**3.4. Inhibition of LMW activator activity by anti-HATKTAK antibody**

Then, to obtain evidence of the existence of HATKTAK in the platelet release products, actions of an anti-HATKTAK rabbit antibody against LMW activator activity were examined.

**Figure 3.** Comparisons of effective dilution of LMW activator in platelet release products and platelet lysate prepared using platelets that were obtained from platelet-rich plasma stored for 0 hours, 72 hours and 120 hours. A black circle means a case with the effective dilution of LMW activator indicated by the horizontal axis. \*, *P*<0.01 with the Mann-Whitney test, and #, *P*<0.01 with the Kruskal-Wallis test. All experiments were performed using the precursor of s-MAPP.

examined.

(compare Figure 4A, 4B and 4C with Figure 2B).

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

Then, to obtain evidence of the existence of HATKTAK in the platelet release products, actions of an anti-HATKTAK rabbit antibody against LMW activator activity were

**Figure 3.** Comparisons of effective dilution of LMW activator in platelet release products and platelet lysate prepared using platelets that were obtained from platelet-rich plasma stored for 0 hours, 72 hours and 120 hours. A black circle means a case with the effective dilution of LMW activator indicated by the horizontal axis. \*, *P*<0.01 with the Mann-Whitney test, and #, *P*<0.01 with the Kruskal-Wallis test. All

experiments were performed using the precursor of s-MAPP.

**3.4. Inhibition of LMW activator activity by anti-HATKTAK antibody** 

**Figure 4.** Comparisons of molecular size of LMW activator in platelet release products prepared using (**A**) platelet release products using fresh platelets stimulated with 0.1 unit/ml thrombin in the presence of Ca++; (**B**) platelet release products using platelets stored for 72 hours stimulated with 0.1 unit/ml thrombin in the presence of Ca++; (**C**) platelet release products using platelets stored for 120 hours stimulated with 0.1 unit/ml thrombin in the presence of Ca++. The LMW activator activity of each fraction was examined at dilutions of x102 (**A**), x103 (**B**) and x106 (**C**). All experiments were performed using the precursor of s-MAPP.

ELISA revealed that anti-HATKTAK reacted strongly to H18-K24 (HATKTAK), K17-K24 and S1-K24 of Apo CIII, but very weakly to Apo CIII and H18-D25 of Apo CIII, whereas anti-Apo CIII goat antibody showed a positive reaction only to Apo CIII (Figure 5A and 5B, respectively).

**Figure 5.** Effects of anti-HATKTAK antibody on LMW activator activity. **A,** ELISA using anti-HATKTAK rabbit IgG and control rabbit IgG against Apo CIII and H18-K24 of Apo CIII. **B,** ELISA using anti-HATKTAK rabbit IgG and anti-Apo CIII gout IgG against peptide associated with Apo CIII, HATKTAAK and Apo CIII. **C,** Effects of control rabbit IgG and anti-HATKTAK rabbit IgG on LMW activator activity in the low-molecular-weight fraction of platelet release products prepared from platelets of fresh platelet-rich plasma. **D,** Effects of control rabbit IgG and anti-HATKTAK rabbit IgG on LMW activator activity in the low-molecular-weight fraction of platelet release products prepared using platelets of platelet-rich plasma stored for 120 hours. In **C** and **D**, figures reveal the result of experiments using the precursor of s-MAPP. The bars in the figure show the average. \*, *P*<0.01; \*\**P*<0.05 by paired *t* test.

Both LMW activators derived from fresh platelet and platelets stored for 120 hours were inhibited by the anti-HATKTAK IgG antibody (Figure 5C and 5D, respectively).

#### **3.5. Evidence of existence of HATKTAK by mass spectrometry**

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ELISA revealed that anti-HATKTAK reacted strongly to H18-K24 (HATKTAK), K17-K24 and S1-K24 of Apo CIII, but very weakly to Apo CIII and H18-D25 of Apo CIII, whereas anti-Apo CIII goat antibody showed a positive reaction only to Apo CIII (Figure 5A and 5B, respectively).

**Figure 5.** Effects of anti-HATKTAK antibody on LMW activator activity. **A,** ELISA using anti-HATKTAK rabbit IgG and control rabbit IgG against Apo CIII and H18-K24 of Apo CIII. **B,** ELISA using anti-HATKTAK rabbit IgG and anti-Apo CIII gout IgG against peptide associated with Apo CIII, HATKTAAK and Apo CIII. **C,** Effects of control rabbit IgG and anti-HATKTAK rabbit IgG on LMW activator activity in the low-molecular-weight fraction of platelet release products prepared from platelets of fresh platelet-rich plasma. **D,** Effects of control rabbit IgG and anti-HATKTAK rabbit IgG on LMW activator activity in the low-molecular-weight fraction of platelet release products prepared using

platelets of platelet-rich plasma stored for 120 hours. In **C** and **D**, figures reveal the result of experiments using the precursor of s-MAPP. The bars in the figure show the average.

inhibited by the anti-HATKTAK IgG antibody (Figure 5C and 5D, respectively).

Both LMW activators derived from fresh platelet and platelets stored for 120 hours were

\*, *P*<0.01; \*\**P*<0.05 by paired *t* test.

In mass spectrometry, HATKTAK showed m/z 756 (Figure 6A). Although it was impossible to show the existence of a substance with m/z 756 in thrombin-digested Apo CIII (10 g/ml) and in platelet release products released from fresh platelets and those stored for 72 hours (data not shown), a substance with m/z 756 was detected in the trypsin-treated Apo CIII (10 g/ml) (Figure 6B) and platelet release products prepared from platelets stored for 120 hours (Figure 6C).

**Figure 6.** Mass spectrometry of HATKTAK, trypsin-digested Apo CIII and platelet release products from platelets of platelet-rich plasma stored for 120 hours. **A**, 1 mg/ml HATKTAK in PBS; **B**, 10 g/ml Apo CIII digested with 1 unit/ml trypsin; and **C**, platelet release products using platelets of platelet-rich plasma stored for 120 hours. Platelets were stimulated with 0.1 unit/ml thrombin in the presence of Ca++.

**Figure 7.** Confocal microscopic images of double staining for the anti-HATKTAK antibody (visualized as red in A), and the anti-CD61 antibody (visualized as green in B). The colocalization of two antibodies is indicated by the conversion of green and red to yellow (C). Scale bar indicate 20 m.

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

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