**2. Materials and methods**

This study was approved by the local institutional review board of our university hospital. The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

### **2.1. Phagocytosis experiment**

The phagocytosis experiments were performed according to a method previously described [30]. Briefly, neutrophils were separated from heparinized venous blood from healthy volunteers by centrifugation on MonoPoly resolving medium (ICN Biochemicals Japan, Tokyo, Japan)**.** After washing of neutrophils in phosphate-buffered saline (PBS) (Sigma, St. Louis, MO), one thousand neutrophils in 5 l of PBS were attached to the surface of a Terasaki microplate well (Nunc, Roskilde, Denmark) by centrifugation at 160 g for 2 minutes. Then, stimulation was performed with 10 l of the test material in PBS supplemented with 1% bovine serum albumin (Sigma) (PBS-BSA) at 37 °C for 15 minutes. After washing, the neutrophils were incubated with 5,000 sheep red blood cells (SRBCs) in 5 l of RPMI 1640 medium supplemented with 1% BSA and a one-hundredth volume of anti-SRBC rabbit IgG at 37 °C for 25 minutes under 5% CO2. After lysing the unphagocytosed SRBCs using a Trisbuffered ammonium chloride solution [31] and fixation with 1% glutaraldehyde (Sigma) in PBS, the phagocytosed SRBCs were counted under a light microscope.

All phagocytosis experiments were performed in triplicate. Mean numbers of ingested SRBCs per neutrophil in a well were calculated. Then, means of three wells were obtained. The results are expressed as phagocytic indices normalized by the phagocytic activity of the PBS-BSA control taken as 100.

#### **2.2. Production of MAPPs in vitro**

722 Lipoproteins – Role in Health and Diseases

dimeric forms, respectively [28].

activity of thrombin [30].

revised in 1983.

**2. Materials and methods** 

**2.1. Phagocytosis experiment** 

PBS-BSA control taken as 100.

directly.

thrombin or trypsin in the presence of the precursors [27]. It has also been suggested that the precursors of l-MAPP and s-MAPP are polymerized transferrins, probably of tetrameric and

As for the production of MAPPs in platelets, it was suggested that GP Ib-bound thrombin reacts with a high-molecular-weight substance (HMW activator) to release a low-molecularweight substance (LMW activator) [29], which can produce MAPPs from the precursors

In an ultracentrifugation study of the platelet lysate, the HMW activator activity was observed in the HDL fraction. Anti-apolipoprotein A1 antibody abolished the HMW activator function from the HDL fraction of the platelet lysate. These findings suggest that the HMW activator belongs to HDL. In an affinity chromatography study of the protein obtained from the HDL-rich fraction of the platelet lysate using an anti-apolipoprotein CIII (Apo CIII) column, it was suggested that LMW activator is derived from Apo CIII. In fact, it was observed that the commercially available Apo CIII could produce LMW activator by the

This study was approved by the local institutional review board of our university hospital. The procedures followed were in accordance with the Helsinki Declaration of 1975, as

The phagocytosis experiments were performed according to a method previously described [30]. Briefly, neutrophils were separated from heparinized venous blood from healthy volunteers by centrifugation on MonoPoly resolving medium (ICN Biochemicals Japan, Tokyo, Japan)**.** After washing of neutrophils in phosphate-buffered saline (PBS) (Sigma, St. Louis, MO), one thousand neutrophils in 5 l of PBS were attached to the surface of a Terasaki microplate well (Nunc, Roskilde, Denmark) by centrifugation at 160 g for 2 minutes. Then, stimulation was performed with 10 l of the test material in PBS supplemented with 1% bovine serum albumin (Sigma) (PBS-BSA) at 37 °C for 15 minutes. After washing, the neutrophils were incubated with 5,000 sheep red blood cells (SRBCs) in 5 l of RPMI 1640 medium supplemented with 1% BSA and a one-hundredth volume of anti-SRBC rabbit IgG at 37 °C for 25 minutes under 5% CO2. After lysing the unphagocytosed SRBCs using a Trisbuffered ammonium chloride solution [31] and fixation with 1% glutaraldehyde (Sigma) in

All phagocytosis experiments were performed in triplicate. Mean numbers of ingested SRBCs per neutrophil in a well were calculated. Then, means of three wells were obtained. The results are expressed as phagocytic indices normalized by the phagocytic activity of the

The purpose of this study was to determine the structure of the LMW activator.

PBS, the phagocytosed SRBCs were counted under a light microscope.

#### *2.2.1. Preparation of the precursors of MAPPs using holo-transferrin*

Precursors of MAPPs were produced artificially using holo-transferrin (Sigma) according to a method described previously [28, 30]. Briefly, the peak fraction of holo-transferrin in Superdex 200 (GE Healthcare UK Ltd., Buckinghamshire, England) gel filtration was incubated on ice for 15 min with 0.2% glutaraldehyde (Sigma) to induce polymerization [32], and subjected to filtration again. The fractions obtained at the same elution volumes as the conventional l-MAPP (tetramer transferrin-rich fraction) and s-MAPP (dimer transferrinrich fraction) were adjusted to an optical density of 0.04 at 280 nm with PBS and used as precursors of l-MAPP and s-MAPP, respectively.

#### *2.2.2. Preparation of LMW activators*

Apo CIII (Chemicon International Inc., Temecula, CA) was adjusted to a concentration of 10 g/ml in PBS and incubated with 0.1 unit/ml human thrombin (thrombin) (Sigma) or 1 unit/ml bovine trypsin (trypsin) (Sigma) at 37 °C for 30 minutes, and then applied on a PD10 column (GE Healthcare UK Ltd.) to obtain the low-molecular-weight fraction, which was serially diluted x10 and used as the LMW activator.

Another Apo CIII (10 g/ml) was incubated with 0.1 unit/ml thrombin or 1 unit/ml trypsin and gel-filtered through a Superdex peptide column (GE Healthcare UK Ltd.) (elution buffer, PBS; elution speed, 0.5 ml/minute; fraction volume, 1 ml each) to obtain LMW activator fraction.

Samples of the low-molecular-weight fraction of variously prepared platelet-related materials including platelet release products and platelet lysate (see below for details) were obtained using PD-10 columns.

Some peptides associated with Apo CIII were synthesized artificially, and the activity as an LMW activator was examined.

#### *2.2.3. Preparation of MAPPs using LMW activators and precursors of MAPPs*

To form MAPPs, 1 ml of appropriately diluted LMW activator derived from Apo CIII, platelet release products or artificially produced peptides was incubated with 10 l of precursor of l-MAPP or s-MAPP at 37 °C for 30 minutes.

### **2.3. Preparation of platelet release products using platelets from platelet-rich plasma**

To prepare fresh and stored platelets, heparinized venous blood was mixed with 13 vol% citrate-phosphate-dextrose (CPD) solution and centrifuged at 60 g for 20 minutes at 20 °C. The platelet-rich plasma, which was contaminated by less than one erythrocyte per 1,000 platelets, was transferred into another plastic tube and stored at 20 °C in a water bath with agitation once a second for 0, 72 or 120 hours.

These platelets were washed twice in PBS supplemented with 6.7 mM EDTA and twice in PBS, and adjusted to a concentration of 4x105/l. The platelet suspension was stimulated with 0.1 unit/ml thrombin at 37 °C for one minute in the presence or absence of 4 mEq/l Ca++, then cooled immediately on an ice-water bath and centrifuged at 960 g for 15 minutes at 4 °C to obtain the platelet release products in the supernatant.
