Prothrombin activation in rabbits.
The suitability of rabbit prothrombin activation fragment F 1.2 as a marker for the activation of the coagulation system was tested. Monoclonal antibodies to rabbit F 1.2 were raised, and a competitive F 1.2 ELISA was developed. Within the detection limit of the ELISA, no increase in rabbit F 1.2 was detected upon recalcification of plasma, whereas human F 1.2 increased 1500-fold. The apparent lack of F 1.2 formation in rabbit serum was confirmed by immunoblotting analysis of endogenous and biotin-labeled prothrombin. Meizothrombin and the B-chain of thrombin were the only prothrombin fragments detectable. In contrast, labeled human prothrombin formed, in addition, prethrombin 2 and F 1.2 in both human and rabbit serum. In contrast, rabbit F 1.2 formation could be demonstrated using purified rabbit prothrombin and factor Xa. These observations raise the possibility that rabbit prothrombin is less susceptible than the human counterpart to factor Xa cleavage at the 271/272 peptide bond. Thus, the primary structure of rabbit prothrombin was deduced by cDNA sequencing. While the 320/321 Xa cleavage site giving rise to meizothrombin was identical in rabbit and human prothrombin, the flanking region of the 271/272 Xa sensitive site contained a six amino acid deletion in the rabbit sequence. Taken together, these observations suggest that the observed differences between human and rabbit prothrombin activation may be due to different susceptibilities of the two Xa cleavage sites rather than plasma or serum cofactor(s).
Department of Chemical Enzymology, DuPont Pharmaceuticals Company, Wilmington, Delaware, USA. Seiffert@dupont.pharma.com
D
D
Seiffert
T J
TJ
Mitchell
Z
Z
Wang
R M
RM
Knabb
F
F
Barbera
T M
TM
Reilly
M
M
Thoolen
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000502:physiology*; D011516:Prothrombin / Q000502:physiology*; D011817:Rabbits; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D018076:DNA, Complementary; D010446:Peptide Fragments; C045879:prothrombin fragment 1.2; D011516:Prothrombin