gastrointestinal tract are needed.

AMI, acute mesenteric ischemia;

MR, Mendelian randomization;

GWAS, genome-wide association study;

SD, standard deviation;

CI, confidence interval;

SNPs, Single-nucleotide polymorphisms;

IVs, instrumental variables;

STROBE-MR, Strengthening the Reporting of Observational Studies in Epidemiology using Mendelian Randomization;

TM, thrombomodulin;

LD, Linkage disequilibrium;

IVW, inverse variance-weighted;

The submitted manuscript is an original contribution not previously published and will not be submitted to any other journal while it is under consideration by Colorectal Disease.

Ethics approval and consent to participate

All data in this study was available publicly thus ethical approval and consent to participate were not required.

Consent for publication

All data in this study was available publicly thus consent for publication was not applicable.

Availability of data and materials

Full summary statistics for the exposures and outcome are available for download from the GWAS catalog website (https://www.ebi.ac.uk/gwas/downloads/summary-statistics).

Competing interests

The authors declare that they have no competing interests.

Funding

This study has no funding support.

Authors' contributions

XLX conducted study conception, analyzed data and draft the manuscript. GY performed the data analysis and revised the manuscript. JP performed the data analysis and revised the manuscript. YW conducted study conception, analyzed data and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Table 1. Causal estimates of plasminogen on AMI by MR analysis.

Exposure	Outcome	SNPs	Method	beta	95% CI	se	p	MR-Egger intercept test	Cochran's Q heterogeneity test
Exposure	Outcome	SNPs	Method	beta	95% CI	se	p	p	p
Plasminogen	AMI	3	IVW	-0.8995	-1.4170 to -0.3820	0.2640	0.0007	0.9479	0.7501
			Weighted median	-0.9026	-1.4579 to -0.3473	0.2833	0.0014
			MR‐Egger	-0.9162	-1.5696 to -0.2628	0.3334	0.2222

Abbreviation: MR, Mendelian randomization; AMI, Acute intestinal ischemia; CI, confidence interval; IVW, inverse-variance weighted.

No competing interests reported.

Association between hemostasis and acute mesenteric ischemia: a Mendelian randomization analysis

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