**3. Results**

#### **3.1. LMW activator is considered to be HATKTAK**

As it was shown in a previous study that both thrombin and trypsin could produce a substance with LMW activator activity from platelet lysate [27] and that incubation of Apo CIII with thrombin resulted in production of LMW activator, Apo CIII was treated with thrombin or trypsin and the LMW activator activities produced were compared to determine whether LMW activators produced by these enzymes are the same substance. Both thrombin and trypsin could produce LMW activator activity, but 1 unit/ml trypsin could produce LMW activator 100 to 1,000 times more effectively than 0.1 unit/ml thrombin (Figure 1A).

The LMW activator activities prepared using thrombin and trypsin appeared at the same NaCl concentration of the MONO S cation exchange chromatography (0.2 M NaCl, pH 8.0) (Figure 1B and Figure 1C, respectively), and at the same elution volume (16 ml) of the Superdex peptide gel filtration (Figure 1D and Figure 1E, respectively), which corresponds to the approximate molecular size of 800 Da (Figure 1F). These findings suggest that the two LMW activators are the same substance.

The fact that LMW activator can be produced by trypsin digestion of Apo CIII suggests that the C terminal of LMW activator consists of K or R [35]. LMW activator is suggested to be one of the basic peptides that would appear after trypsin digestion of Apo CIII (S1-K24, H18-R40, H18-K24, H18-K21, T22-K24) (see Figure 1G for the sequence of Apo CIII, distribution of basic and anionic amino acids and the sugar binding amino acid). These peptides were artificially produced and filtered on the Superdex peptide column to use as molecular size markers. The optical peaks of OPA fluorescence and LMW activator activity by H18-K24 (HATKTAK) appeared at the same elution volume of Superdex peptide gel filtration corresponding to Apo CIII-derived LMW activator (Figure 1D, 1E, 2A and 2B). In MONO S cation exchange chromatography, the action of HATKTAK as LMW activator was recovered at the fraction with the same NaCl concentration (0.2 M NaCl, pH 8.0) (Figure 2C) as the Apo CIII-derived LMW activator (Figure 1B and 1C).

#### **3.2. Only HATKTAK among examined peptides showed MAPP-forming activity**

Among the peptides used for calibration on Superdex peptide gel filtration (Figure 1F), H18-R40 and H18-K21 were separated in the fractions near to HATKTAK (LMW activator). The activity of these peptides as LMW activators were examined. Only HATKTAK showed LMW activator activity with a peak at 1 nM (Figure 2D). Then, some Apo CIII-derived peptides that contain part or all of H18-K24 were examined for their LMW activator activity. All of them were used at a concentration of 1 nM. Only HATKTAK showed MAPP-forming activity (Table 1).

**Figure 2.** HATKTAK functions as the LMW activator. **A.** Superdex peptide gel filtration of HATKTAK (200 g, 0.2 ml). The peptide concentration of each fraction was measured by the OPA fluorescent

method. The inset shows the standard graph for the assay of HATKTAK. **B.** MAPP formation using fractions of Superdex peptide gel filtration of HATKTAK (200 g, 0.2 ml) at a dilution of x106 by incubation with one of the precursors of MAPPs. **C.** MONO S cation exchange chromatography of 1 g/ml HATKTAK. MAPPs were generated by incubation of each fraction from the chromatography at a dilution of x104 and one of the precursors of MAPPs. **D.** MAPP formation using peptides containing part of the sequence of apolipoprotein CIII. Each peptide was serially diluted x10 and incubated with one of the precursors of MAPPs. B, **C** and **D** were performed using the precursor of s-MAPP.

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**Figure 2.** HATKTAK functions as the LMW activator. **A.** Superdex peptide gel filtration of HATKTAK (200 g, 0.2 ml). The peptide concentration of each fraction was measured by the OPA fluorescent


**Table 1.** MAPP formation from precursors of MAPPs with peptides derived from K16 to D25 of Apo CIII (KHATKTAKD)

#### **3.3. LMW activator activity in platelet release products and platelet lysate**

In the next part of the study, the LMW activator activities in variously prepared samples including platelet release products and platelet lysate were compared to determine whether platelets release LMW activator. Platelet release products were prepared using platelets from fresh and stored platelet-rich plasma. The reason why we examined platelet release products prepared from stored platelet-rich plasma along with those prepared from the fresh equivalent is that platelet releasate from stored platelet-rich plasma was expected to contain a higher concentration of LMW activator because platelets lose the precursors of MAPPs during storage [26]. From all of these samples, the low-molecular-weight fraction was separated using PD10 columns, and the activity corresponding to LMW activator was compared by the largest dilution from the original sample for which a phagocytic index higher than 150 was recorded (effective dilution).

1 ng/ml HATKTAK and platelet release products from fresh platelets stimulated with 4 mEq/l Ca++ and 0.1 unit/ml thrombin showed an effective dilution of 102 to 103, whereas fresh platelets stimulated only with 4 mEq/l Ca++ or 0.1 unit/ml thrombin for 1 minute showed far lower effective dilution, and platelet release products prepared from platelets after storage for 72 hours and 120 hours in the form of platelet-rich plasma showed extremely high effective dilution (103 to 105, platelets from platelet-rich plasma stored for 72 hours; 107 to 108, those stored for 120 hours). None of the platelet lysate produced from platelets in fresh and stored platelet-rich plasma showed LMW activator activity (Figure 3).

To confirm whether these LMW activator activities were by the same substance, Superdex peptide gel filtrations of the low-molecular-weight fractions of platelet release products prepared from fresh platelets (A), and those stored for 72 hours (B) and 120 hours (C), with stimulation with 0.1 unit/ml thrombin in the presence of 4 mEq/l Ca++ were performed. Fractions of the gel filtrations were diluted x102 (A), x104 (B) and x106 (C). All of these samples showed LMW activator activity at the fraction corresponding to HATKTAK (compare Figure 4A, 4B and 4C with Figure 2B).
