Discovery of an uncovered region in fibrin clots and its clinical significance

Abstract

Despite the pathological importance of fibrin clot formation, little is known about the structure of these clots because X-ray and nuclear magnetic resonance (NMR) analyses are not applicable to insoluble proteins. In contrast to previously reported anti-fibrin monoclonal antibodies (mAbs), our anti-fibrin clot mAb (clone 102-10) recognises an uncovered region that is exposed only when a fibrin clot forms. The epitope of the 102-10 mAb was mapped to a hydrophobic region on the Bβ chain that interacted closely with a counterpart region on the γ chain in a soluble state. New anti-Bβ and anti-γ mAbs specific to peptides lining the discovered region appeared to bind exclusively to fibrin clots. Furthermore, the radiolabelled 102-10 mAb selectively accumulated in mouse spontaneous tumours, and immunohistochemistry using this mAb revealed greater fibrin deposition in World Health Organization (WHO) grade 4 glioma than in lower-grade gliomas. Because erosive tumours are apt to cause micro-haemorrhages, even early asymptomatic tumours detected with a radiolabelled 102-10 mAb may be aggressively malignant.

Figure 1. Characterisation of the 102–10 mAb.

(a) The 102–10 mAb was reactive to fibrin clots only (n = 6). The results are presented as the means ± s.d., ** P < 0.01. (b) Comparing CBB staining with western blot, the 102–10 mAb reacted exclusively with the Bβ chain of denatured whole fibrinogen (Fng). (c) Western blot was conducted for a sample of the digested Bβ chain, and all positive bands detected with the 102–10 mAb are shown. The amino acid sequence of the 10-kDa peptide detected by the 102–10 mAb was analysed in the next step. (d) Only the No. 5 peptide inhibited the binding of 102–10 mAb to fibrin clots (n = 3; P < 0.01, No. 4 vs. No. 5). (e) The epitope of 102–10 (Bβ201–216; red) interacted with the γ chain (blue), and Bβ203–205 (red ball) interacted with the region around γ216–218 (blue ball) in a hydrophobic manner. The whole structure of fibrinogen is shown (lower panel).

Figure 2. Discovery of a unique region in fibrin clots.

Hydrophobic, ionic, and hydrogen bonds were detected between the Bβ chain and γ chain in the unique region. (a) The anti-Bβ mAb and (b) anti-γ mAb were reactive only to fibrin clots (n = 8). The results are presented as the means ± s.d., ** P < 0.01. (c) Image of the structural change that occurred as fibrinogen transformed into a fibrin clot; the unique region was formed only when fibrinogen transformed into a fibrin clot (blue arrow). The epitope of the 102–10 mAb (red) on the Bβ chain (silver) and residues 206–220 (yellow) of the γ chain (light blue) were the components of the unique region. (d) The unique region within the fibrin clot was proposed to be large enough for both mAbs to bind to the region simultaneously. (e) Electrostatic surface representations (−5 kT/e, red, to +5 kT/e, blue) of the Bβ chain and γ chain in the unique region. Yellow characters indicate the epitope of the 102–10 mAb and the counterpart on the γ chain. (f) Hydrophobic interaction between the Bβ chain (yellow dots) and γ chain (black dots) of the unique region. (g) Amino acids (blue characters) on the Bβ chain (yellow) and amino acids (black characters) on the γ chain (blue) form hydrogen bonds in the epitope region. (f, g) Red indicates the epitope of 102–10.

Figure 3. The kinetics of fibrin clot deposition in several non-malignant disease models and PET/CT with ⁸⁹Zr-labelled 102–10 in spontaneous tumour models.

(a) The kinetics of fibrin clot deposition in rat cerebral infarction (upper), mouse incisional wound (middle), and mouse arthritis (lower) were evaluated immunohistochemically. Fibrin clot deposition was observed at the acute phase (left and middle) but not at the late phase (right). Scale bar, 100 μm. (b) The PET probe was composed of the 102–10 mAb, a linker, and ⁸⁹Zr. (c) The ⁸⁹Zr-labelled 102–10 mAb probe showed clearer and more specific accumulation in tumours as compared to the control (cetuximab). The yellow arrows indicated tumours. (d) The 102–10 mAb probe accumulated within fibrin-positive tumour stroma, as represented by the dashed line.

Figure 4. Immunostaining of fibrin clots in various human tissues and the fibrin clot status according to WHO-classified glioma grade.

(a, b) Fibrin deposition was strongly detected in human cancer tissues but not in normal human tissues or during the late or chronic phases of non-malignant diseases. Scale bar, 100 μm. (c) Immunohistochemical analysis of glioma. The left panels show HE staining, and the right panels show fibrin clot staining. The level of fibrin deposition was graded into 3 subgroups: (−) undetectable fibrin clot (upper panel, grade1); (+) faint but clear deposition (middle panel, grade3); or (++) strong heterogeneous or diffuse deposition (lower panel, GBM). (d) The percentage of fibrin deposition is shown. The clinical stages of the patients were classified as grade 1/2, grade 3, or grade 4 (GBM) according to the WHO grade classification. Each group consisted of 20 cases.