Figure 4: pCRP binding to microvesicles induces structural changes.

pCRP was incubated with liposomes (PC or PC/LPC) and microvesicles (MV) from THP-1 and Jurkat cells. The supernatant (SN) of the last wash step after MV purification was used as control. Vesicles were pelleted and subjected to BN–PAGE. (a) pCRP bound to PC/LPC liposomes, THP-1 and Jurkat cell-derived MV. No binding occurred to PC-only liposomes and no CRP could be found in the pellet fraction of the supernatant. mCRP was not found on the surface of MV and liposomes. (b) Ca²⁺-depletion (EDTA) or 1,6-bis-PC (bisPC) reduces binding of pCRP to MV. (c) Quantification of pCRP binding. There was no significant difference in the amount of pCRP that bound to either MV species or PC/LPC liposomes (n=5). EDTA and 1,6-bis-PC significantly reduced binding (n=2). P values were calculated with a Student’s t-test. **P value of <0.01, NS=not significant. Displayed are means and s.e.m. (d) Kinetics of the pCRP–MV interaction were determined by measuring binding of Alexa Fluor 488-labelled pCRP to immobilized THP-1 MV. (e) Binding of pCRP to THP-1 cell-derived MV was analysed by flow cytometry with conformation-specific antibodies for pCRP (anti-pCRP-8D8/FITC). P values were calculated with a paired t-test. *P value of <0.05 to control MV and shaded areas display the s.e.m. (n=4). (f) Binding of pCRP to liposome mimics of THP-1 MV (P 16,000 g and SN 100,000 g) was analysed as described above. Binding was Ca²⁺-dependent and we did not observe dissociation of pCRP into its monomeric subunits. Displayed are means and s.e.m. (n=3). (g) Binding of pCRP to different cell-derived MV was analysed on BN–PAGE as described in (c). Monocytes (Mono), polymorphonuclear leukocytes (PMNL), phorbol 12-myristate 13-acetate (PMA), monophosphoryl lipid A (MPLA), N-formylmethionyl-leucyl-phenylalanine (fMLP). Displayed are means and s.e.m. (n=3). P values were calculated with a Student’s t-test. *P value <0.05 and **P value <0.01. (h) Conformational changes in pCRP on binding to THP-1 cells and MV were assessed with conformation-specific antibodies by flow cytometry. CRP bound to THP-1 cells could only be detected by anti-pCRP-8D8 antibodies, whereas CRP on MV was recognized by anti-pCRP-8D8 and anti-mCRP-9C9 antibodies. Displayed are means and s.d. (n=3). (i) Deposition of CRP on MV of different cellular origin in the circulation of patients with ST-elevation myocardial infarction (STEMI) was analysed by FACS with anti-pCRP-8D8/FITC and anti-mCRP-9C9/FITC antibodies. Displayed are the percentages of FITC-positive MV of each subset. Bars indicate means and s.d. (n=4). P values were calculated with an unpaired t-test.