Microparticle Surface Proteins Are Associated With Experimental Venous Thrombosis: A Preliminary Study

Abstract

SUMMARYMicroparticles (MPs) are small membrane vesicles released from activated cells and are associated with thrombosis and inflammation. Microparticles contain a unique subset of surface proteins derived from the parent cell and may be responsible for their diverse biological functions. To identify these proteins juvenile baboons (Papio anubis, n=4) underwent iliac vein thrombosis with six-hour balloon occlusion. Plasma samples were taken at baseline and at 2 days post thrombosis for MP analysis. Microparticles were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all primate sequences. For protein identity, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein level of day-2 to baseline. The proteomic analysis was performed twice for each blood sample, totaling 8 experiments. Proteins were considered elevated or depressed if the iTRAQ ratio deviated by 20% change from normal and a p-value less than 0.05. Significantly, 7 proteins were differentially expressed on day-2 compared to baseline, and appeared in at least two animals and regulated in at least 4 experiments, and appeared in at least three animals and regulated in at least four experiments. Among these 7 proteins, up-regulated proteins include various forms of fibrinogen and alpha-1-antichymotrypsin, and down-regulated proteins include immunoglobulins. These proteins influence thrombosis and inflammation through hemostatic plug formation (fibrinogen), inhibiting neutrophil adhesion (alpha-1-antichymoptrypsin), and immunoregulation (immunoglobulins). Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis.

The induction of thrombus generation on nanostructured neutral polymer brush surfaces

“…The role that this collection of 'minor' plasma proteins might play with respect to biocompatibility needs to be investigated to obtain a clearer picture of their functions [13,14,24]. Also, the presence of cell surface related and intercellular proteins indicates that the blood cell derived microparticles may also play a role [41,42]. The interaction of such particles with surfaces may be another contributing factor to thrombogenicity.…”

“…Apart from this, intracellular proteins such as isoforms of myosin-10, myosin-14, Arfaptin-1 and the alpha isoform of afilamin A are also found. Since we used thoroughly centrifuged platelet poor plasma for protein adsorption studies, the origins of the cytoskeletal proteins may be from microparticles derived from red blood cells, platelets and or white blood cells [41,42] which were not removed by the centrifugation and thus were available to interact with the surface (see Supplementary information). We have seen the presence of microparticles like substances in our plasma preparation.…”

A review of studies of the proteomes of circulating microparticles: key roles for galectin-3-binding protein-expressing microparticles in vascular diseases and systemic lupus erythematosus

“…This links both G3BP and gal-3 to inflammatory changes in VTE. In experimental VTE using four baboons, the same research group showed that fibrinogen and α 1 -anti-chymotrypsin were enriched and some immunoglobulins were down-regulated two days after a 6 h occlusion of the iliac veins [59]. G3BP was not enriched.…”

Platelet Receptors

“…These microvesicules are captured onto the thrombus through the interaction between P-selectin expressed on the surface of activated platelets and PSGL-1 present on microvesicules, hence delivering TF to the growing thrombus. Mice deficient in either PSGL-1 or P-selectin display thrombi with little TF and reduced thrombin generation, resulting in hampered thrombus size (Abdullah et al 2009;Morel et al 2008;Ramacciotti et al 2009). …”