Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.
BACKGROUND
Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro.


METHODS
CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay.


RESULTS
CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor.


CONCLUSIONS
TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro.
Laboratory for Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan. Electronic address: tawara.sc@om.asahi-kasei.co.jp.
Shunsuke
S
Tawara
Laboratory for Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan.
Takumi
T
Sakai
Laboratory for Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan.
Osamu
O
Matsuzaki
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D025901:Carboxypeptidase B2 / Q000276:immunology*; D054441:Cell Migration Assays, Leukocyte; D002464:Cell Migration Inhibition / Q000187:drug effects; D015936:Complement C5a / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects; D063285:Fibrin Clot Lysis Time; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000276:immunology; D018180:Thrombomodulin
C472045:ART123; D000893:Anti-Inflammatory Agents; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D018180:Thrombomodulin; D015936:Complement C5a; D025901:Carboxypeptidase B2; D013917:Thrombin
C5a; Carboxypeptidase B2; Clot lysis; Migration; Thrombomodulin