Fibrinogen Sialic Acid Residues Are Low Affinity Calcium-binding Sites That Influence Fibrin Assembly

C V Dang,C K Shin,W R Bell,C Nagaswami,J W Weisel

doi:10.1016/s0021-9258(18)63817-7

C V Dang, C K Shin + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(18)63817-7

Copy DOI

Abstract

Calcium ions occupy low (n congruent to 10; Kd congruent to 1 mM) and high (n = 3; Kd congruent to 1 microM) affinity sites on fibrinogen and facilitate fibrin monomer polymerization. We have previously localized two of the three high affinity Ca2+ sites to gamma 311-gamma 336. However, optimal enhancement of fibrin monomer polymerization occurs only at physiological millimolar Ca2+ concentrations which are two orders of magnitude higher than the concentration required for occupancy of the high affinity Ca2+-binding sites. In this study, we show that removal of fibrinogen sialic acid residues results in loss of low affinity Ca2+-binding sites. Clotting of asialofibrinogen appears to be Ca2+-independent and results in fiber bundles thicker in diameter than normal fibrin bundles as determined by turbidometry and scanning and transmission electron microscopy. By using a Ca2+-sensitive electrode, free sialic acid is shown to bind Ca2+ (Kd congruent to 1 mM). These observations suggest that the high affinity fibrinogen D-domain Ca2+-binding sites may play a role in the tertiary structure of the D-domain, whereas, sialic acid residues are low affinity sites whose occupancy by Ca2+ at physiological calcium concentration facilitates fibrin polymerization.

Highlights

Calcium ions occupy low ( n = 10; Kd = 1 mM) and it has been known for several decades that calhigh ( n = 3; K d 1 pM) affinity sites on fibrinogen and cium facilitates fibrin monomer polymerization [4], the mofacilitate fibrin monomer polymerization
Unclear are the functions of these low affinity sites and thebiochemical basis for thicker in diameter than normal fibrin bundles as de- the calcium-dependent rate of fibrin polymerization
Calcium-independent Clotting of Asialofibrinogen-We examined the calcium-dependent clotting rate of AF and untreated F that were depleted of calcium by EDTA treatment

Summary

RESULTS

Calcium-independent Clotting of Asialofibrinogen-We examined the calcium-dependent clotting rate of AF and untreated F that were depleted of calcium by EDTA treatment (see"Materials and Methods"). AF displays essentially calcium concentration-independent polymerization between no added (-0.1 PM; Ref. 7) and 1mM Ca2* (Fig. 1B). Where n is the solution refractive index, dn/dc is the refractive index increment, X is the wavelength, C is the concentration of fibrinogen in g/ml, N is Avogadro's number, and p is the mass/length ratio. Calcium Titration-Solutions of AF and F were prepared as described above and diluted to a final concentration of 1.1% (w/v). In the presence of calcium-sensitive and reference electrodes, 10pl aliquots of 0.01 M CaC12dissolved in 25 mM Tris, 0.15 M NaCl, pH 7.26, were added to 2 mlof protein solution in a 5-ml beaker. The beaker was gently swirled manually, and the solution was allowedto reach steady state (-1 min) before readings were taken. Time to reach half-maximal ( t d turbidity of clots formingfrom untreated or Factor XIZI-free normal and asialofibrinogen t, was the time inmin to reach half-maximal turbidity determined by absorbance a t 350 nm (Figs. 1and 2)

FAFD FD AF mM

Relative fibrin fiberradii determined by turbidometry

DISCUSSION

The hypothesis that fibrinogen sialic acid residues are low