Skip to main content
BMC is moving to Springer Nature Link. Visit this journal in its new home.
Download PDF

Procoagulant effect of phosphatidylserine-exposed blood cells, endothelial cells and extracellular vesicles in patients with aortic stenosis

Thrombosis Journal volume 23, Article number: 70 (2025) Cite this article

Abstract

Background

See More

The mechanism of thrombotic complications in patients with aortic stenosis (AS) is unclear so far. Our aim was to evaluate the levels of phosphatidylserine (PS) exposed on blood cells, endothelial cells (ECs), and extracellular vesicles (EVs) and its procoagulant activity (PCA) in mild to severe AS patients.

Methods

Exposed PS on blood cells, ECs and EVs were analyzed by flow cytometry. PCA was evaluated by clotting time (CT), intrinsic factor Xa (FXa), extrinsic FXa, thrombin and fibrin formation assays. We also evaluated the inhibitory effects of lactadherin (Lact) on PCA in AS patients.

Results

Our results demonstrated that patients with AS had significantly higher percentages of positive phosphatidylserine (PS+) red blood cells (RBCs), platelets (PLTs), white blood cells (WBCs) and ECs compared to controls. Total EVs with PS+, platelet EVs (PEVs), endothelial-derived EVs (EEVs) and positive tissue factor EVs (TF+EVs) levels were significantly higher in mild to severe AS. In addition, we further confirmed that PS+ blood cells, ECs and EVs significantly contributed to shortened CT and dramatically increased FXa, thrombin and final fibrin generation in mild to severe AS compared to controls. Furthermore, lactadherin significantly inhibited the PCA of PS exposure on blood cells, ECs and EVs in AS patients, whereas anti-TF had no this effect.

Conclusion

Our study revealed a previously unrecognized association between exposed PS levels on blood cells, ECs and EVs and PCA in AS. Lactadherin promises to be a new therapy by blocking PS to prevent thrombosis in AS patients.

Introduction

As the age grows, the incidence of aortic stenosis (AS) is increasing. Study found that AS occurs in 10% of people over the age of 80 and is often associated with a poor prognosis [1]. Thromboembolic complications are major determinants of prognosis and quality of life in AS patients [1, 2]. AS associated thromboembolic complications can lead to thromboembolic stroke, myocardial infarction, pulmonary embolism and heart failure [1, 3]. Thromboembolic complications in patients with severe AS is a major risk factor for stroke and death [1, 4]. The pathophysiology of thromboembolism in AS is multifactorial. Hypercoagulable state of blood is the potential pathogenesis [3]. Previous studies showed that there was no significant difference between anticoagulation and antiplatelet therapy in terms of cardiovascular thromboembolic events [5, 6]. The exact mechanism of the formation of hypercoagulable state in AS has not been clarified, and the optimal anticoagulation mechanism has not been perfected, so further studies on the mechanism of the prothrombotic state in AS patients are needed.

It is well known that inflammation, infection, tumors and immunodeficiency can activate circulating blood cells and inflammatory cells [7,8,9]. Activated blood cells such as erythrocytes, leukocytes and platelets have been shown to induce a prothrombotic state in the body [10,11,12]. This phenomenon has been observed in diseases such as coronary atherosclerosis [13] and atrial fibrillation [14]. There is increasing evidence that erythrocytes, leukocytes and platelets are activated in AS [1, 15, 16]. In addition, other evidence showed that ECs were also activated in AS [17]. Previous studies found that high shear in AS promoted the activation of circulating cells and ECs [18]. However, the extent of activation of blood cells and ECs in these patients is not fully understood.

Circulating EVs are plasma membrane-derived vesicles shed by various types of activated or apoptotic cells, including PLT, monocytes, ECs, erythrocytes and granulocytes [19]. Recent evidence suggest that they may play important functions in cell-cell interactions, homeostasis and pathogenesis of a number of diseases [19]. In addition, EVs play a role in the procoagulant activity in diseases by expressing PS, tissue factor (TF) and other coagulation factors [20]. PS and TF are known to be associated with the prothrombotic state in cardiovascular diseases [21, 22]. However, there are few studies on EVs levels in patients with AS. It is not entirely clear the level changes of PS and TF in AS and their role in PCA.

In this study, we investigated the differences in the levels of total PS+EVs, PEVs, EEVs and TF+EVs as well as the levels of PS exposure on blood cells and ECs between mild to severe AS and controls. In addition, we evaluated the contribution of PS exposure to PCA and further analyzed the disturbance of lactadherin and anti-TF on coagulation activity in AS patients. Our study may contribute to the discovery of new therapeutic targets to effectively intervene in the thrombotic complications of AS and reduce the risk of death.

Methods and materials

Study population

In our study, patients diagnosed with AS by thoracic echocardiography were continuously selected for the study from the Heart Center of (Qingdao Hospital) University of Health and Rehabilitation Sciences from November 2020 to September 2022. A total of 166 patients met the inclusion and exclusion criteria, including 55 mild AS, 51 moderate AS and 60 severe AS patients. Fifty healthy volunteers with normal physical examination were recruited as healthy controls in the same time.

Exclusion criteria for all patients were congenital heart disease, bicuspid aortic valve stenosis, history of aortic valve surgery, history of stroke, history of heart failure, history of thromboembolism, acute and chronic inflammatory diseases, history of surgical procedure within 3 months of presentation, hepatic or renal disease, autoimmune or malignant diseases, and receiving or about to receive anticoagulant or antiplatelet therapy within 2 weeks. We graded the aortic valve stenosis according to the valve orifice area, mild stenosis was that the valve orifice area was reduced but greater than or equal to 1.5 square centimeters; moderate stenosis was that the valve area was between 1.0 and 1.5 square centimeters and severe stenosis was that the valve area was less than or equal to 1.0 square centimeters [23].

In accordance with the Declaration of Helsinki, approval was obtained from the.

Research Ethics Committee of our institution (Interim Audit Document NO. Y12), and written informed consent was obtained from all participants.

Ethical statement

This study has passed the ethical review of Qingdao Municipal Hospital.

Experimental procedures

Materials

We used a polyclonal antibody to human TF from American Diagnostica Inc. (Stamford, CT, USA) and labeled all monoclonal antibody with Alexa Fluor 647 or 488. CD142 (clone HTF-1), CD41a (clone HIP8), purified CD31 (clone L133.1) and TruCount Tube from Becton Dickinson (San Jose, CA, USA) were also used. Human FXa, thrombin, and prothrombin were obtained from Haematologic Technologies. Alexa Fluor 647 or 488 conjugated lactadherin and Tyrode’s buffer containing 1 mM HEPES were prepared and filtered through a 0.22 mm syringe filter from EMD Millipore. Chromogenic substrates S-2765 and S-2238 were purchased from DiaPharma Group. Calibrated polystyrene latex beads (1.0 μm) were purchased from Sigma (UK).

Protein purification and marking

The ratio of lactadherin to fluorescein in this study was 1/ 1.1–1.2, and we used Alexa Fluor 647 or Alexa Fluor 488 to label lactadherin purified from milk [24].

Endothelial cell culture and Recombinant experiments

Human umbilical vein endothelial cells (HUVECs) were cultured using ECs medium without EV (Science Cell, San Diego, CA, USA) at 37 °C in a 5% CO2 humidified atmosphere. ECs were processed from media containing 20% pool serum from mild AS, moderate AS, severe AS patients and healthy controls at room temperature for 24 h. Using the flow cytometer to define PS exposure [25].

Preparation of evs, flow cytometric and ELISA analysis

Blood samples were collected from controls and AS with different degrees of stenosis. Within 30 minutes after blood collection, the samples were centrifuged for 20 minutes at 1500 g. The upper plasma layer was aspirated and centrifuged again for 30 minutes at 20,000 g. We then removed the plasma supernatant, collected the remaining EV at the bottom of the test tube and stored at -80°C. To quantify PS exposure on cells, blood cells or ECs were diluted in Tyrode’s buffer to 0.5–1 × 106/ml. PLT, RBC, WBC or cultured ECs were stained with 6 nM Alexa Fluor 488-labeled lactadherin. Cells were diluted and evaluated by flow cytometry after incubation for 15 min [26]. Lactadherin-positive (lac+), lac+CD41a+, lac+CD31+41a−, and lac+CD142+ were used to characterize PS+EVs, PEVs, EEVs, and TF+EVs. The number of EVs of each type per microliter was calculated using a Trucount tube after accumulation of 10,000 gated events using the following formula: n = (C × Beads added) / (Beads counted × Sample volume). In the formula, ‘C’ stands for the number of positive events after subtraction of the background signal.

To measure the level of p-selectin. Fasting venous blood samples were drawn from an elbow vein, placed in tubes containing anticoagulant (EDTA-K2) and immediately centrifuged at 3000 rpm for 10 min at 4 °C (Anke, DL-4000B). The upper plasma layer was removed and stored at -80 °C. ELisa kits (R&D company, Mibio Hailian Bio, China) were used to measure plasma levels of p-seletion.

Coagulation time and Inhibition assays of EVs

KC4A coagulometer was used to evaluate the PCA of EVs. We mixed EV-enriched suspension and Tyrode’s buffer in a ratio of 1:9 to obtain EV-containing suspension, and incubated 100 µl of EV-containing suspension with 100 µl of EV-depleted plasma at 37 °C for 3 min. 100 µl of warmed CaCl2 (25 mM) was added to start the reaction and record the coagulation time (CT). In the inhibition assay, we incubated 100 µl of EVs suspension with 50 µl of lactadherin or anti-TF for 10 min at 37 °C. The coagulation time was recorded after the addition of 100 µl of EV-free human plasma and 50 µl of warmed CaCl2 (50 mM).

Intrinsic, extrinsic FXa and thrombin formation and Inhibition assays of EVs

We first performed FXase and prothrombinase activity assays on all samples. To prepare intrinsic Xa, we incubated 10 µL EVs suspension with FX (130 nm), Fixa (1 nm), thrombin (0.2 nm), and CaCl2 (5 mm) in FXa buffer (10 ml 1 × incubate in TBS containing 0.2% BSA) at 25 °C for 5 min, then used EDTA (7 mm final concentration) to stop the reaction. FXa production was measured using a universal microplate spectrophotometer (PowerWave XS; Bio-Tek, Winooski, VT, USA) at 405 nm after incubation with 10 µl of S-2765 (0.8 mM). Except for EVs cultured with FX (130 nM), FVIIa (1 nM) and CaCl2 (5 nM), the formation of extrinsic FXa was similar to that of intrinsic FXa. In the prothrombinase assay, EVs with 0.05 nM FXa (0.05 nM), FVa (1 nM), prothrombin (1 µM), and CaCl2 (5 nM) were incubated in prothrombinase buffer (10 ml 1 × TBS with 0.05% BSA) for 5 min at 25 °C and then stopped with EDTA. For inhibition assays, EVs were preincubated with lactadherin (128 nM) or anti-TF at their peak time at 25 °C in Tyrode’s buffer for 10 min and then incubated with the specified coagulation factors. The formation of FXa or thrombin was assessed as described above.

Fibrin formation assays

Fibrin formation was evaluated by turbidity. EVs were added to recalcified (10 mM, final) EV-depleted plasma (88% EV-depleted plasma, final) in the absence or presence of lactadherin/anti-TF. A SpectraMax 340PC plate reader was used to measure fibrin production by turbidity at 405 nm.

Statistical analysis

SPSS 27.0 software and GraphPad Prism 9.0 were used to analyze statistics. Data conforming to a normal distribution are expressed as mean ± standard deviation and were statistically analyzed using Student’s t-test or ANOVA, as appropriate. Post-hoc tests between groups using the least significant difference (LSD). Categorical variables were compared using the χ2 test. Pearson or Spearman’s rank correlation analysis was used to express correlations between two continuous variables. P < 0.05 was considered statistically significant.

Results

Participants characteristics

We analyzed the clinical characteristics of 50 healthy controls and 166 AS patients (Table 1). There were no significant differences in gender, current smoking, current alcohol, hypertension, diabetes mellitus, atrial fibrillation and stroke between control and mild to severe AS patients. Age, body mass index (BMI), fasting glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), triglyceride (TG), serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) levels were significantly higher in AS than control. While high-density lipoprotein cholesterol (HDL-c) levels and aortic valve area (AVA) were significantly lower in AS than controls. In addition, the levels of P-Selectin, white blood cells (WBC), neutrophils, red blood cells (RBC), platelets (PLT), fibrinogen, D-dimer (D-D) and thrombin-antithrombin complex (TAT) levels were markly higher in AS compared to controls. Conversely, prothrombin time (PT) and activated partial thromboplastin time (APTT) levels were significantly lower in all AS patients than controls. Further analysis showed that severe and moderate AS patients had significantly different levels of WBC, neutrophils, RBC, PLT, fibrinogen, D-D, TAT, PT, APTT, AST, ALT, Cr, TG, TC and LDL-c than mild AS groups. Further analysis of confounding factors, the effects of gender, age, hypertension, diabetes mellitus, atrial fibrillation, and WBC count on total PS+EVs levels were not statistically significant (P > 0.05), and mild, moderate or severe AS groups had effects on total PS+EVs levels. This means that after adjustment for sex, age, hypertension, diabetes mellitus, atrial fibrillation and WBC count by multivariate linear regression analysis, this association remained statistically significant (P < 0.05) (Table 2).

Table 1 Clinical features and laboratory findings of the study participants
Full size table
Table 2 Multiple linear regression on total PS+EVs levels in mild to severe AS patients
Full size table

The percentages of LAC+RBC/PLT/WBC/ECs and their association with hypercoagulable markers in mild to severe AS and controls

We analyzed the percent of lactadherin-binding blood cells and ECs by flow cytometry (Fig. 1 and Supplementary Fig. 1–2). Compared to control groups, the percentage of RBC, PLT, WBC and serum-cultured ECs were significantly higher in mild to severe AS patients, PS exposure levels gradually increased from mild to severe AS. Among all AS groups, severe AS had the highest PS exposure rate of RBC, PLT, WBC and ECs. The relationships between the percentages of LAC+ RBC, LAC+ PLT, LAC+ WBC and LAC+ ECs and hypercoagulable markers (D-dimer or TAT ) in AS were also analyzed (Table 3). From mild to severe AS patients, D-dimer (D-D) and TAT had a significant positive correlation with the percentages of LAC+RBC, LAC+PLT, LAC+WBC and LAC+ECs.

Fig. 1
figure 1

The percentage of lactadherin-binding (Lact +) blood cells from healthy subjects (n = 50), mild AS patients (n = 55), moderate AS patients (n = 51) and severe AS patients (n = 60) were measured by flow cytometry. (A, B, C, D) RBCs, WBCs, PLTs and ECs were incubated separately with Alexa Fluro 488-lactadherin and calculated their percentage. RBC: red blood cell; WBC: white blood cell; PLT: platelet; ECs: endothelial cell. *p < 0.05

Full size image
Table 3 Correlation between total PS+ evs/lac++ blood cells/LAC + ECs and hypercoagulability markers in mild to severe AS patients
Full size table

Dynamics of PS+EVs/EEVs/PEVs/TF+EVs in mild to severe AS and controls

The total number of EVs and their phenotypic features were evaluated by flow cytometry in Fig. 2. Circulating total EVs levels were significantly elevated in mild to severe AS groups compared with controls, and there was a statistically remarkable difference among AS groups (Fig. 2 and Supplementary Fig. 2). Furthermore, compared to controls, the levels of CD14a+ PEVs, CD31+ CD41a EEVs and TF+ EVs were also significantly higher in patients from mild to severe AS (Fig. 2). As the degree of AS stenosis increased, total EVs, CD14a+ PEVs, CD31+ CD41a, EEVs and TF+ EVs levels were gradually increased, with most pronounced elevations in patients with severe AS. As shown in Table 3, total EVs levels were positively correlated with markers of hypercoagulability (D-dimer or TAT ), and particles played at least a partial procoagulant role.

Fig. 2
figure 2

The EVs in healthy controls (n = 50), mild AS patients (n = 55), moderate AS patients (n = 51) and severe AS patients (n = 60) were analysed by flow cytometry. (A) Alexa-Fluor 488-lactadherin binding was used to determine that EVs gated in this way were PS+. (B, C, D) PEVs (Alexa Fluro 647-CD41a+), EEVs (Alexa Fluro 488-CD31+/Alexa Fluor 647-CD41a− ) and TF+EVs (Alexa Fluor 647-CD142+) were also measured and counted by co-labeling with Alexa Fluor 488− and Alexa Fluor 647-labeled antibody. PS+EVs: phosphatidylserine-positive extracellular vesicles; EEVs: endothelial-derived extracellular vesicles; PEVs: platelet derived extracellular vesicles; TF+EVs: tissue factor-positive extracellular vesicles. *p < 0.05

Full size image

PCA of PS+ blood cells, EVs and ECs in AS patients

As we predicted, from mild to severe AS, the PS+ EVs, RBC, PLT and WBC showed significantly shorter CT than those from controls, and the ECs pretreated with each AS group serum also had a shorter CT than the controls (Fig. 3A). We further analyzed the coagulation activity of PS+ EVs, blood cells and ECs by intrinsic FXa, extrinsic FXa and thrombin generation assays (Fig. 3B, C). We found that from mild to severe AS, the production of the intrinsic FXa, extrinsic FXa and thrombin production were increased in PS+ EVs, PLT, WBC and ECs, and the production of intrinsic FXa and thrombin were increased in RBC than in controls (Fig. 3B, C). To further verify whether this increase in PCA was due to PS exposure, in an inhibition assay of the severe AS group, we used lactadherin (128 nM) and anti-TF to block available PS (Fig. 3D, E, F, G). We found that preincubation with lactadherin significantly prolonged the CT of PS+ EVs, blood cells and ECs in severe AS compared with anti-TF in severe AS and controls (Fig. 3D). In addition, lactadherin significantly reduced intrinsic FXa, extrinsic FXa and thrombin production in the severe AS patients, whereas anti-TF had no difference (Fig. 3E, F, G).

Fig. 3
figure 3

The PCA of EVs, RBC, PLT, WBC and ECs from mild to severe AS patients and healthy controls and inhibition assays. EVs, PLT, RBC, WBC and ECs were acted on the plasma of the controls and measured for the CT and the production of intrinsic Xa, extrinsic Xa, and thrombin. (A) CT was detected in EVs, RBC, PLT, WBC and ECs of the mild to severe AS patients and healthy controls. (B) EVs and PLT were evaluated for the production of intrinsic Xa, extrinsic Xa and thrombin; RBC was evaluated for the production of intrinsic Xa and thrombin. (C) WBC and ECs were evaluated for the production of intrinsic Xa, extrinsic Xa and thrombin. (D) CT was detected in EVs, RBC, PLT, WBC and ECs of severe AS patients with the addition of Lact or anti-TF disrupting the binding of procoagulant enzyme complexes. (E, F, G)intrinsic Xa, extrinsic Xa and thrombin were detected in EVs, RBC, PLT, WBC and ECs of severe AS patients with the addition of Lact or anti-TF disrupting the binding of procoagulant enzyme complexes. PCA: Procoagulant activity. Lact: lactadherin. Anti-TF: anti-tissue factor. Thr: thrombin. For A, B, C, &P < 0.05, vs. Controls; *P < 0.05, vs. Mild AS; #P < 0.05, vs. Moderate AS. For D, E, F, G, **p < 0.01 severe with Anti-TF and severe with Lact vs. control

Full size image

Fibrin formation assays of EVs, blood cells and ECs

To evaluate the role of PS exposure on fibrin production, we measured the final fibrin production of EVs, RBC, PLT, WBC and ECs in each AS group and examined the effects of lactadherin and anti-TF on the final fibrin content of severe AS group (Fig. 4A, B). The final fibrin production content was significantly increased in each AS group of EVs, hemocytes and ECs compared with the normal control group, and the increase was most marked in the severe AS group (Fig. 4A). The addition of lactadherin significantly reduced the final fibrin levels in severe AS patients, whereas anti-TF had no effect on the final fibrin formation (Fig. 4B).

Fig. 4
figure 4

Final fibrin formation assays and inhibition assays of blood cells, EVs and ECs in mild to severe AS and controls. (A) Final fibrin formation assays to test the PCA of EVs, RBCs, PLTs, WBCs and ECs in control and mild to severe AS. (B) The capacity of Lact or anti-TF to disrupting the final fibrin formation assays in Severe AS. For A, &P < 0.05, vs. Controls; *P < 0.05, vs. Mild AS; #P < 0.05, vs. Moderate AS. For B, **P < 0.01, Severe with Anti-TF and Severe with Lact vs. Severe AS groups

Full size image

Discussion

The level of PS exposure in haemocytes, ECs or EVs and its role in AS coagulation is unclear. In this study, from mild to severe AS groups, our data showed that the percentages of PS + blood cells, ECs and EVs were significantly higher, D-D and TAT were significantly positively correlated with PS levels. In addition, there was a significant shortening of CT, a significant increase in FXa, prothrombin and finally fibrin formation in the mild to severe AS group compared to the normal group. The procoagulant inhibition test showed that lactadherin significantly prolonged CT, reduced the levels of procoagulant enzyme complexes and final fibrin formation. This suggests that the levels of PS exposure in EVs, RBCs, PLT, WBCs and ECs related to prothrombinase assembly and fibrin formation in mild to severe AS patients.

Study found that high shear stress was an inducer of blood cell activation in the development of arterial disease [27]. Diehl et al. [28] demonstrated a correlation between shear stress and platelets or leukocytes in AS patients. In addition, some studies found that the presence of high shear stress in AS activated the expression of ECs [17, 29]. In our study, the activation numbers of RBC, PLT, WBC and ECs were significantly higher in all AS groups than controls, which is consistent with previous findings. This was mainly caused by altered hemodynamics due to stenotic aortic orifices and thickened valves, resulting in ECs and circulating blood cells activated by abnormal shear forces [12, 17, 30]. It is noteworthy that our study found that among all AS patients, the number of activated RBCs, PLT, WBCs and ECs was significantly higher in severe AS patients than in mild and moderate AS patients. In addition, our study further demonstrated that EVs and PS exposure of these activated cells and ECs were significantly elevated in AS. From this, we can conclude that the high shear stress generated in AS patients affected the activation and release of hemocytes and ECs, and further increased EVs and PS levels, the degree of valve stenosis played a crucial role in the activation of blood cells and ECs.

EVs are small cell membrane vesicles of 0.1–1 μm released by apoptosis, abnormal activation of blood cells and endothelial cells of membrane remodeling, during states of inflammation, hypoxia and high shear levels, EVs shedding can increase [22, 31]. Research has shown that high shear stress in AS is associated with the release of circulating leukocyte, platelet and endothelial cell derived EVs [28, 32]. In this study, we demonstrated that the total levels of PS+ EVs, EEVs, PEVs and TF+EVs were significantly higher in all AS groups than in healthy controls. Previous studies have shown that the levels of EVs from WBC, PLT and ECs were increased in severe AS compared to controls, which was consistent with our findings [28]. In addition, studies have shown that high levels of EVs were a major contributor to hypercoagulability [25]. Which is consistent with our findings. This is mainly because circulating EVs provide an additional procoagulant phospholipid surface for activation of the coagulation cascade reaction [33]. Therefore, it can be concluded that the shear stress caused by AS may contribute to the coagulation process by activating the generation of EVs from hemocytes and ECs. More importantly, the PCA of EVs in AS is highly dependent on the level of PS exposure [33].

On the plasma membrane, PS was preferentially found in the inner leaflet [34]. Earlier studies showed that Annexin V a certain threshold of PS exposure (2-8%) is required for binding to take place. Lactadherin was found to detect a significantly higher percentage of PS-positive PLTs than Annexin V [35]. Lactadherin is a more effective and sensitive probe for detecting EVs than Annexin V [36]. During biological processes such as apoptosis and activation, PS could be exposed on the cell surface [37]. Research has shown that the increased levels of PS+ ECs occur in some diseases [38]. In AS, leukocytes, lymphocytes, neutrophils, macrophages and monocytes were abnormal activation, PS exposure on the plasma membrane surface was increased [39, 40]. Our study showed that patients with mild AS to severe AS showed higher levels of PS+ RBC, WBC, PLT and ECs were significantly higher in AS groups than controls. This was similar to previous findings. In addition, studies have confirmed that PS exposed to blood cells, ECs and EVs can provide sites for the coagulation cascade reaction, which promoted the process of coagulation [38,39,40]. This further confirms our findings. Activated blood cells, ECs, and EVs exposed PS play a procoagulant role in AS patients.

Previous studies have shown that thrombin generation was increased in AS [41]. In our study, we identified the role of PS exposure in thrombin generation in AS patients and additionally analyzed the relationship between PS exposure and markers of high cohesion. Early studies have shown that AS patients had increased levels of coagulation markers [1], which is consistent with the results of our study. However, our study further explained the impact indicators of elevated levels of hypercoagulable markers in AS. Our findings suggested that PS played a key role in influencing the levels of coagulation markers and the final fibrin formation in AS patients. The rate of PS exposure of total EVs, blood cells and ECs were positively correlated with D-D and TAT in AS patients, which further confirmed the relation to PS exposure and AS thrombosis.

In previous studies, PS has been shown to promote a hypercoagulable state in the body under pathological conditions [1]. Our study further demonstrates the role of PS in AS, compared with the control group, the CT of PS + EVs, RBC, PLT, WBC, and ECs in AS patients was shortened, the content of plasminogen complex was increased, and the body was in a hypercoagulable state. In addition, TF may also played a role in the procoagulant process [1]. However, the contribution of PS and TF to the procoagulant process in AS is unclear. For further analysis, we used anti-TF antibody and lactadherin to disrupt the exposure levels of TF and PS. Our results showed that the inhibitory effect of lactadherin on PS led to a significant decrease in the coagulation activity of PS in the samples, whereas the coagulation activity was not significantly altered in the group with the inclusion of the anti-TF antibody. This did not fully explain our finding of increased TF + EVs in AS, but our study also found that TF + EVs were a smaller proportion of total EVs. Previous studies also did not find a critical role for TF + EVs in PCA [42, 43]. This could be caused by the fact that cycling TF are mostly in an encoded state and often cycle in a coagulated inactive or cryptic form, clusters of PS can be used to decode for TFs and increase TF activity [33, 44]. Another finding that TF was in contact with the circulation but did not induce significant coagulation suggested that TF circulated in a coagulation-inactive or cryptic form in most cases and rarely played a role, which further supported our inhibition experimental results [44, 45]. Thus, activated blood cells, ECs, and EVs exposed PS play a procoagulant role in AS patients.

Conclusions

In conclusion, our results surmised that AS patients had a hypercoagulable state compared to controls. Elevated levels of PS exposure in blood cells, ECs and EVs may significantly related to hypercoagulability in AS patients. PS exposure levels in blood cells, ECs and EVs and coagulation activity were significantly higher in AS patients as the severity of AS increased. Furthermore, the addition of lactadherin to block PS exposure resulted in a significant decrease in PCA in AS patients, thus lactadherin is expected to be a new therapy for thromboprophylaxis in AS patients.

Limitation

One of the main limitations of our study is that it is cross-sectional, a single-center study. In addition, our sample size was small and externally there is a need for further studies with multi-center, large samples.

References

  1. Trimaille A, Hmadeh S, Matsushita K, Marchandot B, Kauffenstein G, Morel O. Aortic stenosis and the haemostatic system. Cardiovasc Res. 2023;119:1310–23. https://doi.org/10.1093/cvr/cvac192

    Article  CAS  PubMed  Google Scholar 

  2. Kaul H, Gutzwiller JP, Schneider K, Dirsch O, Häusermann M. Aortic valve stenosis as a cause of major systemic embolism–a case report. Angiology. 1998;49:231–4. https://doi.org/10.1177/000331979804900310

    Article  CAS  PubMed  Google Scholar 

  3. Alajaji W, Hornick JM, Malek E, Klein AL. The characteristics and outcomes of native aortic valve thrombosis: A systematic review. J Am Coll Cardiol. 2021;78. https://doi.org/10.1016/j.jacc.2021.06.023. 811– 24.

  4. Natale F, Aronne L, Credendino M, Siciliano A, Allocca F, Weizs SH, Martone F, di Marco GM, Calabrò P, Tedesco MA, Russo MG, Calabrò R. Which is the correct management of patients with asymptomatic severe calcific aortic stenosis after symptomatic spontaneous calcium cerebral embolism? J Cardiovasc Med. 2011;12:428–9. https://doi.org/10.2459/JCM.0b013e328344bcc7

    Article  Google Scholar 

  5. Soodi D, VanWormer JJ, Rezkalla SH. Aspirin in primary prevention of cardiovascular events, clin. Med Res. 2020;18:89–94. https://doi.org/10.3121/cmr.2020.1548

    Article  CAS  Google Scholar 

  6. Brouwer J, Nijenhuis VJ, Delewi R, Hermanides RS, Holvoet W, Dubois C, Frambach P, De Bruyne B, van Houwelingen GK, Van Der Heyden J, Toušek P, van der Kley F, Buysschaert I, Schotborgh CE, Ferdinande B, van der Harst P, Roosen J, Peper J, Thielen F, Veenstra L, Chan PYD, Swaans MJ, Rensing B, van T HA, Timmers L, Kelder JC, Stella PR, Baan J. Ten, aspirin with or without clopidogrel after transcatheter Aortic-Valve implantation, N. Engl. J Med. 2020;383:1447–57. https://doi.org/10.1056/NEJMoa2017815

    Article  CAS  Google Scholar 

  7. Tognarelli EI, Retamal-Díaz A, Farías MA, Duarte LF, Palomino TF, Ibañez FJ, Riedel CA, Kalergis AM, Bueno SM, González PA. Pharmacological Inhibition of IRE-1 alpha activity in herpes simplex virus type 1 and type 2-Infected dendritic cells enhances T cell activation. Front Immunol. 2021;12:764861. https://doi.org/10.3389/fimmu.2021.764861

    Article  CAS  PubMed  Google Scholar 

  8. Cervantes F, Arellano-Rodrigo E, Alvarez-Larrán A. Blood cell activation in myeloproliferative neoplasms. Haematologica. 2009;94:1484–8. https://doi.org/10.3324/haematol.2009.013375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Matte A, Federti E, De Franceschi L. Erythrocyte pyruvate kinase activation in red cell disorders. Curr Opin Hematol. 2023;30. https://doi.org/10.1097/MOH.0000000000000758. 93– 8.

  10. Keating FK, Butenas S, Fung MK, Schneider DJ. Platelet-white blood cell (WBC) interaction, WBC apoptosis, and procoagulant activity in stored red blood cells. Transfusion. 2011;51:1086–95. https://doi.org/10.1111/j.1537-2995.2010.02950.x

    Article  PubMed  Google Scholar 

  11. Morel O, Morel N, Freyssinet JM, Toti F. Platelet microparticles and vascular cells interactions: a checkpoint between the haemostatic and thrombotic responses. Platelets. 2008;19:9–23. https://doi.org/10.1080/09537100701817232

    Article  CAS  PubMed  Google Scholar 

  12. Vahidkhah K, Cordasco D, Abbasi M, Ge L, Tseng E, Bagchi P, Azadani AN. Flow-Induced damage to blood cells in aortic valve stenosis. Ann Biomed Eng. 2016;44:2724–36. https://doi.org/10.1007/s10439-016-1577-7

    Article  PubMed  PubMed Central  Google Scholar 

  13. Swirski FK, Nahrendorf M. Leukocyte behavior in atherosclerosis, myocardial infarction, and heart failure. Science. 2013;339:161–6. https://doi.org/10.1126/science.1230719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang L, Bi Y, Yu M, Li T, Tong D, Yang X, Zhang C, Guo L, Wang C, Kou Y, Dong Z, Novakovic VA, Tian Y, Kou J, Shammas MA, Shi J. Phosphatidylserine-exposing blood cells and microparticles induce procoagulant activity in non-valvular atrial fibrillation. Int J Cardiol. 2018;258:138–43. https://doi.org/10.1016/j.ijcard.2018.01.116

    Article  PubMed  Google Scholar 

  15. Coté N, Mahmut A, Bosse Y, Couture C, Pagé S, Trahan S, Boulanger MC, Fournier D, Pibarot P, Mathieu P. Inflammation is associated with the remodeling of calcific aortic valve disease. Inflammation. 2013;36:573–81. https://doi.org/10.1007/s10753-012-9579-6

    Article  CAS  PubMed  Google Scholar 

  16. Álvarez-Heredia P, Domínguez-Del-Castillo JJ, Reina-Alfonso I, Gutiérrez-González C, Hassouneh F, Batista-Duharte A, Trujillo-Aguilera A, López-Romero R, Muñoz I, Solana R, Pera A. A straightforward Cytometry-Based protocol for the comprehensive analysis of the inflammatory valve infiltrate in aortic stenosis. Int J Mol Sci. 2023;24. https://doi.org/10.3390/ijms24032194

  17. Baratchi S, Zaldivia M, Wallert M, Loseff-Silver J, Al-Aryahi S, Zamani J, Thurgood P, Salim A, Htun NM, Stub D, Vahidi P, Duffy SJ, Walton A, Nguyen TH, Jaworowski A, Khoshmanesh K, Peter K. Transcatheter aortic valve implantation represents an Anti-Inflammatory therapy via reduction of shear Stress-Induced, Piezo-1-Mediated monocyte activation. Circulation. 2020;142:1092–105. https://doi.org/10.1161/CIRCULATIONAHA.120.045536

    Article  CAS  PubMed  Google Scholar 

  18. Jhun CS, Newswanger R, Cysyk JP, Ponnaluri S, Good B, Manning KB, Rosenberg G. Dynamics of blood flows in aortic stenosis: mild, moderate, and severe. ASAIO J. 2021;67:666–74. https://doi.org/10.1097/MAT.0000000000001296

    Article  PubMed  PubMed Central  Google Scholar 

  19. Wu ZH, Ji CL, Li H, Qiu GX, Gao CJ, Weng XS. Membrane microparticles and diseases. Eur Rev Med Pharmacol Sci. 2013;17:2420–7.

    PubMed  Google Scholar 

  20. Noubouossie DF, Henderson MW, Mooberry M, Ilich A, Ellsworth P, Piegore M, Skinner SC, Pawlinski R, Welsby I, Renné T, Hoffman M, Monroe DM, Key NS. Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways. Blood. 2020;135:755–65. https://doi.org/10.1182/blood.2019001643

    Article  PubMed  PubMed Central  Google Scholar 

  21. Grover SP, Mackman N, Factor T. An essential mediator of hemostasis and trigger of thrombosis, arterioscler. Thromb Vasc Biol. 2018;38:709–25. https://doi.org/10.1161/ATVBAHA.117.309846

    Article  CAS  Google Scholar 

  22. Tan X, Shi J, Fu Y, Gao C, Yang X, Li J, Wang W, Hou J, Li H, Zhou J. Role of erythrocytes and platelets in the hypercoagulable status in polycythemia vera through phosphatidylserine exposure and microparticle generation. Thromb Haemost. 2013;109:1025–32. https://doi.org/10.1160/TH12-11-0811

    Article  CAS  PubMed  Google Scholar 

  23. Falk V, Baumgartner H, Bax JJ, De Bonis M, Hamm C, Holm PJ, Iung B, Lancellotti P, Lansac E, Muñoz DR, Rosenhek R, Sjögren J, Tornos MP, Vahanian A, Walther T, Wendler O, Windecker S, Zamorano JL. 2017 ESC/EACTS guidelines for the management of valvular heart disease. Eur J Cardiothorac Surg. 2017;52. https://doi.org/10.1093/ejcts/ezx324. 616– 64.

  24. Shi J, Heegaard CW, Rasmussen JT, Gilbert GE. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature. Biochim Biophys Acta. 2004;1667:82–90. https://doi.org/10.1016/j.bbamem.2004.09.006

    Article  CAS  PubMed  Google Scholar 

  25. Chi H, Shao Y, Xie F, Zhang J, Zhang G, Jiang G, Tong D, Li J. Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention. J Thromb Thrombolysis. 2023;56:264–74. https://doi.org/10.1007/s11239-023-02835-5

    Article  CAS  PubMed  Google Scholar 

  26. He Z, Zhang Y, Cao M, Ma R, Meng H, Yao Z, Zhao L, Liu Y, Wu X, Deng R, Dong Z, Bi Y, Kou J, Novakovic V, Shi J, Hao L. Increased phosphatidylserine-exposing microparticles and their originating cells are associated with the coagulation process in patients with IgA nephropathy. Nephrol Dial Transpl. 2016;31. https://doi.org/10.1093/ndt/gfv403. 747– 59.

  27. Nomura S, Tandon NN, Nakamura T, Cone J, Fukuhara S, Kambayashi J. High-shear-stress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells. Atherosclerosis. 2001;158:277–87. https://doi.org/10.1016/s0021-9150(01)00433-6

    Article  CAS  PubMed  Google Scholar 

  28. Diehl P, Nagy F, Sossong V, Helbing T, Beyersdorf F, Olschewski M, Bode C, Moser M. Increased levels of Circulating microparticles in patients with severe aortic valve stenosis. Thromb Haemost. 2008;99:711–9. https://doi.org/10.1160/TH07-05-0334

    Article  CAS  PubMed  Google Scholar 

  29. Vion AC, Ramkhelawon B, Loyer X, Chironi G, Devue C, Loirand G, Tedgui A, Lehoux S, Boulanger CM. Shear stress regulates endothelial microparticle release. Circ Res. 2013;112:1323–33. https://doi.org/10.1161/CIRCRESAHA.112.300818

    Article  CAS  PubMed  Google Scholar 

  30. Kanda H, Yamakuchi M, Matsumoto K, Mukaihara K, Shigehisa Y, Tachioka S, Okawa M, Takenouchi K, Oyama Y, Hashiguchi T, Imoto Y. Dynamic changes in platelets caused by shear stress in aortic valve stenosis. Clin Hemorheol Microcirc. 2021;77:71–81. https://doi.org/10.3233/CH-200928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Anselmo A, Frank D, Papa L, Viviani AC, Di Pasquale E, Mazzola M, Panico C, Clemente F, Soldani C, Pagiatakis C, Hinkel R, Thalmann R, Kozlik-Feldmann R, Miragoli M, Carullo P, Vacchiano M, Chaves-Sanjuan A, Santo N, Losi MA, Ferrari MC, Puca AA, Christiansen V, Seoudy H, Freitag-Wolf S, Frey N, Dempfle A, Mercola M, Esposito G, Briguori C, Kupatt C, Condorelli G. Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release. Eur Heart J. 2021;42:2780–92. https://doi.org/10.1093/eurheartj/ehab247

    Article  CAS  PubMed  Google Scholar 

  32. Sangha GS, Weber CM, Sapp RM, Setua S, Thangaraju K, Pettebone M, Rogers SC, Doctor A, Buehler PW, Clyne AM. Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles. Front Physiol. 2023;14:1246910. https://doi.org/10.3389/fphys.2023.1246910

    Article  PubMed  PubMed Central  Google Scholar 

  33. Morel O, Toti F, Hugel B, Bakouboula B, Camoin-Jau L, Dignat-George F, Freyssinet JM. Procoagulant microparticles: disrupting the vascular homeostasis equation? Arterioscler. Thromb Vasc Biol. 2006;26:2594–604. https://doi.org/10.1161/01.ATV.0000246775.14471.26

    Article  CAS  Google Scholar 

  34. Kay JG, Grinstein S. Sensing phosphatidylserine in cellular membranes. Sensors. 2011;11:1744–55. https://doi.org/10.3390/s110201744

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Albanyan AM, Murphy MF, Rasmussen JT, Heegaard CW, Harrison P. Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with Annexin V. Transfusion. 2009;49:99–107. https://doi.org/10.1111/j.1537-2995.2008.01933.x

    Article  PubMed  Google Scholar 

  36. He Z, Si Y, Jiang T, Ma R, Zhang Y, Cao M, Li T, Yao Z, Zhao L, Fang S, Yu B, Dong Z, Thatte HS, Bi Y, Kou J, Yang S, Piao D, Hao L, Zhou J, Shi J. Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease. Thromb Haemost. 2016;115. https://doi.org/10.1160/TH15-09-0710. 738– 51.

  37. Nagata S, Suzuki J, Segawa K, Fujii T. Exposure of phosphatidylserine on the cell surface. Cell Death Differ. 2016;23:952–61. https://doi.org/10.1038/cdd.2016.7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tong D, Yu M, Guo L, Li T, Li J, Novakovic VA, Dong Z, Tian Y, Kou J, Bi Y, Wang J, Zhou J, Shi J. Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients. Ann Hematol. 2018;97:605–16. https://doi.org/10.1007/s00277-018-3228-6

    Article  CAS  PubMed  Google Scholar 

  39. Li S, Xu L, Liu B. The neglected role of neutrophils in the severity of aortic valve stenosis. J Cardiovasc Pharmacol. 2019;74:367–8. https://doi.org/10.1097/FJC.0000000000000737

    Article  CAS  PubMed  Google Scholar 

  40. Conte M, Petraglia L, Campana P, Gerundo G, Caruso A, Grimaldi MG, Russo V, Attena E, Leosco D, Parisi V. The role of inflammation and metabolic risk factors in the pathogenesis of calcific aortic valve stenosis, aging clin. Exp Res. 2021;33:1765–70. https://doi.org/10.1007/s40520-020-01681-2

    Article  Google Scholar 

  41. Dimitrow PP, Hlawaty M, Undas A, Sniezek-Maciejewska M, Sobień B, Stepień E, Tracz W. Effect of aortic valve stenosis on haemostasis is independent from vascular atherosclerotic burden. Atherosclerosis. 2009;204:e103–8. https://doi.org/10.1016/j.atherosclerosis.2008.12.029

    Article  CAS  PubMed  Google Scholar 

  42. Li J, Tong D, Chen F, Song B, Wang Y, Liu Y, Zhang X, Liu N, Xu Y, Li Y, Yin X, Liu W. Inflammatory cytokines enhance procoagulant activity of platelets and endothelial cells through phosphatidylserine exposure in patients with essential hypertension. J Thromb Thrombolysis. 2021;51:933–40. https://doi.org/10.1007/s11239-020-02342-x

    Article  CAS  PubMed  Google Scholar 

  43. Usta AH, Akbas F, Aral H. Relationship between Circulating microparticles and hypertension and other cardiac disease biomarkers in the elderly. BMC Cardiovasc Disord. 2019;19:164. https://doi.org/10.1186/s12872-019-1148-6

    Article  Google Scholar 

  44. Chen VM, Hogg PJ. Encryption and decryption of tissue factor. J Thromb Haemost. 2013;11(1):277–84. https://doi.org/10.1111/jth.12228

    Article  PubMed  Google Scholar 

  45. Kay JG, Grinstein S. Phosphatidylserine-mediated cellular signaling, Adv. Exp. Med. Biol. 991 (2013) 177– 93. https://doi.org/10.1007/978-94-007-6331-9_10

Download references

Acknowledgements

We would like to specially thank Qingdao Municipal Hospital Heart Center and Qingdao Municipal Hospital Central Laboratory, Qingdao, China.

Funding

This study was supported by the Technological Benefits to the Public by Qingdao Municipal Government, the grant from National Key Research and Development Program of China (2022YFC3602500) and Beijing High-level Public Health Technical Talents Construction Project (Discipline Leader-03-24) and Beijing Municipal Administration of Hospitals’ Ascent Plan（Code:DFL20240601.

Author information

Author notes

  1. Haiyang Wang and Zhaona Du are first authors, these authors have contributed equally to this work.

Authors and Affiliations

  1. Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, No. 2 Anzhen Road, Chao Yang District, Beijing, 100029, China

    Haiyang Wang & Yujie Zhou

  2. Department of Cardiology, Jining NO.1 People’s Hospital, Jining, China

    Zhaona Du

  3. Department of Cardiology, Qingdao Municipal Hospital, University of Health and Rehabilitation Sciences, NO. 5 Donghai Middle Road, Qingdao, 266071, China

    Haiyang Wang, Yibing Shao, Fangyu Xie, Jihe Li & Wei Xia

  4. Qingdao Municipal Hospital, Qingdao University, Qingdao, China

    Wei Wu

  5. Department of Oncology, Qingdao Municipal Hospital, University of Health and Rehabilitation Sciences, Qingdao, China

    Dongxia Tong

Authors
  1. Haiyang Wang
    View author publications

    Search author on:PubMed Google Scholar

  2. Zhaona Du
    View author publications

    Search author on:PubMed Google Scholar

  3. Yibing Shao
    View author publications

    Search author on:PubMed Google Scholar

  4. Wei Wu
    View author publications

    Search author on:PubMed Google Scholar

  5. Dongxia Tong
    View author publications

    Search author on:PubMed Google Scholar

  6. Fangyu Xie
    View author publications

    Search author on:PubMed Google Scholar

  7. Jihe Li
    View author publications

    Search author on:PubMed Google Scholar

  8. Wei Xia
    View author publications

    Search author on:PubMed Google Scholar

  9. Yujie Zhou
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Z.D., H.W., Y.S., J.L., W.X. and Y.Z. designed the experiments, analyzed data, and wrote the paper. W.W. and D.T. performed the experiments. F.X. helped with the experiments.

Corresponding authors

Correspondence to Jihe Li, Wei Xia or Yujie Zhou.

Ethics declarations

Consent for publication

Written informed consent was obtained from the individual(s) for the publication of any data included in this article.

Competing interests

The authors have no conflicts of interest to declare.

Data availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, H., Du, Z., Shao, Y. et al. Procoagulant effect of phosphatidylserine-exposed blood cells, endothelial cells and extracellular vesicles in patients with aortic stenosis. Thrombosis J 23, 70 (2025). https://doi.org/10.1186/s12959-025-00755-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12959-025-00755-3

Keywords

Thrombosis Journal

ISSN: 1477-9560

Contact us