Hanna Kälvegren

Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation

Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam3CSK4 stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam3CSK4-induced platelet aggregation and secretion. Platelet interaction with Pam3CSK4 and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+]i mobilisation and thromboxane B2 (TxB2) production. The results show that Pam3CSK4 but not MALP-2 induces [Ca2+]i increase, TxB2 production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam3CSK4-induced responses. The ATP-secretion and aggregation in Pam3CSK4-stimulated platelets was impeded by the purinergic P2X1 inhibitor MRS 2159, the purinergic P2Y1 and P2Y12 antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB2 production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam3CSK4 did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam3CSK4-induced platelet aggregation and secretion depends on a P2X1-mediated Ca2+ mobilisation, production of TxA2 and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.

Chlamydia pneumoniae Binds to Platelets and Triggers P-Selectin Expression and Aggregation: A Causal Role in Cardiovascular Disease?

Objective—Evidence linking Chlamydia pneumoniae to atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction between C pneumoniae and platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis. Methods and Results—The effects of C pneumoniae on platelet aggregation and secretion were assessed with lumiaggregometry, and the ability of C pneumoniae to bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found that C pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reduced C pneumoniae-induced platelet aggregation. Exposure of C pneumoniae to polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions. Conclusion—Our findings demonstrate a new concept of how C pneumoniae activates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.

Increased Levels of Leukocyte-Derived MMP-9 in Patients with Stable Angina Pectoris

Objective There is a growing interest for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in plasma as novel biomarkers in coronary artery disease (CAD). We aimed to identify the sources of MMP-8, MMP-9, TIMP-1 and TIMP-2 among peripheral blood cells and further explore whether gene expression or protein release was altered in patients with stable angina pectoris (SA). Methods In total, plasma MMP-9 was measured in 44 SA patients and 47 healthy controls. From 10 patients and 10 controls, peripheral blood mononuclear cells (PBMC) and neutrophils were isolated and stimulated ex vivo. MMPs, TIMPs and myeloperoxidase were measured in plasma and supernatants by ELISA. The corresponding gene expression was measured by real-time PCR. Results Neutrophils were the dominant source of MMP-8 and MMP-9. Upon moderate stimulation with IL-8, the neutrophil release of MMP-9 was higher in the SA patients compared with controls (p<0.05). In PBMC, the TIMP-1 and MMP-9 mRNA expression was higher in SA patients compared with controls, p<0.01 and 0.05, respectively. There were no differences in plasma levels between patients and controls except for TIMP-2, which was lower in patients, p<0.01. Conclusion Measurements of MMPs and TIMPs in plasma may be of limited use. Despite similar plasma levels in SA patients and controls, the leukocyte-derived MMP-9 and TIMP-1 are significantly altered in patients. The findings indicate that the leukocytes are more prone to release and produce MMP-9 in symptomatic and angiographically verified CAD—a phenomenon that may have clinical implications in the course of disease.

Release of matrix metalloproteinases-1 and -2, but not -9, from activated platelets measured by enzyme-linked immunosorbent assay

Matrix metalloproteinases (MMPs), in particular MMP-9, have been introduced as novel biomarkers in coronary artery disease. Activated platelets are considered to be a major source of the highly elevated levels of MMPs that are detected in serum compared to plasma. The aim of this study was to clarify if activated platelets release MMPs-1, -2 and -9 as measured by enzyme-linked immunosorbent assays (ELISA). Isolated platelets (separated by several procedures) or platelet-rich plasma (PRP) were stimulated by collagen, thrombin or the TLR2 agonist Pam3CSK4. The concentrations of MMPs-1,-2 and -9 in supernatants were determined by ELISA. In addition, a MMP-9 enzyme activity assay was used as well as immunofluorescent staining of MMPs-1,-2 and 9 in platelets. Isolated platelets stimulated by collagen, thrombin or Pam3CSK4 released significant amounts of MMP-1 to the supernatant measured as either pro- or total-MMP-1. However, there was no detectable release of MMP-2 or -9 from isolated platelets. Collagen-stimulated platelets in PRP released MMP-2, but not -9. Before stimulation; platelets were positive for MMPs-1 and -2, but not -9, as assessed by immunofluorescence. Acting as positive controls, neutrophils were found to release significant amounts of MMP-9. Our findings indicate that activated platelets may be a major source of MMP-1 and to a minor extent MMP-2, in peripheral blood. However, in contrast to what has been argued in previous literature, platelets appear to be only negligible contributors to circulating MMP-9.

The role of plasma adenosine deaminase in chemoattractant-stimulated oxygen radical production in neutrophils

OBJECTIVES Adenosine deaminase (ADA) has a role in many immunity mediated disorders, such as asthma, tuberculosis and coronary artery disease. This study aims to investigate the ability of plasma ADA to modulate reactive oxygen species (ROS) production in neutrophils, and examine the involvement of adenosine and the cyclic AMP signaling pathway in this process. METHODS Neutrophils were stimulated, in the absence or presence of plasma, with the chemotactic peptide fMLP (formyl-methionyl-leucyl-phenylalanine), and the ROS production was determined with luminol-enhanced chemiluminescence. Activity of ADA was measured spectrophotometrically. RESULTS Plasma dose-dependently amplified the ROS generation in fMLP-stimulated neutrophils. In parallel, incubation of neutrophils in plasma elevated the total ADA-activity approximately 10 times from 1.3 U/ml to 12 U/ml. Inhibition of ADA, or type IV phosphodiesterases, significantly lowered the plasma-mediated ROS production. Furthermore, the high-affinity adenosine A(1) receptor antagonists DPCPX and 8-phenyltheophylline markedly inhibited the plasma-induced respiratory burst in neutrophils, suggesting an A(1) receptor-mediated mechanism. CONCLUSIONS This study suggests that plasma ADA amplifies the release of toxic oxygen radicals from neutrophils through a downregulation of the inhibitory adenosine/cAMP-system and an enhanced activation of the stimulatory adenosine A(1)-receptor. This mechanism has probably a crucial role in regulating neutrophil function and in the defence against microbial infections. However, a sustained neutrophil activation could also contribute to inflammatory disorders such as atherosclerosis.

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll-like receptors (TLR) and protease-activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet-bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg-gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1-agonist Pam3 CSK4 , which evoked a time-dependent release of the chemokine. Interestingly, the TLR2/1-evoked response was abolished by a following addition of viable P. gingivalis wild-types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam3 CSK4 -stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor-1, and that also these responses were antagonized by P. gingivalis. These results supports immune-modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.

Hepatocyte growth factor in patients with coronary artery disease and its relation to periodontal condition

Hepatocyte growth factor (HGF) is an angiogenic, cardioprotective factor important for tissue and vascular repair. High levels of HGF are associated with chronic inflammatory diseases, such as coronary artery disease (CAD) and periodontitis, and are suggested as a marker of the ongoing atherosclerotic event in patients with CAD. Periodontal disease is more prevalent among patients with CAD than among healthy people. Recent studies indicate a reduced biological activity of HGF in different chronic inflammatory conditions. Biologically active HGF has high affinity to heparan sulfate proteoglycan (HSPG) on cell-membrane and extracellular matrix. The aim of the study was to investigate the serum concentration and the biological activity of HGF with ELISA and surface plasmon resonance (SPR), respectively, before and at various time points after percutaneous coronary intervention (PCI) in patients with CAD, and to examine the relationship with periodontal condition. The periodontal status of the CAD patients was examined, and the presence of P. gingivalis in periodontal pockets was analyzed with PCR. The HGF concentration was significantly higher, at all time-points, in patients with CAD compared to the age-matched controls (P< 0.001), but was independent of periodontal status. The HGF concentration and the affinity to HSPG adversely fluctuated over time, and the biological activity increased one month after intervention in patients without periodontitis. We conclude that elevated concentration of HGF but with reduced biological activity might indicate a chronic inflammatory profile in patients with CAD and periodontitis.

Toll like receptor 2/1 mediated platelet adhesion and activation on bacterial mimetic surfaces is dependent on src/Syk-signaling and purinergic receptor P2X1 and P2Y12 activation

Platelets are considered to have important functions in inflammatory processes as key players in innate immunity. Toll like receptors (TLRs), expressed on platelets, recognize pathogen associated molecular patterns and trigger immune responses. Pathogens are able to adhere to human tissues and form biofilms which cause a continuous activation of the immune system. The authors aimed to investigate how immobilized Pam3CSK4 (a synthetic TLR2/1 agonist) and IgG, respectively, resembling a bacterial focus, affects adhesion and activation of platelets including release of two cytokines, regulated on activation normal T-cell expressed and secreted (RANTES) and macrophage migration inhibitory factor (MIF). The authors also aim to clarify the signaling downstream of TLR2/1 and FcγRII (IgG receptor) and the role of adenine nucleotides in this process. Biolayers of Pam3CSK4 and IgG, respectively, were confirmed by null-ellipsometry and contact angle measurements. Platelets were preincubated with signaling inhibitors for scr and Syk and antagonists for P2X1 or P2Y1 [adenosine triphosphate (ATP), adenosine diphosphate (ADP) receptors] prior to addition to the surfaces. The authors show that platelets adhere and spread on both Pam3CSK4- and IgG-coated surfaces and that this process is antagonized by scr and Syc inhibitors as well as P2X1 and P2Y antagonists. This suggests that Pam3CSK4 activated platelets utilize the same pathway as FcγRII. Moreover, the authors show that ATP-ligation of P2X1 is of importance for further platelet activation after TLR2/1-activation, and that P2Y12 is the prominent ADP-receptor involved in adhesion and spreading. RANTES and MIF were secreted over time from platelets adhering to the coated surfaces, but no MIF was released upon stimulation with soluble Pam3CSK4. These results clarify the importance of TLR2/1 and FcγRII in platelet adhesion and activation, and strengthen the role of platelets as an active player in sensing bacterial infections.