Purification and Characterization of Macromolecular Phagocytosis Activators Released From Platelets

Abstract

Substantial amounts of macromolecular activators of phagocytosis from platelets (MAPPs) were released in response to exposure of platelets to the specific agonists thrombin and collagen and to calcium ionophore A23187. Centrifugation of the platelets in culture medium also resulted in a release of MAPPs, but not when the platelets were frozen and thawed prior to centrifugation. In an experiment using outdated platelet concentrates, larger and smaller MAPPs (1-MAPP and s-MAPP, respectively) were purified from the thrombin stimulated supernatant (PRPr-plasma) by sequential steps of ammonium sulfate precipitation, delipidation with ethylacetate, ConA-Sepharose affinity chromatography, MONO Q anion exchange chromatography, and Superose 12 gel filtration. This procedure yielded 59,500-fold and 63,600-fold purified 1-MAPP (0.95 mg) and s-MAPP (0.41 mg), respectively, from 1,320 ml PRPr-plasma containing 84,500 mg protein. By gradient polyacrylamide gel electrophoresis the respective molecular weights (MW) of 1-MAPP and s-MAPP were 290-320 kd and 140-160 kd; isoelectric points were 5.6 and 4.6. Both MAPPs activated neutrophil phagocytosis of IgG-SRBC at concentrations in the range of 1 pM-100 nM. Indirect enzyme-linked immunosorbent assay (ELISA) and comparisons of the concentrations required for phagocytosis activation suggested that the MAPPs were not derived from fibronectin.

Effects of platelet release products on neutrophilic activity in human whole blood

“…In addition, these platelets produce and release prostaglandins [21]. It has been confirmed that MAPPs are glycoproteins (MAPPs: l-MAPP, 300kDa; s-MAPP, 150kDa) [13]. The methods to stimulate platelets included centrifugation, or incubation with thrombin, collagen, and calcium ionophore A23187.…”

“…The methods to stimulate platelets included centrifugation, or incubation with thrombin, collagen, and calcium ionophore A23187. These treatments gave a substantial release of MAPPs [13].…”

“…L-MAPP and s-MAPP rich fractions were determined by a phagocytosis assay using separated neutrophils as shown in our previous reports [13]. Both peak fractions were concentrated 10-fold with a Microcon centrifugal filter device (Millipore, Bedford) employing an YM 30 Diaflow membrane which limited molecules larger than approximately 30,000 Daltons, and were mixed together with 10-fold volume of RPMI-BSA medium before use.…”

On the Phagocyting Properties of Human Platelets

“…This process differs from ropalocytosis, expressed by an active penetration of one cell into another, without a consequent detriment of the invading one. Platelet phagocytosis, even of inert particles such as latex, appears to be an active process accompanied by an increased production of adenoside diphosphate [1] and protein synthesis [4], as well as a release of macromolecular activators capable of further phagocytic promotion [9,10]. Being in contact with bacteria and protozoa, platelet phagocytosis is associated with a bactericidal activity [3], even though there are opponents to the existence of that platelet function [7].…”