TY - JOUR
T1 - Sequence of plasmin proteolysis at the NH2-terminus of the Bβ-chain of human fibrinogen
AU - Koehn, James A.
AU - Hurlet-Jensen, Anne
AU - Nossel, Hymie L.
AU - Canfield, Robert E.
N1 - Funding Information:
* This research has been supported by grants from the National Institutes of Health (HL 15486, HL-2 1006, and HL-0746 1). r To whom all correspondence should be addressed
PY - 1983/9
Y1 - 1983/9
N2 - Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen Bβ-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. Bβ1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of Bβ1-21 and of FPB followed the production of Bβ1-42. Using purified Bβ1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: Bβ1-14 (FPB) and β22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the Bβ-chains of a variety of fibrinogen derivatives and have found similar patterns of Bβ1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the β21-22 bond to be 1.8 × 10-5 m and of the β14-15 bond to be 2.8 × 10-5 m. In another report (A. Hurlet-Jensen, J. A. Koehn, and H. L. Nossel, Thromb. Res. 29, 609-617 (1983)), we estimated the Km for plasmic cleavage of the β42-43 bond in fibrinogen to be 7.0 × 10-7 m. Taken together, these data indicate a sequence of events in which Bβ1-42 was initially cleaved from fibrinogen during plasmin digestion. Once released from its parent molecule, Bβ1-42 can undergo further plasmin attack preferentially at the 21-22 bond to yield Bβ1-21 and β22-42. Plasmin can then attack Bβ1-21 at the 14-15 bond to release FPB. The kinetic data presented here indicate that these secondary cleavages are likely to be very limited in vivo.
AB - Employing high-performance liquid chromatography (HPLC), we have isolated and quantified the peptides that are released from the NH2-terminus of human fibrinogen Bβ-chains by plasmin proteolysis. The peptides were identified by amino acid composition and by a radioimmunoassay developed for fibrinopeptide B detection. Bβ1-42 was the earliest fragment released during limited plasmin proteolysis. The level of this peptide reached a maximum and then began to decline during the course of the digestion. In addition, increasing levels of Bβ1-21 and of FPB followed the production of Bβ1-42. Using purified Bβ1-42 as a substrate, preferential cleavage was shown to occur at the 21-22 bond, with a minor cleavage at the 14-15 bond. Exhaustive digestion yielded two major components which were separated by HPLC: Bβ1-14 (FPB) and β22-42. The rate of cleavage at the 14-15 bond, which is the customary site of thrombin proteolysis, was not affected by the addition of hirudin indicating that this was not the result of trace contamination with thrombin. We have also examined plasmin proteolysis at the NH2-terminal region of the Bβ-chains of a variety of fibrinogen derivatives and have found similar patterns of Bβ1-42 release. Using HPLC data, we have estimated the Km for plasmic cleavage of the β21-22 bond to be 1.8 × 10-5 m and of the β14-15 bond to be 2.8 × 10-5 m. In another report (A. Hurlet-Jensen, J. A. Koehn, and H. L. Nossel, Thromb. Res. 29, 609-617 (1983)), we estimated the Km for plasmic cleavage of the β42-43 bond in fibrinogen to be 7.0 × 10-7 m. Taken together, these data indicate a sequence of events in which Bβ1-42 was initially cleaved from fibrinogen during plasmin digestion. Once released from its parent molecule, Bβ1-42 can undergo further plasmin attack preferentially at the 21-22 bond to yield Bβ1-21 and β22-42. Plasmin can then attack Bβ1-21 at the 14-15 bond to release FPB. The kinetic data presented here indicate that these secondary cleavages are likely to be very limited in vivo.
UR - http://www.scopus.com/inward/record.url?scp=0020619006&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(83)90116-1
DO - 10.1016/0003-2697(83)90116-1
M3 - Article
C2 - 6227262
AN - SCOPUS:0020619006
SN - 0003-2697
VL - 133
SP - 502
EP - 510
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -