The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin.
Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.
Siebenlist
K R
KR
Sinai Samaritan Medical Center, University of Wisconsin Medical School, Milwaukee 53233.
DiOrio
J P
JP
Budzynski
A Z
AZ
Mosesson
M W
MW
D012103:Batroxobin / Q000494:pharmacology; D001777:Blood Coagulation; D005337:Fibrin / Q000502:physiology* / Q000648:ultrastructure; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000502:physiology*; D005344:Fibrinopeptide A / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism*; D011108:Polymers; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; D010446:Peptide Fragments; D011108:Polymers; C066064:des-(Bbeta1-42)-fibrin; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D005337:Fibrin; D005340:Fibrinogen; D012103:Batroxobin; D013917:Thrombin