Page 33 - M. Immunology

Inhibitory Activity of Blood Coagulation Induced by Pig Tissue Factor Comparison Pig
Tissue Factor Pathway Inhibitor α Fusion Immunoglobulin with Human Counterpart

Chang-Hee Lee, Taehoon Chun
Department of Biotechnology, College of Life Sciences and Biotechnology,
Korea University, Seoul, 02841, Republic of Korea

BACKGROUND AIM

Organ transplantation is a method to change a recipient’s damaged organ with a We developed pig tissue factor pathway inhibitor fusion immunoglobulin
healthy one (Denner 2014). Pig to human xenotransplantation could be a good (TFPI-Ig) and human TFPI-Ig. These two therapeutic approaches were tested
solution for the shortage of donor’s organs suitable for transplantation (Denner for their ability for inhibiting pig TF activity in human plasma. Results showed
2014). Pig islet xenotransplantation has been considered to treat type I diabetes that Pig TFPI-Ig has higher efficiency for reducing the activity of pig TF.
patients (Shaprio et al. 2000). However, xenotransplantation has some huddles
that are induced by both innate and adaptive immunity in humans. These reactions METHODS
are much faster and more severe than allograft rejection (Zhu et al. 2014). Among
various types of pig islet graft rejection in humans, instant blood mediated Production and purification of soluble pig and human TFPI-Ig
inflammatory reaction (IBMIR) is considered as the most important one to Fusion immunoglobulin proteins were purified from culture supernatants
overcome because IBMIR contributes more than half of pig islet xenograft loss using protein A column.
within a few hours (Nilsson et al. 2011). IBMIR caused by pig to human
xenotransplantation can occur if pig tissue factor (TF) expressed on pig islets is Inhibition test of pig TF/human FVIIa activity
exposed to human plasma protein such as factor VII (FVII) and FX that can initiate Pig TF activity inhibition test was conducted using tissue factor chromogenic
thrombin generation and coagulation cascade (Van Der Windt et al. 2007). The activity kit according to the manufacturer’s instructions.
coagulation reaction occurs further severe inflammation via complement activation
and neutrophil infiltration to human blood (Zhu et al. 2014) Thrombin generation assay
The amount of thrombin generation was measured using a Thrombinoscope
software.

RESULTS

Figure 1 Fig.1. Construct and expression of Figure 2
pig TFPI-Ig and human TFPI-Ig
(a) Schematic diagram of soluble pig
a TFPI-Ig (pTFPI-Ig) or human TFPI-Ig
(hTFPI-Ig) construct. The cDNA
segment encodes full length of TFPIα
(unshaded region), hinge (dotted
region), and constant region of human
b IgG1 heavy chain (shaded region).
Displayed sequences show the junction
between TFPIα and the hinge region.
Additional proline-glycine (PG) residue
is underlined. CH2, CH2 domain of
human IgG1 heavy chain; CH3, CH3
c domain of human IgG1 heavy chain. (b)
Purification of soluble pTFPI-Ig and
hTFPI-Ig. Vectors for each soluble
pTFPI-Ig and hTFPI-Ig were transfected
into CHO cells. The protein purified with
protein A-affinity chromatography.
d Purified proteins (10μg) were analyzed
by SDS-PAGE analysis in the reducing
condition using 2-mercaptoethanol and Fig. 2. Pig TFPI-Ig inhibits pig TF activity more
visualized by Coomassie Blue staining. effectively than human TFPI-Ig.
Molecular weight markers were
indicated as Mw. (c) Western blot Cleavage of FXa substrate by FXa was measured in the
analysis with anti-human IgG1 antibody presence of pig TF, human FVII, and various
after purification of soluble pTFPI-Ig concentrations of TFPI-Igs. Human IgG1 (100nM) was
and hTFPI-Ig proteins. (d) Alignment of treated as control. Results are expressed as percentage
amino acid sequences within mature
pig TFPIα (GenBank accession of control Ig (% control). All results are shown as mean
NM_001135258) and human TFPIα ± SE. Significant differece between pig TFPI-Ig (pTFPI-
(GenBank accession NM_006287). N- Ig) and human TFPI-Ig (hTFPI-Ig) is indicated by
linked glycosylation sites are indicated asterisk (P<0.05). All results are representative of three
in bold. Cleavage sites from mature independent experiments)
TFPIα are boxed.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
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