Ca2+-related regulatory function of fibrinogen.

Abstract

Fibrinogen displays a regulation of considerable physiological significance by lowering the Ca2+ requirement for the conversion of the fibrin-stabilizing factor (Factor XIII) zymogen to the range of concentrations of this ion found in plasma. Fibrinogen modulates both Ca2+-dependent steps in the complex process of zymogen activation, involving the heterologous dissociation of subunits of the thrombin-modified zymogen (Factor XIII') species (a'2 a'2) and the unmasking b2 of iodoacetamide titratable sites during generation of transamidating activity (a'2 --a*2). It is interesting that a thrombinindependent pathway of zymogen activation (a2b2 X a -A), b2 which we found to operate at Ca2+ concentrations above 50 mM, is not affected by the presence of fibrinogen. Regulation by fibrinogen thus appears to be specific for controlling only the physiological pathway of zymogen conversion.Fibrinogen has always been characterized as the plasma protein that serves as the precursor for the fibrin network formed during the clotting of blood. Accordingly, only the reaction of fibrinogen with thrombin (1-3) and that of fibrin with fibrinoligase (coagulation Factor XIIIa; refs. 4-6) were thought to be of physiological importance. Our recent work reveals that the fibrinogen molecule displays a hitherto unrecognized regulatory property of considerable physiological significance. It will be shown in the present paper that fibrinogen greatly enhances the generation of fibrinoligase activity and lowers the Ca2+ requirement for this process. As such, modulation by fibrinogen is somewhat reminiscent of the biochemical functions of troponin C in muscle (7) and of the various Ca2+-dependent regulatory proteins in other tissues (see refs. 7-11).Fibrinoligase, a transamidase that is responsible for the covalent fusion of fibrin molecules by 'y-glutamyl-elysine bridges The fibrin-stabilizing factor zymogen (Factor XIII) and the pure b subunits were isolated from outdated human plasma by published procedures (24). These purified materials were stored at 40 in 50 mM Tris1HCl, pH 7.5/1 mM EDTA. Concentrations of the zymogen and the b protein were calculated on the basis of E "m = 13.8 at 280 nm (25). The functional molarity of Factor XIII was measured by titration with iodoacetamide as described by Curtis et al. (14). The molarity of thrombin was determined from pre-steadystate kinetics for the hydrolysis of p-nitrophenylguanidobenzoate (26).Factor XIII zymogen was hydrolytically converted from the a2b2 native state to the a'2b2 one (Factor XIII') at 25°, pH 7.5, in solutions of 0.15 ml containing 10MM zymogen and 0.17,gM thrombin, as well as 50 mM Tris-HCl and 0.5 mM EDTA. After a reaction time of 30 min, sufficient amount of hirudin (Sigma, lot 27B-2330) was added to provide a 2-fold excess of hirudin over the clotting units of thrombin, in terms of the thrombinneutralizing potency of the hirudin preparation as specified by the supplier. Total conversion of the a subunits to a' was verified by sodium dodecyl sulfate disc gel polyacryl...

Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

“…The B values for these residues are not much different from those observed for the ion free structures. These observations are consistent with fXIII's millimolar binding affinity (15) for Ca 2ϩ ions. The Yb 3ϩ ion, which binds more tightly, is in a slightly different location and has more coordinating protein atoms with fewer water ligands.…”

“…In the blood, activation of circulating fXIII requires thrombin cleavage, calcium ions (1.5 mM) (12)(13)(14), and fibrin(ogen) (15). High levels of calcium (Ͼ50 mM) can activate fXIII without the use of thrombin (15), and it has recently been shown that platelet fXIII can be activated nonproteolytically in vivo (16).…”

The activation peptide cleft exposed by thrombin cleavage of FXIII-A2 contains a recognition site for the fibrinogen α chain

“…[7][8][9] This process is significantly enhanced in the presence of fibrinogen, an effect that has been ascribed to the AaC region 242-424. 10,11 Fibrinogen is a 340 000-Da glycoprotein composed of 2 sets of disulfide-linked nonidentical polypeptide chains: Aa, Bb, and g. 12,13 Thrombin catalyzes the polymerization of fibrinogen to fibrin by sequentially cleaving fibrinopeptide A and fibrinopeptide B initiating lateral aggregation of protofibrils and fiber formation. [14][15][16] FXIII-A 2 * stabilizes the forming protofibril by introducing cross-links between adjacent g chains.…”

Interactions between factor XIII and the αC region of fibrinogen

“…The existence of an interaction between FXIII-A 2 B 2 and the ␣C region of fibrinogen is predicted by work from Credo et al 22 which reported that the presence of fibrinogen accelerates activation of FXIII, and more specifically the ␣C residues 242-424 enhanced the activation of FXIII to almost the same degree as full-length fibrinogen. 23 For this enhancement to occur an interaction between FXIII-A 2 B 2 and the ␣C regions of fibrinogen must take place at an early stage of clot formation.…”

“…Hornyak and Shafer 21 compared activated, nonactivated platelet FXIII-A and nonactivated plasma FXIII-A 2 B 2 for binding to fibrin clots (K d of 2.1M, 14M, and 200nM, respectively). In addition, Hornyak and Shafer examined the effect of fibrin on the activation of platelet FXIII-A, a phenomenon previously described by Credo et al 22, 23 Hornyak and Shafer found that fibrin did not promote activation of platelet FXIII-A alone, but it did enhance FXIII-A 2 B 2 activation, suggesting that this effect might be mediated by the dissociation of the B chains. 21 Immunoblotting of fibrinogen plasmin degradation products identified binding regions in the A␣ and B␤ chains for platelet FXIII-A.…”