Blood platelets and C-reactive protein (CRP) are both used clinically as markers of ongoing inflammation, and both participate actively in inflammatory responses, although the biological effects are still incompletely understood. Rapidly adhering platelets express receptors for complement factor 1q (C1q) and the Fc part of immunoglobulin G (IgG), and CRP is known to activate/regulate complement via C1q binding, and to ligate FcγRs. In the present study, we used normal human IgG pre-adsorbed to a well-characterized methylated surface as a model solid-phase immune complex when investigating the effects of CRP and C1q on platelet adhesion and activation. Protein adsorption was characterized using ellipsometry and polyclonal antibodies, and human serum albumin (HSA) and non-coated surfaces were used as reference surfaces. Platelet adhesion to IgG and HSA was inhibited by both C1q and CRP. Furthermore, CRP (moderately) and C1q (markedly) decreased the spreading of adhering platelets. The combination of C1q and CRP was slightly more potent in reducing cell adhesion to IgG, and also impaired the adhesion to HSA and non-coated surfaces. Platelet production of thromboxane B2 (TXB2) was also reduced by C1q both in the presence and absence of CRP, whereas CRP alone had no effect on TXB2 production. We conclude that CRP and C1q regulate the behaviour of platelets, and that this may be an important immunoregulatory mechanism during inflammatory conditions. © 2008 Australasian Society for Immunology Inc. All rights reserved.

Blood platelets are emerging as important immunomodulatory cells, but complement interaction with platelets is not well understood. Several platelet structures have been described as complement protein 1q (C1q) binding receptors, such as C1qRp/CD93 and gC1qR. However, there are conflicting results whether these receptors are C1q binding structures, or even at all expressed on the cell surface. Recently, the collagen-binding integrin alphaIIbetaI was reported to bind C1q on mast cells, and this receptor is also present on platelets. The aim of this study was to further characterize the effects of C1q on platelets, by quantifying the platelet surface expression of P-selectin (CD62P) and monitoring the formation of platelet-neutrophil aggregates. Using flow cytometry, we found that C1q dose-dependently triggered a rapid but moderate and transient up-regulation of P-selectin already within 5s of C1q exposure. Pre-incubation with an antibody directed against gC1qR significantly inhibited (with 57% compared to control) the up-regulation, whereas an antibody towards the alphaIIbetaI-integrin showed no effect. Stimulation with C1q did not change the cytosolic calcium-levels, as measured with the fluorescent ratiometric probe Fura-2, however, a protein kinase C inhibitor (GF109203x) blocked the C1q-induced P-selectin expression. Furthermore, pre-incubation of platelets with C1q diminished both the collagen as well as the collagen-related peptide-induced up-regulation of P-selectin, most evident after 90s of stimulation. This indicates that C1q may regulate platelet activation via the GPVI receptor, which is a novel finding. Moreover, C1q antagonized the collagen-induced formation of platelet-neutrophil aggregates, indicating a reduced interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1(PSGL-1/CD162). In summary, C1q induces a moderate rapid platelet P-selectin expression, modulates subsequent collagen and collagen-related peptide stimulation of platelets, and inhibits the formation of platelet-neutrophil aggregates. These immuno-regulatory effects of C1q may have a crucial role in innate immunity and inflammation.

Blood platelets are nowadays recognized as cells with immuno‐modulatory properties as they express receptors involved in immunity (e.g. complement‐, toll‐like‐ and Fcγ‐receptors) and release inflammatory mediators. Furthermore, formation of plateletleukocyte aggregates has an important role during inflammatory conditions, e.g. coronary artery disease. We have previously reported regulatory effects of complement protein 1q (C1q) on platelet activation in experimental setups using isolated cells. In the present study we have continued by investigating the effect of C1q on collagen‐induced aggregation and production of reactive oxygen species (ROS), formation of plateletleukocyte aggregates and levels of soluble P‐selectin in whole blood. Impedance measurements showed that C1q, at physiological concentrations, inhibited collageninduced aggregation in whole blood, whereas it potentiated the collagen‐provoked production of ROS in a luminal‐dependent chemiluminescence assay. The potentiation was dependent on platelets, as the effect was not seen when the platelet fibrinogen binding receptor GpIIb/IIIa was blocked by Reopro. Moreover, the formation of large platelet‐leukocyte aggregates in collagen‐stimulated whole blood was inhibited by C1q. This may be explained by the finding that C1q antagonized the collagen‐induced activation, revealed by lowered levels of soluble P‐selectin. In conclusion, C1q may have an important role in regulating platelet activation and associated leukocyte recruitment during vessel wall injury and thus be involved in inflammatory disorders such as coronary artery disease.

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 Pam(3)CSK(4) stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam(3)CSK(4)-induced platelet aggregation and secretion. Platelet interaction with Pam(3)CSK(4) and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+](i) mobilisation and thromboxane B2 (TxB(2)) production. The results show that Pam(3)CSK(4) but not MALP-2 induces [Ca2+](i) increase, TxB(2) production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam(3)CSK(4)-induced responses. The ATP-secretion and aggregation in Pam(3)CSK(4)-stimulated platelets was impeded by the purinergic P2X(1) inhibitor MRS 2159, the purinergic P2Y(1) and P2Y(12) 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 TxB(2) 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, Pam(3)CSK(4) did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam(3)CSK(4)-induced platelet aggregation and secretion depends on a P2X(1)-mediated Ca2+ mobilisation, production of TxA(2) and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.