Elsevier

Thrombosis Research

Volume 129, Issue 5, May 2012, Pages e251-e256
Thrombosis Research

Regular Article
Monomeric C-reactive protein alters fibrin clot properties on endothelial cells

https://doi.org/10.1016/j.thromres.2012.03.014Get rights and content

Abstract

Elevated plasma levels of C-reactive protein (CRP) are independently associated with increased risk of atherothrombosis. Several lines of evidence suggest that CRP has prothrombogenic effects on injured vessel wall(s) by enhancing tissue factor (TF) expression. Abnormal fibrin formation is correlated with increased thrombotic risk. However, the impact of localized, cell surface-driven in situ tissue factor generation by CRP on clot dynamics and fibrin architecture has not previously been evaluated. We examined the impact of native CRP and modified or monomeric CRP (mCRP) on the fibrin formation and structure in Human Umbilical Vein Endothelial Cells (HUVECs). Fibrin formation and structure were examined using laser scanning confocal microscopy. Incubation with mCRP on the cell surface had faster fibrin polymerization by the analysis of turbidimetry. Confocal microscopy of fibrin clots showed a significantly increased density in the treatment of mCRP compared with native CRP and control in the proximal versus distal relationship to the cell surface. The increased expression and activity of TF on the cell surface was observed by addition of mCRP. Blockage of tissue factor and lipid rafts significantly reduced the density of fibrin network produced by mCRP-stimulated endothelial cells. mCRP changes clot dynamics and alters fibrin architecture by enhancing TF on the endothelial cell surface. These results support the concept that elevated CRP levels may induce fibrinolytic resistance and endothelial dysfunction by altering fibrin clot structure.

Introduction

Fibrin is the end-product of coagulation, and the formation and degradation of fibrin are essential for hemostasis and thrombosis. Altered fibrin structure is associated with clot stability in thrombotic disease, including premature myocardial infarction, and venous thromboembolism [1], [2]. Furthermore, fibrin clot properties are influenced by environmental and genetic factors, especially as they relate to vascular wall cells [3], [4]. The mechanisms are involved in the exposure of extravascular (smooth muscle cell and fibroblast) tissue factor (TF) activity or induction of intravascular (endothelial cell) TF activity by inflammatory factor [4]. Therefore, besides numerous components present in plasma, the interaction of fibrin with surrounding vessel wall cells is also thought to be important in the clot development and stability.

C-reactive protein (CRP) is a member of the pentraxin protein family which is widely known as a contributing factor in vascular diseases [5], [6], [7], [8], [9], [10]. CRP has been associated with activating the blood coagulation system and increasing the risk of thrombosis. It has been suggested that CRP induces procoagulant [11] and anti-fibrinolytic activity [12] in vascular endothelial cells. While circulating CRP exists in the native pentameric form (pCRP), it can also exist in a modified monomeric form (mCRP). These two distinct forms of CRP have been shown to have diverse effects on biological activity [13], [14], [15], [16], [17]. The conformation of CRP might play a role in controlling platelet aggregation [18], [19]. Evidence indicates that mCRP, but not pCRP enhances thrombus growth [16]. However, it is not known whether the CRP is associated with altered fibrin clot properties that might contribute to this complication regardless of platelet reactivity. In the present study, we compared the relative impact of mCRP and pCRP on endothelial cell to modulate fibrin properties.

Section snippets

Reagents

Purified human recombinant natCRP (Trichem Resources) was dialyzed extensively against 0.1 mmol/L Tris–HCl, pH 7.5, containing 0.2 mmol/L NaCl, 2 mmol/L CaCl2, and passed over Detoxigel (Pierce). Endotoxin concentration in CRP preparations was < 0.06 endotoxin units/mL. mCRP was obtained by urea chelation from purified human CRP as described by Kresl et al. [20]. AlexaFluor-488-labeled fibrinogen was purchased from Sigma-AldrichCorporation. Anti-TF antibody was obtained from America Dignostica Inc.

mCRP, not pCRP, increases fibrin formation

To observe the impact of CRP-stimulated endothelial cells in fibrin formation and stability, we pretreated mCRP or pCRP (range from 1 to 50 μg/mL) for 16 hours, and incubated HUVECs monolayer with normal pooled PPP and used turbidity to examine the fibrin formation. A dose-dependent change in fibrin clot variables on stimulation with mCRP but not with pCRP was observed (Fig. 1). Compared with vehicle and pCRP, mCRP had a shorter onset time, and greater maximum △ Abs (Table 1). In a plasma clot

Discussion

This study demonstrates that altered clot properties are associated with CRP conformational change. mCRP, but not pCRP, can induce TF activity in endothelial cells and trigger events involved in the alteration of fibrin formation and properties. The results showed that CRP may promote atherothrombosis through the alteration of the fibrin properties by affecting endothelial cells.

Recent data indicate that CRP upregulates TF activity on vascular cells, which includes monocytes, endothelial cells

Funding Sources

This work was supported by the AHA National Scientist Development Grant (10SDG2570037), a Research Council Grant from the University of Missouri (URC-10-001-Wu) (to J. Wu.) and the National Natural Science Foundation of China (81172050) (to J. Wu.).

Conflict of interest statement

The authors state that they have no conflict of interest.

Acknowledgments

We wish to thank Dr. William P. Fay from the University of Missouri-Columbia for providing all the necessary facilities and environment got carrying out research and for providing valuable suggestions for the completion of the manuscript.

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    R. Li. and M. Ren contributed equally to this study.

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