Figure 5.

Antigenic peptide domains recognized by anti-TPO peptide Abs and thrombin cleavage sites of rhTPO. The selected antigenic peptide domains of HT1 (D⁸ to Q²⁸), HT2 (S⁴⁷ to D⁶²), HT3 (L¹⁰⁸ to A¹²⁶), HT4 (N¹⁷² to A¹⁹⁰), HT5 (S²⁶² to T²⁸⁴) and HT6 (P³⁰⁶ to G³³²) are denoted by shaded boxes. Each amino acid residue in the sequence is shown in the one-letter amino acid code. N-glycosylation sites are indicated by a closed circle with an N. Thrombin cleavage sites (14) are indicated along a TPO amino acid sequence. In human TPO, there is no site of [P4]-[P3]-[Pro]-[Arg or Lys]-[P1′]-[P2′], where P3 and P4 are hydrophobic amino acids and P1′ and P2′ are non-acidic amino acids. On the other hand, the predicted thrombin sites, which are [P2]-[Arg or Lys]-[P1′], where P2 or P1′ is Gly and the [Arg or Lys]-[P1′] bond is cleaved, were found in human TPO at AR⁷⁸–G⁷⁹, LR¹³⁶–G¹³⁷, GK¹³⁸–V¹³⁹, and TR²³⁷–G²³⁸. In addition, the AR¹⁹¹–T¹⁹² is one of the other predicted thrombin cleavage sites containing [Ala]-[Arg]-[P5], where the [Arg]-[P5] bond is cleaved, as reported to occur in fibrinogen, chymotrypsinogen A, and antibody λ chain (14). These predicted sites were designated as “Tbn” sites, indicated by triangles. Among them, N-terminal amino acid sequence analyses revealed actual thrombin cleavage sites to be at GR¹¹⁷–T¹¹⁸ and AR¹⁹¹–T¹⁹², both indicated by closed triangles. The N-terminal amino acid sequences obtained from the major thrombin-cleaved peptide fragments (FA, FB, FC, and FD) and intact rhTPO were as follows: T¹⁹²TGSGLLKWQQGFRAKIPGL, X(or S¹) PAPPAX(or C)DLRVLSKLLRDSH, T¹¹⁸TAHKDPNAIFLSFQHLLRGK, X(or S¹) PAPPAX(or C)DLRVLSKLLRDSH, and X(or S¹) PAPPAX(or C)DLRVLSKLLRDSH, respectively. Additionally, amino acid composition analysis was conducted to determine each C-terminal end. All results indicated that the major peptide fragments (FA, FB, FC, and FD) consisted of T¹⁹² to G³³², S¹ to R¹⁹¹, T¹¹⁸ to R¹⁹¹, and S¹ to R¹¹⁷, respectively, as indicated by the open boxes. Both thrombin sites found in human TPO (2, 15, 16) (GR¹¹⁷–T¹¹⁸ and AR¹⁹¹–T¹⁹²) are conserved among TPOs, including mice (16, 18) (GR¹¹⁷–T¹¹⁸ and AR¹⁹¹–T¹⁹²), rats (17) (GR¹¹⁷–T¹¹⁸ and AR¹⁹¹–T¹⁹²), and dogs (16) (GR¹¹⁵–T¹¹⁶ and AR¹⁸⁹–T¹⁹⁰).