Preparation and Properties of Human Prothrombin Complex

Abstract

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Purification of Blood Coagulation Factors II, VII, IX and X from Bovine Citrated Plasma

“…Under our conditions, DEAE Sephadex chromatography resulted in sepa ration of coagulation factor activity from many other plasma proteins, in- eluding albumin, y-globulin and fibrinogen. In accordance with other authors [13,18,34,45], a partial separation was effected between the factors II and X, the latter having a much higher affinity for the anion exchanger. The presence of DFP appeared to be necessary for prevention of acti vation of coagulation factors due to chromatographic procedures, as has been established by Jackson and Hanahan [17], Even in the presence of DFP, factor X eluted in a double peak as was observed previously by Jackson et al [18] and by M ilstone et al [31], We can confirm the finding of Jackson and Hanahan [17] that both kinds o f factor X have the same mobility on polyacrylamide electrophoresis.…”

“…Factor II and factor IX could not be separated by DEAE cellulose chromatography [9,35,45], Partial separation of these factors was achieved by hydroxylapatite chromatography [27], as has been shown also for the human coagulation factors [5,43]. In our experiments, factor II eluted at a lower phosphate concentration than was found for human prothrombin.…”

“…The molecular weights of bovine factors II and IX were determined by G-100 gel filtration to be approximately 82,000 for both factors [9,45]. Controversially, no indication of multiple factor-II elution patterns or factor-II polymorphism was obtained in the present study [2,3,22,28],…”

Human Prothrombin

“…The initial prothrombin separation on ECTEOLA represents a concentration of approximately 300 fold and provides a product with an average specific activity of 1690 u/mg and, although not homogeneous , is much superior to preparations presently developed for therapeutic purposes (23) . An additional chromatographic procedure, using DEAE cellulose or Sephadex G200, increased specific activity comparable to that of the best products available including the recently described barium crystalized bovine preparation of Tishkoff and co-workers (24).…”

Studies on Plasma Factor Involved in 5-Hydroxytryptamine Release From Blood Platelets by Bacterial Endotoxin

“…Adsorption on and elution from barium citrate This was carried out by methods of Tishkoff et al (8). Forty-four ml of 1 M barium chloride solution was added to 443 ml of rabbit plasma at 4'C.…”