Abstract
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.
Highlights
Fibrinogen containing the FPA sequence but lacking the Bfil-42 sequence (“des-(Bfil-42)-fibrinogen”), was compared to native fibrinogen to investigate the role played by Bfll-42 in the polymerization of a-fibrin, 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 (w, 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/31-42)-a-fibrin, indicating that the Bj31-42 sequence contributes to the A polymerization site
Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent a-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent a-fibrin monomers
Summary
The rate of fibrinopeptide release from fibrinogen was determined by incubating fibrinogen (3-5 mg/ml) in 0.15 M ammonium acetate buffer, pH 7.5, or in 0.05 M ammonium acetate buffer, pH 7.5, with thrombin (0.05 to 0.2 unit/ml) or reptilase (0.075-0.2 unit/ml) at room temperature for periods of up to 360 min. Thrombin binding by native and des-(B@l-42)-fibrin was measured in the 50 mM Tris buffer system (CL, 0.04 or 0.14) by incubating fibrinogen (0.50 or 1.85 mg/ml) with thrombin (l-25 units/ml, final concentration) at room temperature for 2 h. Des-(BOl-42)-fibrin ing electron microscopy were formed at room temperature (p, 0.14) as 50.~1 drops (0.1 mg/ml fibrinogen) in a Petri dish, incubated for O-20 min with thrombin or reptilase (0.1 unit/ml, final), picked up on glow discharged carbon/formvar-coated grids, and negatively contrasted with 2% (w/v) uranyl acetate. All fibers within randomly selected areas of each photograph were measured and averaged
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
