Synergic effect of GPIBA and von Willebrand factor in pathogenesis of deep vein thrombosis

Introduction

Deep vein thrombosis (DVT) is one of the leading cardiovascular diseases after acute myocardial infarction and stroke. DVT is a major cause of pulmonary thromboembolism since the unstable thrombus detaches and travels to the lungs.^1,2 The pathogenesis of DVT is still not clearly known although platelet activity, blood thickness, and coagulation are thought to be the main reasons leading to DVT. ³ Besides, recent findings indicated that other factors such as inflammation ⁴ and critical molecules including von Willebrand factor (vWF), ⁵ tissue factor, ⁶ and 17α-estradiol ⁷ are involved in the experimental DVT.

platelet glycoprotein Ib alpha chain (GPIBA), glycoprotein Ib platelet subunit beta (GPIBB), glycoprotein IX (GPIX), glycoprotein V (GPV) encode platelet membrane proteins, playing vital roles in normal platelet aggregation. ⁸ The GPIBA gene codes for the platelet GPIbα subunit of the glycoprotein GPIb-IX complex, composed of the above-mentioned four platelet membrane proteins. ⁹ GP1BA is critical in the process of platelet aggregation. Mutation in the GPIbα protein coded by GPIBA gene leads to blooding disorders, such as Bernard–Soulier Syndrome ¹⁰ and platelet type von Willebrand disease. ¹¹ GPIbα carries the binding site for vWF, an adhesive protein, mediating platelet adhesion to the vessel wall and platelet recruitment to the growing thrombus.^12,13 vWF is critical for DVT in mouse models. ⁵ Thus, the correlation among GPIBA, coagulation factor, and thrombosis needs to be investigated since it may provide a potential target to the therapies against cardiovascular disease.

In this study, we elucidated the alteration in expression of GPIBA and concentration of coagulation factors in DVT model. Meanwhile, heparin treatment could reverse the GPIBA and coagulation factors in thrombosis model. Other proteins critical in adhesion and binding of platelets such as epithelial membrane protein 2 (EMP2), vascular cell adhesion protein 1 (VCAM1), immunoreceptor tyrosine-based activation motif 1 (ITAM1), integrin subunit alpha M (ITGAM), or fibronectin were also affected in thrombosis model and with heparin treatment. Thus, this work demonstrated the potential synergic role of GPIBA and vWF in the formation of DVT.

Materials and methods

Results

Dynamic expression of GP1BA in vein blood of thrombosis model with heparin treatment

We first determined alteration of platelet surface membrane protein GP1BA level in deep vein from thrombosis model. Flow cytometry data indicated that GP1BA level was significantly increased in venous blood from thrombosis model in contrast to that in negative control group (Figure 1(a)). Besides, treatment with heparin drastically decreased the GP1BA level in thrombosis model (Figure 1(a)). Meanwhile, we also found apparent increase in GP1BB, GP5, or GP9 level in thrombosis model (Figure 1(b) to (d)). However, treatment with heparin did not significantly change GP1BB, GP5, or GP9 level (Figure 1(b) to (d)). Thus, we concluded GP1BA level in venous blood was significantly increased in thrombosis model, while heparin could reverse this process.

OPEN IN VIEWER

Figure 1.

Heparin treatment decreased GPIBA level in thrombosis model. Flow cytometry analysis of GP1BA (a), GP1BB (b), GP5 (c), and GP9 level (d).

Heparin treatment reduced the level of coagulation factor vWF in thrombosis model

Next, we determined whether heparin could affect the level of coagulation factor in thrombosis model. ELISA analysis showed that level of vWF, F11, or F12 was significantly increased in thrombosis model (p < 0.001, Figure 2). By contrast, heparin treatment drastically decreased the level of vWF (p < 0.001, Figure 2). However, the concentration of F11 or F12 in venous blood did not significantly alter in the presence of heparin (p > 0.05, Figure 2). Therefore, heparin reduced the concentration of vWF in thrombosis model.

OPEN IN VIEWER

Figure 2.

ELISA analysis showed the alteration of coagulation factor vWF, F11, and F12 in heparin-treated thrombosis model.

Heparin lowered the expression of ITAM1, ITGAM, or fibronectin in the thrombus tissue

Since several proteins such as ITAM1, ITGAM, or fibronectin is critical in mediating the adhesion and binding of platelets and lymphocytes to the matrix of endothelial cells, we then determined if heparin could affect the expression of these proteins. Immunohistochemical analysis demonstrated that expression of ITAM1, ITGAM, or fibronectin was enhanced in thrombosis model (Figure 3(b)) compared to that in normal controls (Figure 3(a)). In the presence of heparin, expression of ITAM1, ITGAM, or fibronectin was weaker (Figure 3(c)) than that in model group. Collectively, these data indicated that heparin treatment decreased the expression of ITAM1, ITGAM, or fibronectin in thrombosis model.

OPEN IN VIEWER

Figure 3.

Immunohistochemical analysis showed the enhancement of ITAM1, ITGAM, and fibronectin in thrombosis model (b) compared to that in normal control (a), while heparin treatment weakened the expression of these proteins (c).

Heparin decreased expression level of EMP2 or VCAM1 in thrombus tissue

Then, we determined whether heparin could affect EMP2 or VCAM1 level in thrombosis model. Through western blot, we found the expression level of EMP2 or VCAM1 was significantly increased in thrombosis models compared to that in normal controls (Figure 4). By contrast, the treatment with heparin could decrease the EMP2 or VCAM1 level in thrombus tissues (Figure 4). Thus, heparin could reduce the expression level of EMP2 or VCAM1, which was increased in thrombosis models.

OPEN IN VIEWER

Figure 4.

Western blot showed alteration in EMP2 and VCAM1 expression level in thrombosis models.

Discussion

In this study, we determined the role of GPIBA and vWF in thrombosis models. This indicated the potential interaction between GPIBA and vWF in the pathogenesis of thrombosis and provided a potent therapeutic target.

Until now, the specific role of platelet in DVT is still not well-understood. A recognized mechanism for platelet recruitment is adhesion to vWF. GPIBA provides binding site for the vWF, directly showing the involvement of GPIBA in biological function of platelet. This is consistent with the previous finding that GAIBA is associated with bleeding. ¹⁵ Platelet GPIbα subunit coded by GPIBA regulated thrombosis. ¹⁶ This suggested that GPIBA might involve in the formation of thrombus. This was further supported by the findings that mutation in GPIBA was present in a family with venous thrombosis. ¹⁷ Based on these works, we found that GPIBA and vWF were aberrantly expressed in DVT models, further showing their role in occurrence of thrombosis. Furthermore, we also showed the alteration in proteins critical in platelet adhesion, suggesting participation of GPIBA in thrombus formation.

The clinical effect of heparin was evaluated in thrombosis. ¹⁸ Heparin has a preventive effect against DVT.^19,20 In this study, we demonstrated the effect of heparin in offsetting the aberrant expression level of GPIBA and other proteins in DVT models. This is the first report to associate the GPIBA and heparin in thrombosis model, showing the antiplatelet effect of heparin was possibly mediated by GPIBA. However, the molecular mechanism underlying this synergic effect is not investigated in the current study. Further study will be focused on GPIBA mutant or knockout models to determine the direct correlation between GPIBA and curative effect of heparin.

Conclusion

This study elucidated the role of GPIBA and vWF in DVT models and evaluated the effect of heparin on these proteins. This work may provide a therapeutic target against thrombosis.