**2.8. Assessment of peptide concentrations using the o-phthalaldehyde (OPA) fluorescent method**

Peptide concentrations were assessed according to the OPA fluorescent method with intact protein [34]. Fluorescence was measured with an excitation wavelength of 340 nm and an emission wavelength of 450 nm using a spectrofluorometer (FP-6300, JASCO, Tokyo, Japan).

#### **2.9. Mass spectrometry**

Matrix-associated laser desorption/ionization time-of-flight mass spectrometry was performed with a Voyager System 4314 (Applied Biosystems, Foster City, CA) in the reflector time-offlight configuration at an acceleration voltage of 25 kV with delayed ion extraction. Samples were diluted 1:1 with a freshly prepared matrix solution consisting of 10 mg/ml -cyano-4 hydroxycinnamic acid (Sigma) in 20% acetonitrile with 0.1% trifluoroacetic acid. Aliquots of 1 l were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air. Mass scale calibration was performed externally.

#### **2.10. Immunohistochemistry**

Coagulation of the fresh peripheral blood taken without anticoagulants was induced in a glass tube for 3 hours. This coagulum was formalin-fixed, and paraffin sections of 3 m thickness were prepared. After deparaffinization and hydration, antigen retrieval was performed by heating in 10 mM citrate buffer (pH 6.0) for 40 minutes. After rinsing in PBS and incubation with 2% BSA in PBS at room temperature for 60 minutes, incubation with a mixture of rabbit anti-HATKTAK IgG (x1,000 diluted) and murine anti-CD 61 monoclonal IgG (Dako Japan, Tokyo, Japan) (x100 diluted) at 4 °C overnight was performed followed by incubation with a mixture of Alexa Fluor 594-conjugated anti-rabbit IgG (x200 diluted) and Alexa Fluor 488-conjugated anti-murine antibody (x200 diluted) (Molecular Probes, Eugene, OR, USA). The fluorescent signals were viewed under a confocal microscope (Bio-Rad Radiance 2100). As a control experiment, an identical immunohistochemical procedure with omission of the primary antibodies was performed.
