Fig. 7. Removal of G3-containing fragments from human plasma or blocking with PSGL fusion proteins reduces leukocyte aggregation. (A) Human plasma was subjected to western blot analysis probed with 2B1 to detect endogenous G3-containing fragments on 4% and 10% SDS gels. (B) Human plasma was incubated with protein G-bound 2B1 or serum-treated protein G alone. After centrifugation, treated plasma and the protein G beads were analyzed on a western blot probed with 2B1. Treatment with 2B1 reduced the amount of versican in the plasma (left panel, immunodeprivation). The binding of versican to the protein G bead-2B1 complex was confirmed (right panel). (C) The effects of anti-G3 antibody treated (immunodepleted) and untreated human plasma on human leukocyte aggregation were examined. Freshly isolated human leukocytes did not aggregate when maintained in RPMI medium containing 10% FBS. The addition of human plasma or protein G bead-treated plasma induced leukocyte aggregation. This effect diminished greatly when the plasma was immunodeprived by 2B1 treatment (n=3; ^**P<0.01). (D) Human plasma was added to human leukocytes in the presence or absence of purified GST, GST-PSGL, GST(279-402). Both PSGL fusion proteins blocked human plasma-induced leukocyte aggregation (n=3; ^**P<0.01). (E) Human plasma and plasma mixed with polyclonal antiserum against versican G3 domain were injected into the mouse peritoneal cavity. Two days after the injection, ascites was recovered and monocyte aggregation was examined immediately. Plasma-induced monocyte aggregation was neutralized in the presence of anti-G3 antibody.