Peter Gunnarsson

The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs).

We studied whether the acute‐phase protein α1‐acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura‐2‐loaded neutrophils, and this response was markedly enhanced by pretreatment with anti‐L‐selectin antibodies. (The EC50 value of the AGP‐induced Ca2+ response was 9 μg/ml.) Activation of phospholipase‐C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP‐mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin‐like lectin 5 (Siglec‐5) and oligosac‐charide 3′‐sialyl‐lactose both antagonized the AGP‐in‐duced response and caused an immediate increase in [Ca2+]i in anti‐L‐selectin‐treated neutrophils, which indicates a signaling capacity of Siglec‐5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase‐treated AGP, bound to Siglec‐5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre‐engagement of L‐selectin significantly enhanced this signaling capacity.— Gunnarsson, P., Levander, L., Påhlsson, P., Grenegård, M. The acute‐phase protein α1‐acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin‐like lectins (Siglecs). FASEB J. 21, 4059–4069 (2007)

α1-acid glycoprotein (AGP)-induced platelet shape change involves the Rho/Rho kinase signalling pathway

alpha(1)-acid glycoprotein (AGP) is an acute-phase protein that contributes to inflammation processes. The role of AGP in platelet activation and thrombosis is, however, largely unknown. Therefore, we thoroughly investigated the effects of AGP on human platelets. Platelets were isolated from healthy volunteers and subsequently exposed to AGP. Platelet responses were monitored as change in light transmission, intracellular calcium concentration, light microscopy and protein phosphorylation by Western blot. We found that AGP induced platelet shape change independently of a second release of adenine nucleotides or thromboxane A(2), and that effect was abolished by endothelium-derived platelet inhibitors such as nitric oxide (NO) and adenosine. Furthermore, AGP triggered a minor calcium response and a pronounced Rho/Rho-kinase-dependent increase in Thr696 phosphorylation of myosin phosphatase target subunit 1 (MYPT1). Moreover, the Rho/Rho-kinase inhibitor Y-27632 significantly decreased the AGP-induced shape change. The results also showed that the AGP-elicited shape change was antagonised by pretreatment with low doses of collagen and thrombospondin-1. Our results describe a novel mechanism by which AGP stimulates platelet shape change via activation of the Rho/Rho-kinase signalling pathway. Physiological important platelet inhibitors, such as NO, completely counterbalance the effect of AGP. Hence, the present study indicates that AGP directly contributes to platelet activation, which in turn might have an impact in physiological haemostasis and/or pathological thrombosis.