A novel hirudin derivative characterized with anti-platelet aggregations and thrombin inhibition.
BACKGROUND
Hirudin is an anti-coagulative product of the salivary glands of the medicinal leech Hirudo medicinalis. It is a powerful and specific thrombin inhibitor. Peptides containing the RGD motif competitively inhibit the binding of fibrinogen to GP IIb/IIIa on the platelets, thus inhibiting platelet aggregation.


RESULTS
We have constructed a recombinant RGD-hirudin (r-RGD-hirudin) by fusing the tripeptide RGD sequence to the native hirudin (wt-hirudin). The r-RGD-hirudin was expressed at high levels in Pichia pastoris, and was purified to approximately 97% homogeneity. The specific anti-thrombin activity of purified r-RGD-hirudin is 12,000 ATU/mg, which is equivalent to wt-hirudin, but only r-RGD-hirudin can inhibit platelet aggregation. The biological effects of r-RGD-hirudin on Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Bleeding Time (BT), maximum platelet aggregation (PAGm) induced by ADP were studied in rabbit model and compared with that of wt-hirudin. The rabbits were infused r-RGD-hirudin had prolonged TT, PT, and aPTT which were similar to that of wt-hirudin; but only r-RGD-hirudin was capable of inhibiting PAGm. Histopathological analyses showed that r-RGD-hirudin was two to three times more effective than wt-hirudin in preventing thrombosis.


CONCLUSIONS
r-RGD-hirudin can potentially be used as a novel anti-coagulant for the prevention of thrombosis after carotid artery anastomosis or in other thrombotic events.
Mo
Wei
W
The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, China. weimo1025@shmu.edu.cn
Zhang
Yan-Ling
YL
Chen
Hong-Shan
HS
Wang
Long-Sheng
LS
Song
Hou-Yan
HY
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D001760:Bleeding Time; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D008297:Male; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry; D010974:Platelet Aggregation / Q000187:drug effects*; D015202:Protein Engineering; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*
D000925:Anticoagulants; D006629:Hirudins; C544959:RGD-hirudin; D011994:Recombinant Proteins; D013917:Thrombin