**3.7. Increases in concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate**

As the LMW activator activity in the products released from platelets increased markedly if stored platelets were used, concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate prepared from platelet-rich plasma stored for 120 hours at 20 °C were compared with those prepared from fresh equivalent plasma to determine whether lipoproteins are internalized by platelets during storage of platelet-rich plasma. All of Apo CIII, Apo AI and Apo B100 in the lysate from 120 hour-stored platelets were high (20 ng/ml, 18 ng/ml and 252 ng/ml in average, respectively), whereas those from platelet-rich plasma before storage were very low (1.4 ng/ml, 0.6 ng/ml and 130 ng/ml on average, respectively) (Figure 8).

**Figure 8.** Concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate. Concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate, which were prepared from platelet-rich plasma before (0 hours) and after storage for 120 hours (120 hours), were determined using commercially available ELISA kits. The bars in the figure show the average. \*\*, *P*<0.05 by Wilcoxon test.

### **4. Discussion**

Since we found MAPPs in platelet release products, we have investigated their structure and the mechanisms of production and release by platelets. When fresh platelets are stimulated by thrombin, collagen or centrifugation in the presence of Ca++, MAPPs are released. When platelets are stored longer than 72 hours in the form of platelet-rich plasma, they lose the capacity to release MAPPs. It is speculated that this is because platelets lose their contents of thrombin and precursors of MAPPs during storage in the form of plateletrich plasma. They recover the MAPP-releasing function by washing with PBS and incubation with plasma-derived precursors of MAPPs and thrombin in the presence of Ca++ [26]. This suggests that some platelet factor other than thrombin and the precursors of MAPPs is necessary for MAPP production. To analyze this factor, the action of the platelet lysate obtained by freeze-thaw of stored platelets on MAPP formation was investigated. By incubation of the platelet lysate with thrombin and the plasma-derived precursors, it was possible to produce MAPPs. When producing MAPPs using plasma-derived precursors and the platelet lysate, we found that trypsin instead of thrombin can produce MAPPs [27].

734 Lipoproteins – Role in Health and Diseases

**platelets** 

**lysate** 

**4. Discussion** 

**3.6. Immunohistochemical evidence of existence of HATKTAK in activated** 

both anti-HATKTAK and anti-CD 61 antibodies simultaneously (Figure 7).

To show the existence of HATKTAK in activated platelets, an immunohistochemical study of the blood coagula was performed. Platelets in the coagula showed positive reactions with

**3.7. Increases in concentrations of Apo CIII, Apo AI and Apo B100 in the platelet** 

As the LMW activator activity in the products released from platelets increased markedly if stored platelets were used, concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate prepared from platelet-rich plasma stored for 120 hours at 20 °C were compared with those prepared from fresh equivalent plasma to determine whether lipoproteins are internalized by platelets during storage of platelet-rich plasma. All of Apo CIII, Apo AI and Apo B100 in the lysate from 120 hour-stored platelets were high (20 ng/ml, 18 ng/ml and 252 ng/ml in average, respectively), whereas those from platelet-rich plasma before storage were

very low (1.4 ng/ml, 0.6 ng/ml and 130 ng/ml on average, respectively) (Figure 8).

**Figure 8.** Concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate. Concentrations of Apo CIII, Apo AI and Apo B100 in the platelet lysate, which were prepared from platelet-rich plasma before (0 hours) and after storage for 120 hours (120 hours), were determined using commercially available ELISA kits. The bars in the figure show the average. \*\*, *P*<0.05 by Wilcoxon test.

Since we found MAPPs in platelet release products, we have investigated their structure and the mechanisms of production and release by platelets. When fresh platelets are After we found that the plasma precursors of MAPPs are dimer and tetramer transferrins [28], we produced precursors of MAPPs by glutaraldehyde treatment of commercially available holo-transferrin and used them to produce MAPPs in vitro.

In the former study [30], we found that Apo CIII associated with the high-density lipoprotein in platelet lysate can be the source of LMW activator. In this study, we compared the LMW activators produced from Apo CIII by the actions of thrombin and trypsin. It was revealed that both LMW activators appeared in the fractions of the same elution volume of Superdex peptide gel filtration and in the fractions with the same NaCl concentration of MONO S cation exchange chromatography. These findings suggest that the LMW activators produced by thrombin and trypsin are the same substance. The fact that trypsin can form LMW activator suggests that LMW activator is a peptide with a C-terminal amino acid of lysine or arginine and that the N-terminal is an amino acid next to lysine or arginine in the amino acid sequence of Apo CIII. It can be asserted that the action of thrombin in LMW activator formation occurs by its trypsin-like activity, although the activity of thrombin to release LMW activator from Apo CIII is far lower than that of trypsin. Cation exchange chromatography in this study revealed that the LMW activator is cationic, suggesting that it is a peptide rich in basic amino acids. In Apo CIII, K17 to K24 is a region rich in basic amino acids, containing four basic amino acids among eight amino acids in total. Several candidate peptides for LMW activator that contain all or part of the K17 to K24 peptide were raised. By comparing the elution volumes of peaks of these peptides with that of LMW activator function on Superdex peptide gel filtrations and by examining MAPP formation using these peptides, it was strongly suggested that HATKTAK is the LMW activator.

As for the reaction of thrombin, the most abundant natural substrate of thrombin is fibrinogen [36], whereas O'Mullan et al. [37] reported that the action of thrombin to various proteins including Apo CIII is more variable and various peptides cleaved from Apo CIII appear by the action of thrombin on Apo CIII. Our study revealed that the trypsin-like thrombin activity that digests Apo CIII to release HATKTAK is weak, but does in fact exist. We have shown that GP-1b-bound thrombin functions in MAPP production [29]. GP-Ib is

a high-affinity thrombin receptor on platelets [38-39]. Binding of thrombin with GP-1b might enhance the trypsin-like activity of thrombin in platelets.

To confirm that LMW activator, HATKTAK, is produced by the platelets, we examined the LMW activator activity in the low-molecular-weight fractions of variously prepared platelet release products. We found that, in the platelet release products prepared using 4x105/l fresh platelets (0.1 unit/ml thrombin in the presence of 4 mEq/l Ca++), the LMW activator involved is as much as 1 ng/ml HATKTAK.

By storage of the platelet-rich plasma, release of LMW activator from platelets induced by thrombin in the presence of Ca++ increased prominently. It was suggested that the concentration of LMW activator released from platelets stored for 120 hours is 10,000 times as much as that from fresh platelets.

One reason for the tremendous increase in LMW activator, HATKTAK, in the released products during storage is probably the loss of the precursor of MAPPs during storage, as shown in a previous report [26]. It is speculated that, in activated fresh platelets, LMW activator (HATKTAK) is produced as much as in stored platelets, but it decreases markedly because precursors of MAPPs remove it in fresh platelets. Another possible reason is that lipoproteins, apolipoproteins or fragments of apolipoproteins might be transported at high levels into platelets from the plasma during storage. In fact, it was shown that the concentrations of Apo CIII and Apo A1 in the platelet lysate increased markedly after storage of platelets in the form of platelet-rich plasma, but this was still too small to explain the observed increase in the effective dilution by as much as 1,000 times.

Indirect ELISA of the platelet release products using anti-HATKTAK antibody was undertaken to prove the existence of HATKTAK. The results were satisfactory if synthesized pure peptides were used, and it was shown that the anti-HATKTAK rabbit antibody reacted positively to Apo CIII-derived peptides with C-terminal HATKTAK. However, we have not succeeded in establishment of a method to analyze HATKTAK in platelet release products. It is postulated that some substances derived from the platelet release products interfere with the adherence of HATKTAK on the wall of microtiter plate. Therefore, we examined the effect of the antibody on the LMW activator function. It was confirmed that the anti-HATKTAK antibody cancels the activity of the LMW activator in the platelet release products from fresh platelet-rich plasma and that stored for 120 hours. Mass spectrometry study revealed the presence of a substance corresponding to HATKTAK (m/z 756) in the platelet release products from platelets stored for 120 hours. Immunohistochemistry of the blood coagula revealed the existence of platelets with double-positive reaction to anti-HATKTAK and anti-CD61 antibodies. These findings strongly suggest that the LMW activator is HATKTAK and is produced and released by platelets.

A schematic illustration of the probable mechanism of production and release of MAPPs and HATKTAK by platelets is depicted in Figure 9.

At present, the mechanism of how MAPP contributes to neutrophilic phagocytosis enhancement after binding to neutrophils is not known. Because MAPPs possess transferrin molecules and anti-transferrin receptor antibody inhibits the action of MAPPs [28], it is suggested that the action of MAPPs occurs via the transferrin receptor. As shown previously, treatment of neutrophils with MAPP does not result in an increase in the number of Fc receptors on neutrophils [40]. This suggests that the effect of MAPP is to strengthen the affinity of Fc receptors with the Fc portion of IgG and to internalize more foreign materials inside the cell. The necessity of HATKTAK for MAPP function suggests that the transferrin receptor might have a site for binding to both transferrin and HATKTAK, and the fact that MAPPs consist of multimers (dimer and tetramer) of transferrin suggests that transferrin receptors must be fixed at an appropriate distance when MAPPs induce enhancement of phagocytosis via the Fc receptors. The mechanisms of how the transferrin receptor, which is stimulated by HATKTAK and transferrin, transfers the information to Fc receptors remain to be elucidated.

**Figure 9.** A schematic illustration of the mechanism of production of MAPPs and HATKTAK in platelets and their subsequent release.

In conclusion, we could show that HATKTAK is the LMW activator, which is produced in activated platelets and activates MAPPs, and that the residual HATKTAK is released with other platelet-related substances including MAPPs.
