Calcium ion binding to human and bovine factor X.

Human and bovine factor X contain 11 and 12 glutamyl residues, respectively, within the first 40 amino terminal residues that are post-translationally modified to gamma-carboxyglutamyl (Gla) residues. We have measured calcium ion binding to human factor X by equilibrium dialysis. This is the first examination of calcium ion binding to human factor X. We have also re-examined the equilibrium dialysis binding of calcium ions to bovine facor X in order to compare the two species. The data was analysed using a variety of models that allow for more than one class of binding site and for co-operativity among binding sites. Calcium ion binding to human factor X fits a model that had two classes of sites: one class with a single site that had an affinity of 0.1 mM and a second class with 19 equivalent, non-interacting sites with an average affinity of 3.5 mM. There was no evidence for co-operativity in calcium ion binding. Calcium ion binding to bovine factor X was best stimulated by a model that assumed one tight site, four co-operative sites, and 18 equivalent, non-interacting sites. To examine the co-operativity seen in calcium ion binding to bovine factor X, calcium ion binding to isolated Gla region (residues 1-44) and Gla-domainless factor X was measured by equilibrium dialysis. Calcium ion binding to Gla-domainless factor X was simulated by a model that had two classes of sites: one class with a single site that had an affinity of 0.25 mM, and a second class that had 15 sites with very low affinity sites (greater than 15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin.

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.

Antithrombin action of phosvitin and other phosphate-containing polyanions is mediated by heparin cofactor II.

We have examined the antithrombin effects of various phosphate-containing polyanions (including linear polyphosphates, polynucleotides and the phosphoserine glycoprotein, phosvitin) on the glycosaminoglycan-binding plasma proteinase inhibitors, antithrombin III (ATIII) and heparin cofactor II (HCII). These phosphate-containing polyanions accelerate the HCII-thrombin reaction, as much as 1600-fold in the case of phosvitin. The HCII-thrombin reaction with both phosvitin and polynucleotides appears to follow the ternary complex mechanism. The HCII-thrombin complex is rapidly formed in the presence of these phosphate polyanions (each at 10 micrograms/ml) when 125I-labeled thrombin is incubated with human plasma (ex vivo). None of these phosphate polyanions accelerate the ATIII-thrombin reaction. Our results suggest that the antithrombotic effect of these phosphate-containing polyanions is mediated by HCII activation and not by ATIII.

Carboxylate polyanions accelerate inhibition of thrombin by heparin cofactor II.

The heparin cofactor II (HCII)/thrombin inhibition reaction is enhanced by various carboxylate polyanions. In the presence of polyaspartic acid, the HCII/thrombin reaction is accelerated more than 1000-fold with the second-order rate constant increasing from 3.2 x 10(4) M-1 min-1 (in the absence of polyAsp) to 3.6 x 10(7) M-1 min-1 as the polyAsp concentration is increased from 1 to 250 micrograms/ml. This accelerating effect was observed for HCII/thrombin, though to varying degrees, with other carboxylate polyanions. In contrast to HCII, the rate of antithrombin III inhibition of thrombin was decreased in the presence of polyAsp. The HCII/thrombin complex is rapidly formed in the presence of 10 micrograms/ml polyAsp when 125I-labeled-thrombin is incubated with plasma. It is possible that at physiological sites rich in carboxylate polyanions, thrombin may be preferentially inhibited by HCII.