The Regulatory Activities of recombinant Properdin and TSP1 on Platelet and Complement activation

The complement system provides a first line defence against microbial pathogens and cooperates with platelet activation of haemostasis/thrombosis system in a physiologically meaningful way by forming a physiological barrier to prevent blood loss following vascular injury. Due to chondroitin sulphate (CS) secretion from activated platelets, some pattern recognition molecules, i.e. MBL, CL-11 and ficolins of the lectin pathway (LP), and C1q of the classical pathway (CP) can drive complement activation on the surface of activated platelets. Properdin is, a positive regulator of the AP, is entirely composed of thrombospondin-like domains (TSR) sharing a high degree of identity with TSR Type-I repeat of Thrombospondin-1 (TSP1). TSP1 is an adhesive homo-trimeric glycoprotein that is re-leased during platelet activation. The structural similarities between properdin and TSP1 are striking. The current result showed a highly significant difference of P-selectin expression level which was observed in washed activated human platelets triggered by 10μg/ml (w/v) of highly oligomerised properdin using ELISA in comparison with other known agonists, including TSP1. Platelet activation resulted in α-thrombin release as shown by ELISA as well as thrombin generation monitored using platelet microparticles (PMPs). My work demonstrated for the first time that highly oligomerised properdin can bind to in-tact platelets while the binding of the physiologically occurring lower grade properdin oligomers; i.e. dimers, trimers and tetramers, to resting platelets was not observed. These lower grade oligomers only bind to activated platelets. That demonstrated the ca-pability of highly oligomerised properdin to promote platelet adhesion. The activation was suggested through binding directly to platelet receptors or indirectly through sup-porting extracellular matrix (ECM) in sub-endothelial layer. The binding was observed with Collagen Type-I, von Willebrand factor (vWF) and Fibronectin (Fn), also, with atherogenic particles such as Low-density lipoprotein (LDL), cholesterol and triglycerides. The crosstalk between activated platelets and the complement system was investigated by screening for a possible regulatory role of TSP1 during complement activation. 10μg/ml (w/v) of TSP1 were found to significantly down-regulate complement activation using C3 and C4 deposition assays under alternative pathway or lectin pathway specific assay conditions. While the down-regulation of lectin pathway functional activity was due to direct TSP1 binding to mannan and Chondroitin sulphate (CS) on the activator surface competing for the binding of MBL and/or CL-11, TSP1 was shown to inhibit the alternative pathway through direct binding to factor B (fB) and/or to C3 with surprisingly high binding affinity. TSP1 binding led to a competitive inhibition of properdin functional activity. These results suggest that TSP1 acts as a negative regulator which significantly shortens the half-life of the C3 and C5 convertase complexes of the AP. Therefore, native TSP1 may act as a negative fluid-phase regulatory component of complement activation to protect activated-platelets from overshooting complement activation. TSP1 is believed to influence the colonisation and dissemination of microbial pathogens such as Streptococcus pneumoniae. My results demonstrate that TSP1 release may in part protect S.pneumoniae (D39) from complement mediated killing, as demonstrated in a serum bactericidal assay (SBA). That may suggest that for colonisation/dissemination, TSP1 may help the pathogen to escape from elimination by the complement system. In conclusion, my results propose a so far unknown physiological role of TSP1 as a negative regulator of the alternative pathway activation route of complement by shorting the half-life of the LP and/or AP C3 and C5 convertases and as such may provide a new example of synergisms and antagonisms between the platelet and the complement system.