C-Reactive Protein Isoforms Differ in Their Effects on Thrombus Growth
Objective— We studied the impact of native (natCRP) and modified CRP (mCRP) isoforms on platelet adhesion and thrombus growth under arterial flow.
Methods and Results— Blood was perfused over type I collagen at a wall shear rate of 1500 s−1, and platelet deposition and thrombus growth were evaluated by confocal microscopy. natCRP and mCRP were either incubated with blood before perfusion experiments or immobilized in the collagen surface and exposed to flowing blood. mCRP significantly increased platelet adhesion and thrombus growth when directly incubated with blood and when immobilized on a collagen surface (P<0.05). In contrast, natCRP did not exert any effect. Confocal immunohistochemistry revealed the presence of CRP on the surface of adhered platelets and within the thrombus and showed an upregulation of P-selectin and CD36 in effluent platelets preincubated with mCRP (P<0.05). Flow cytometry analysis of agonist-induced platelet activation demonstrated that mCRP, but not natCRP, significantly increased platelet surface P-selectin (P<0.05) without modifying CD63 and PAC-1.
Conclusions— Our data indicate that whereas serum natCRP may not affect thrombus growth, mCRP displays a prothrombotic phenotype enhancing not only platelet deposition, but also thrombus growth under arterial flow conditions.
In recent years, C-reactive protein (CRP), long associated with inflammation, has emerged as a clinical marker of future cardiovascular events among apparently healthy subjects and of worse prognosis in acute coronary patients.1–3
Thrombus formation on rupture of an atherosclerotic plaque is believed to be the responsible event for most of the coronary syndromes, in a process mainly mediated by platelet adhesion, activation, and aggregation. The first response to vascular injury consists of platelet adhesion to the damaged vessel wall or to exposed tissue components, and is mediated by flow-regulated interactions that have a key influence on subsequent thrombus growth, often culminating in life-threatening complications.4,5
Long considered merely a bystander in vascular disease, new evidence indicates that CRP may be not only a marker, but also an active player in the development of cardiovascular pathology.6 The role of CRP as a modulator of inflammation and thrombosis is controversial, because both proinflammatory and antiinflammatory properties have been ascribed to the molecule.7–9 For instance, CRP inhibits neutrophil activation and adhesion,9 and blocks platelet aggregation in vitro,10,11 whereas arterial injury in CRP-transgenic mice is associated with increased thrombosis.12 Overexpression of the human CRP gene in atherosclerosis-prone mice has also shown contradictory effects on the development of atherosclerosis.13,14 To explain these apparently contradictory actions, it was proposed that distinct isoforms of CRP were formed during inflammation. The classically studied serum CRP is a pentamer composed of five noncovalently bound globular subunits arranged as a cyclic annular disk, the so-called native CRP (natCRP). natCRP can undergo subunit dissociation into individual monomeric units, as when associating with a cell-membrane.15 These subunits undergo a conformational change that significantly modifies CRP structure, solubility, and antigenicity. This form of CRP, called modified or monomeric CRP (mCRP), is found in fibrous tissues of normal and inflamed human blood vessel intima.16 Although the expression of CRP mRNA in both normal and plaque arterial tissues has been reported,17 it remains to be proven whether extrahepatic cells possess the machinery necessary to fold 5 subunits into the native pentamer. In vitro, mCRP can be produced from natCRP by exposing natCRP to heat, urea, or acidic conditions, in the absence of calcium ions.18 mCRP can also spontaneously form from natCRP during storage. Studies directly addressing the distinct isoforms of CRP have reported that mCRP displays proinflammatory effects on neutrophils, endothelial cells, and platelets, whereas natCRP displays antiinflammatory activities.19–21
In the present study, we investigated the relative impact of natCRP and mCRP on the dynamics of platelet adhesion and thrombus growth under defined flow conditions. Our results indicate that mCRP, but not natCRP, enhances platelet adhesion and thrombus growth.
Materials and Methods
See supplemental file (available online at http://atvb.ahajournals.org) for expanded Methods section.
CRP Isoforms Obtention
High purity human natCRP (Calbiochem) was stored in 10 mmol/L Tris, 140 mmol/L NaCl buffer (pH 8.0) containing 2 mmol/L CaCl2 to prevent spontaneous formation of mCRP from the native pentamer. mCRP was obtained by urea chelation from purified human CRP as described by Potempa et al.18
Venous blood from medication-free volunteers was withdrawn in 10 UI/mL sodium heparin. Procedures were approved by the Clinical Research Committee of our Institution.
Blood was then incubated with natCRP, mCRP, or control buffer (37°C, 10 minutes). Platelets were rendered fluorescent by the addition of mepacrine 10 μmol/L (Sigma), unless otherwise specified.
Perfusion Experiments in Flat Chamber
Glass slides were coated with type I collagen (4°C, overnight). When indicated, collagen-coated slides were incubated with 5 μg/mL of natCRP, mCRP, or blocking buffer (1% bovine serum albumin) for 3 hours at 37°C. Coated slides were placed in a parallel plate chamber.22 A peristaltic pump was used to perfuse blood through the chamber at a constant shear rate of 1500 s−1 for 5 minutes.
Imaging of Platelet Thrombi
Platelet deposition was scanned with a Leica TCS SP2 confocal laser scanning microscope. Platelets were viewed with an APO 20X objective. Surface covered by platelets and area of individual thrombi were calculated using NIH Image software (by Dr Wayne Rasband, National Institutes of Health). Average height of platelet thrombi was calculated creating a topographical image from the spatial data set acquired and three-dimensional rotation projections were created from stack series of selected thrombi.
For detection of CRP, a further set of perfusion experiments with unlabeled platelets was performed. Immunodetection of CRP on fixed slides was performed with a monoclonal anti-human CRP antibody (Sigma, clone 8) and with a monoclonal antibody (mAb) which specifically recognizes mCRP isoform (clone 8C10 kindly provided by Dr Potempa). Coverslides were incubated with Alexa Fluor 488 donkey antimouse IgG (H+L).
P-Selectin and CD36 Immunostaining
Effluent blood from perfusion experiments with blood unlabeled with mepacrine was collected and fixed with 3.8% paraformaldehyde for 30 minutes, and platelet-rich plasma was obtained by centrifugation at 200g for 17 minutes. Platelets were then isolated by centrifugation, immobilized on poly-l-lysine-coated coverslides, and incubated either with a phycoerytrin (PE) conjugated anti-CD62P mAb (Pharmingen) or a fluorescein isothiocyanate (FITC) conjugated anti-CD36 mAb (Pharmingen). Platelets were viewed with an APO 63X objective.
CRP-treated samples were diluted 1:10 in modified Tyrode Buffer and activated with collagen (5 μg/mL) or ADP (1 μmol/L). A CD41a – FITC mAb (Pharmingen) was used as an activation-independent marker of platelets for CD62P and CD63 analysis. P-selectin and CD63 were assessed with a PE-conjugated anti-CD62P mAb (Pharmingen) and a PE-conjugated anti-CD63 mAb (Pharmingen), respectively. GPIIb-IIIa conformational change was assessed with a FITC-conjugated PAC-1 mAb (Beckton Dickinson). In additional experiments, the responses to CRP were studied in the presence of 2.5 μg/mL of function-blocking anti-CD16 mAb 3G8 (Pharmingen).
Different conditions were performed at least twice in each subject and 5 subjects of each treatment were assessed. After testing for normal distribution and equality of variances with Levene F test, Student t test or ANOVA as appropriate was used to determine statistical significance between treatments. A value of P<0.05 was considered significant.
Contribution of CRP Isoforms to Thrombus Formation
Preincubation of blood with mCRP increased platelet deposition in a concentration-dependent manner. Statistically significant increase was detected with concentrations higher than 1 μg/mL mCRP. In the presence of 25 μg/mL mCRP, platelet deposition was more than 3-fold higher than in untreated blood (P<0.05). On the contrary, incubation with natCRP did not produce any effect on platelet deposition at any tested concentration (Figure 1A).
To elucidate the effect of CRP on aggregate size, the area of individual thrombi formed on the collagen surface was evaluated. For this purpose we quantified the area of aggregates larger than 100 μm2. Thrombus area significantly increased in samples treated with mCRP in a dose-dependent manner, with a statistically significant increase detected at concentrations higher than 1 μg/mL mCRP (P<0.05). By contrast, blood incubation with natCRP (10 μg/mL) produced inhibition on aggregate size (P<0.05) as shown in Figure 1B.
Three-dimensional topographical imaging of platelet thrombi were obtained from the spatial data set acquired by confocal microscopy. natCRP treatment did not yield any effect on thrombus height. On the contrary, 3D topographical imaging revealed that mCRP increased thrombus height in a concentration-dependent manner, with a statistically significant increase detected at concentrations higher than 1 μg/mL mCRP (P<0.05) as shown in Figure 1C.
CRP immunodetection on the platelet surface with the antibody that recognizes both CRP isoforms was more intense in perfusions ran with mCRP-treated blood than those ran when incubating blood with natCRP, as shown in Figure 2. (P<0.05). Additionally, mCRP immunodetection with anti-mCRP antibody showed lack of mCRP on the surface of control and natCRP-treated platelets, whereas mCRP was strongly immunodetected on the surface of platelets treated with mCRP (see supplemental Figure II).
Because mCRP, but not natCRP, enhanced thrombus growth, CRP distribution within the thrombus volume was evaluated by 3D projections of selected thrombi (Figure 3). CRP labeling in thrombi formed when incubating blood with natCRP appeared to be distributed as diffuse spots with a higher presence on the collagen surface (blue) than in the top edge (red) (Figure 3.I: 1A through 1F). By contrast, CRP immunostaining in mCRP thrombi was found to be distributed as aggregated patches within the entire volume of the thrombus (Figure 3.I: 2A through 2F). Immunodetection of mCRP revealed that mCRP was mainly localized on the thrombus growing edge (Figure 3.II). See supplemental movie-file with 90° rotating animation.
P-Selectin and CD36 Immunodetection
mCRP pretreatment, but not natCRP, enhanced the expression of both P-selectin and CD36 of effluent platelets. The percentage of CD62P expression increased from 36.7±4.3% in control samples, to 67.1±5.0% in mCRP-treated blood (P<0.05). Immunodetection of CD36 significantly increased up to 70% in effluent platelets incubated with mCRP compared to control and natCRP-treated platelets (P<0.05; Figure 4).
Flow Cytometry Studies
Blood incubation solely with CRP isoforms did not induce any significant effect in the expression of the platelet activation markers P-selectin, CD63, and PAC-1. Conversely, in collagen-stimulated platelets, preincubation with mCRP significantly enhanced platelet P-selectin expression (P<0.05), and it did not exert any effect in the expression of neither CD63 nor PAC-1. Preincubation with natCRP did not produce any significant effect on the expression of CD63 and PAC-1 in either resting or collagen-stimulated platelets (Figure 5A). However, although not statistically significant, natCRP increased P-selectin expression on collagen-induced activated platelets because of the partial dissociation of natCRP into its subunits. mCRP was formed from natCRP because of the acidity of the collagen solution needed to induce platelet activation. Indeed, dot blotting of natCRP subjected to the same conditions of the flow cytometry studies confirmed the partial dissociation of natCRP into mCRP, as shown in Figure 5B. In fact, P-selectin expression on ADP-induced platelet activation was not upregulated by natCRP preincubation. On the contrary, mCRP significantly increased P-selectin expression on ADP-induced activated platelets (Figure 5C). Blockade of the FcγRIII receptor (CD16) before blood incubation with mCRP did not suppress P-selectin expression on agonist activated platelets.
Effect of Surface-Immobilized CRP Isoforms on Thrombus Formation
The effect of CRP isoforms on thrombus growth was also evaluated in perfusion experiments on surfaces coated with immobilized collagen and either natCRP or mCRP. Similarly to what we observed when CRP was directly added to blood, immobilized mCRP in the collagen surface significantly enhanced platelet deposition. Mean platelet deposition on collagen/mCRP- coated surface was 2-fold higher than in control collagen-coated surfaces (P<0.05; Figure 6A).
Immobilized mCRP also significantly increased aggregate size. Indeed, the area of individual thrombi deposited on collagen (463±76 μm2) was significantly lower than that on the collagen/mCRP surface (1097±278 μm2; P<0.05; Figure 6B). Platelet deposition and aggregate size on collagen/natCRP-coated surface did not differ significantly from platelet deposition on collagen alone. In fact, increasing concentrations of immobilized natCRP in the collagen surface did not increase platelet deposition nor aggregate size (Figure 6A and 6B).
To investigate whether the presence of circulating natCRP would affect the platelet response to immobilized mCRP we added natCRP (5 μg/mL) to the blood and measured its effects on platelet deposition and aggregate size on collagen/mCRP-coated surfaces. natCRP did not affect platelet deposition on mCRP/collagen surface, measured as mean platelet deposition and aggregate size (Figure 6A and 6B).
Three-dimensional topographical imaging of platelet thrombi revealed that immobilized mCRP significantly enhanced thrombus growth. Platelet aggregates formed on collagen/mCRP were more than 2-fold higher than those formed on collagen alone (6.17±1.07 μm on collagen versus 14.31±2.34 μm on collagen/mCRP, P<0.05; Figure 6C). Immobilized natCRP on collagen affected neither aggregate size nor height of the thrombi.
Numerous epidemiological studies have shown that plasma CRP level is a powerful predictor of future cardiovascular events in seemingly healthy subjects and of worse prognosis in acute coronary patients. However, the pathophysiological importance of CRP is far from being fully understood. Data regarding the exact mechanisms of CRP effects are limited and controversial. In this study we show contrasting effects of CRP on the dynamics of thrombus formation under arterial flow conditions based on its different conformation. Whereas mCRP significantly enhanced platelet activation, adhesion, and thrombus growth, natCRP had no effect.
Clinically, the threshold of CRP plasma concentration associated to cardiovascular risk is more than 3 μg/mL, whereas levels higher than 10 μg/mL are usually attributed to other causes as acute infection or inflammation.23 We therefore used in our experiments physiological and pathophysiological concentrations of CRP, spanning from 1 to 25 μg/mL. Interestingly, mCRP was able to enhance thrombus growth at concentrations higher than 1 μg/mL, which coincides with concentrations predicting cardiovascular risk.
The postperfusion confocal analysis allowed us to study 3-dimensionally thrombus formation, measuring platelet deposition, aggregate size, and thrombus height on a protein-coated surface. Several reported effects of CRP have been shown to be calcium dependent,24 thus we used sodium heparin as anticoagulant instead of calcium chelators. Recent reports have questioned the validity of CRP in vitro studies. Commercial CRP preparations can be contaminated with sodium azide or lipopolysaccharide (LPS) and when dialysed, free of these factors, several of the effects of CRP are lost.25,26 To eliminate these confounding factors we used two different purified commercial preparations and, additionally, perfusion experiments performed with control buffer had no effect on platelet adhesion and thrombus formation.
mCRP, unlike natCRP, was able to induce thrombosis by promoting platelet deposition and thrombus growth on the collagen surface. mCRP not only significantly increased platelet adhesion, but also aggregate size and thrombus height. These observations support a role for mCRP in platelet adhesion, and also on platelet to platelet interaction, which is the responsible event for thrombus growth and further vessel occlusion. Accordingly, P-selectin, which has been shown to stabilize platelet–platelet and platelet–leukocyte aggregates,27,28 was also upregulated by mCRP, as seen in effluent platelets. This increase in P-selectin might explain, partly, the mCRP enhancement of thrombus growth. Indeed, platelet surface P-selectin followed a similar pattern after collagen and ADP stimulation in flow cytometry analysis. However, mCRP was unable to induce GPIIb/IIIa activation and surface CD63 expression. These observations suggest that mCRP enhances platelet recruitment and subsequent thrombus formation by exocytosis of α-granules and platelet agonists release. In contrast to previous data,19 mCRP effects were not mediated through the FcγRIII receptor. mCRP also seemed to modulate platelet CD36 expression, which is reported to act as a receptor of misfolded proteins,29 suggesting a potential role of CD36 in mediation of mCRP activity. Further studies seem warranted to better elucidate the role of mCRP on platelet activation-related signaling pathways. In contrast, natCRP did not enhance platelet adhesion and thrombus height. Interestingly, high concentrations of natCRP reduced thrombus area, consistently with its inhibitory action on platelet aggregation.10,11
These findings raise the possibility that the increased thrombus formation after arterial injury and monocyte-platelet aggregation in human CRP-transgenic mice,12,24 could be attributed to distinct isoforms of CRP rather than natCRP itself. Moreover, the different effects of CRP isoforms found in the present study are in accordance with the opposing effects of CRP isoforms previously reported by Khreiss et al on shear-induced neutrophil-platelet aggregation.19 In agreement with our findings, mCRP has been shown to exert greater proinflammatory effects in endothelial cells and neutrophils.20,30 Paradoxically, Schwedler et al reported that natCRP promoted but mCRP reduced atherosclerosis in ApoE−/− mice.31 These results are not necessarily contradictory with our findings because Schwedler et al studied the effect of CRP isoforms in early atherosclerosis, and designed a model of low dosing of mCRP over a long period of time, which could have heightened immune surveillance, slowing the process of atherosclerotic plaque formation. Conversely, we focused our efforts on studying the direct effect of native and modified CRP on the thrombotic complications, such as those happening on plaque rupture. Our study is, to the best of our knowledge, the first to show a causal and dual role of CRP isoforms on thrombus formation under arterial flow conditions.
How CRP mediates platelet activity still remains unclear. The presence of CRP observed on the platelet surface and within the thrombus structure after blood perfusion suggests a stable and direct interaction between platelets and CRP. When natCRP dissociates into free subunits, it yields monomeric mCRP with a loss of predominantly β-sheet secondary structure and an increase in α-helix,30 forming insoluble aggregates, which could explain the different distribution of natCRP and mCRP within the thrombus. The observed difference in intrathrombus distribution might be attributed to a stronger interaction of mCRP with the platelet surface compared to natCRP. It is important to point out that it is not clear whether monoclonal anti–CRP-clone 8 from Sigma detects both native and modified CRP. According to the manufacturer it recognizes both isoforms. However, Schwedler et al showed that anti–CRP-clone 8 predominantly recognized mCRP rather than natCRP.32 On the other hand, native CRP may likely bind to phosphatidylcholine abundantly expressed on the surface of activated platelets, and it has been shown that cell membranes dissociate natCRP to a structural intermediate (mCRP(m)), which can further detach from the membrane to form mCRP.15 Whether the staining with the anti–CRP-clone 8 antibody on the surface of adhered platelets preincubated with natCRP shows the presence of mCRP, natCRP, or the intermediate mCRP(m) is not clear. However, adhered platelets preincubated with natCRP stained positive for CRP with the anti–CRP-clone 8 and negative for mCRP with the anti-mCRP antibody (clone 8C10), suggesting the lack of presence of mCRP.
In this work we have observed that mCRP not only was able to enhance thrombosis when directly added to blood, but also when immobilized with collagen, a key component of atherosclerotic lesions. Although the presence of CRP mRNA in atherosclerotic tissue has been established,17,33 it is unclear whether the expressed CRP is the pentameric isoform or the monomeric isoform. It remains unknown whether CRP locally produced in the vessel wall34 is, indeed, mCRP that is naturally expressed in the intima.16 It is likely that collagen exposure after mechanical or spontaneous plaque rupture may also result in exposure of mCRP to blood components, leading to platelet aggregation and thrombus formation. Alternatively, inflammation may lead to formation of mCRP from natCRP within the blood stream, thus linking thrombosis and inflammation, key events in acute coronary syndromes. Interestingly, this concept supports the fact that the mere presence of CRP in plasma is not associated to platelet aggregation, but it is conceivable that mCRP, as in vascular tissue, might be one of the thrombogenic triggering factors.
In summary, our data indicate that whereas natCRP may not affect thrombus growth, mCRP displays a prothrombotic phenotype enhancing not only platelet deposition, but also thrombus growth under arterial flow conditions. Further research seems warranted to elucidate more detailed mechanisms by which CRP isoforms regulate thrombus formation, and to clarify the role of CRP in cardiovascular disease.
We thank Dr J. Crespo and M. Pescador for their technical help. We also thank Dr L. Potempa for kindly providing the antibodies against mCRP and natCRP.
Sources of Funding
This work has been possible thanks to funds provided by Ministry of Science and Education of Spain (PNS 2006/10091), Ministry of Health- Instituto Salud Carlos III (CIBEROBN- CB06/03), and Fundación Jesús Serra. B.M. is granted with a fellowship from the Catalan Government (FI2005 DUIE). G.V. is recipient of a grant from the Science and Education Spanish Ministry (JdC).
Original received March 12, 2008; final version accepted August 30, 2008.
Badimon L, Fuster V, Corti R, Badimon JJ. Coronary Thrombosis: Local and Systemic Factors. New York: McGraw Hill; 2004.
Pasceri V, Willerson JT, Yeh ET. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation. 2000; 102: 2165–2168.
Xia D, Samols D. Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxemia. Proc Natl Acad Sci U S A. 1997; 94: 2575–2580.
Fiedel BA, Gewurz H. Effects of C-reactive protein on platelet function. II. Inhibition by CRP of platelet reactivities stimulated by poly-L-lysine, ADP, epinephrine, and collagen. J Immunol. 1976; 117: 1073–1078.
Vigo C. Effect of C-reactive protein on platelet-activating factor-induced platelet aggregation and membrane stabilization. J Biol Chem. 1985; 260: 3418–3422.
Danenberg HD, Szalai AJ, Swaminathan RV, Peng L, Chen Z, Seifert P, Fay WP, Simon DI, Edelman ER. Increased thrombosis after arterial injury in human C-reactive protein-transgenic mice. Circulation. 2003; 108: 512–515.
Kovacs A, Tornvall P, Nilsson R, Tegner J, Hamsten A, Bjorkegren J. Human C-reactive protein slows atherosclerosis development in a mouse model with human-like hypercholesterolemia. Proc Natl Acad Sci U S A. 2007; 104: 13768–13773.
Paul A, Ko KW, Li L, Yechoor V, McCrory MA, Szalai AJ, Chan L. C-reactive protein accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2004; 109: 647–655.
Ji SR, Wu Y, Zhu L, Potempa LA, Sheng FL, Lu W, Zhao J. Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRP (m). Faseb J. 2007; 21: 284–294.
Krupinski J, Turu MM, Martinez-Gonzalez J, Carvajal A, Juan-Babot JO, Iborra E, Slevin M, Rubio F, Badimon L. Endogenous expression of C-reactive protein is increased in active (ulcerated noncomplicated) human carotid artery plaques. Stroke. 2006; 37: 1200–1204.
Khreiss T, Jozsef L, Potempa LA, Filep JG. Opposing effects of C-reactive protein isoforms on shear-induced neutrophil-platelet adhesion and neutrophil aggregation in whole blood. Circulation. 2004; 110: 2713–2720.
Khreiss T, Jozsef L, Potempa LA, Filep JG. Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells. Circulation. 2004; 109: 2016–2022.
Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO, III, Criqui M, Fadl YY, Fortmann SP, Hong Y, Myers GL, Rifai N, Smith SC Jr, Taubert K, Tracy RP, Vinicor F. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: A statement for healthcare professionals from the Centers for Disease Control and Prevention and the Am Heart Association. Circulation. 2003; 107: 499–511.
Taylor KE, Giddings JC, van den Berg CW. C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide. Arterioscler Thromb Vasc Biol. 2005; 25: 1225–1230.
van den Berg CW, Taylor KE, Lang D. C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations. Arterioscler Thromb Vasc Biol. 2004; 24: e168–e171.
Merten M, Thiagarajan P. P-selectin expression on platelets determines size and stability of platelet aggregates. Circulation. 2000; 102: 1931–1936.
Herczenik E, Bouma B, Korporaal SJ, Strangi R, Zeng Q, Gros P, Van Eck M, Van Berkel TJ, Gebbink MF, Akkerman JW. Activation of human platelets by misfolded proteins. Arterioscler Thromb Vasc Biol. 2007; 27: 1657–1665.
Khreiss T, Jozsef L, Hossain S, Chan JS, Potempa LA, Filep JG. Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils. J Biol Chem. 2002; 277: 40775–40781.
Schwedler SB, Amann K, Wernicke K, Krebs A, Nauck M, Wanner C, Potempa LA, Galle J. Native C-reactive protein increases whereas modified C-reactive protein reduces atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2005; 112: 1016–1023.
Schwedler SB, Guderian F, Dammrich J, Potempa LA, Wanner C. Tubular staining of modified C-reactive protein in diabetic chronic kidney disease. Nephrol Dial Transplant. 2003; 18: 2300–2307.
Jabs WJ, Theissing E, Nitschke M, Bechtel JF, Duchrow M, Mohamed S, Jahrbeck B, Sievers HH, Steinhoff J, Bartels C. Local generation of C-reactive protein in diseased coronary artery venous bypass grafts and normal vascular tissue. Circulation. 2003; 108: 1428–1431.
Calabro P, Willerson JT, Yeh ET. Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells. Circulation. 2003; 108: 1930–1932.