Regular ArticleMonomeric C-reactive protein alters fibrin clot properties on endothelial cells
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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2014, Thrombosis ResearchCitation Excerpt :The effect of CRP on fibrin network structure could be via its effect on TF. C-reactive protein has been shown to increase TF expression and activity in blood monocytes, vascular smooth muscle cells as well as endothelial cells (ECs) [56,57]. Recent evidence has associated CRP with faster fibrin polymerisation and consequently greater maximum absorbance in ECs and in the presence of TF expression, CRP produced clots with thicker fibres that were more fibre dense [57].
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2013, Thrombosis ResearchCitation Excerpt :CRP, a systemic marker of inflammation, is considered a risk factor for the occurrence of cardiovascular events [13]. CRP level has been reported to link both higher platelet reactivity and fibrin clot strength [13,14]. Thus, the systemic effects of inflammation may extend beyond the more direct effects on atherosclerotic plaque generation and cellular processes of inflammation.
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R. Li. and M. Ren contributed equally to this study.