The objective of the review is to cover the formation of modern understanding of molecular mechanisms of blood coagulation initiation. It was provided mainly by the research of Professor D.M. Zubairov and his colleagues. Since 1963, he has established that blood coagulation initiation is not connected to the phenomenon of vascular wall moistening and contact complex factors activation. Research of the thromboplastic activity distribution in tissue cells, blood and in the serum phospholipid microparticles allowed to conclude that blood coagulation is initiated by long-term expression of tissue factor and rapid massive alterations in cellular membranes. This was confirmed by the detection of the turned phospholipids mesophases in tissue thromboplastin preparations and heterogeneity of vitamin К-depending factors binding. Based on the results of the research, a functional conception of blood coagulation initiation by phase alteration of bilayer structure of cellular membranes to a mesomorphic structure was developed. It is caused by different agonists through receptor dependant Са 2+-mobilizing cell signal systems or by massive migration of calcium ions into the cell at its damage. An initial bioimitating non-enzymatic proteolysis vitamin of К-dependant factors and their massive enzymatic activating in the ensembles of enzymatic complexes takes place on heterophase phospholipids surface. Clotting is limited by blood and tissue macrophages, removing cells and phospholipids particles with heterophase surface from cell circulation, and also by anticoagulant factors action. Based on this conception, the researches revealed the pathogenetic of role thrombogenic micro vesicles originating form the cellular membranes transformation in the development of disseminated intravascular coagulation syndrome, myocardial infarction, leucosis, autoimmune and infectious diseases. Finding out the basic concepts of blood coagulation initiation mechanism puts D.M. Zubairov in one row with scientists, pawning the bases of modern biology and medicine.

The binding of 125I-labeled prothrombin fragment I. prethrombin I and alpha-thrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 of EDTA was studied. The experimental curves plotted in the Scatchard coordinates testify to the presence in thromboplastin of two types of fragment I binding sites: those with a high (Kd = 7.6 x 10(-6) M) and moderate (Kd = 1.3 x 10(-8) M) binding affinity. The parameters of fragment I binding and their changes reproduced, for the most part, the mode of prothrombin binding observed in previous studies. The experimental results provide indirect evidence in favour of a hydrophobic role of Ca(2+)-dependent binding of prothrombin fragment I to thromboplastin. The binding of prethrombin I was nonspecific and Ca(2+)-independent, whereas alpha-thrombin showed a relatively high level of nonspecific electrostatic binding which was competitively inhibited by Ca2+. Thromboplastin proteins interacted (both directly and in a Ca(2+)-independent fashion) with all the prothrombin derivatives under study.

: According to the conception proposed, blood coagulation is initiated by Ca(2+)-induced rearrangement of the bilayer structure of native cell membranes into heterophasic. The thrombogenic action is realized through Ca2+ entering the cell cytoplasm. This leads to phosphatidyl serine translocation into the surface monolayer of the membrane, its clustering and the arising of phosphatidyl ethanolamine mesophases. Vitamin K-dependent coagulation factors are bound at the boundaries of clusters and mesophases of these phospholipids through Ca2+ ions. They form enzyme complexes functioning due to the matrix structure of phospholipid surface as units of thrombin generation.

The binding of 125I-labeled human prothrombin to native and papain-treated tissue thromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard plots for the protein binding suggest the presence at thromboplastin surface of two types of binding sites, high affinity [Kd(app) = 7.4.10(-8) M] and moderate affinity [Kd(app) = 7.9.10(-5) M]. The removal of Ca2+ did not influence the Kd (values for these) sites but markedly reduced their number. Proteolysis by papain caused a decrease in the affinity of high affinity sites without affecting the Kd values of the moderate affinity sites yet caused a proportional increase in the number of both high and moderate affinity sites in the presence of Ca2+. At low prothrombin concentrations a positive cooperativity of protein binding at high affinity sites in the presence of Ca2+ was observed.

The role of protein moiety of tissue thromboplastin under its specific enzymatic modification and the effects of some protease inhibitors were studied. Treatment with HCl, pepsin and papain was followed by a decrease in the biological activity of thromboplastin, which was unaffected by the inhibitors of some proteolytic enzymes (DFP, monoiodoacetate and o-phenanthroline). It was assumed that the protein component of thromboplastin fulfils a structural function in the assembly of the lipoprotein matrix, on which surface the enzymatic reactions of blood coagulation are known to occur.

Canine fibrinogen was digested by a complex of proteases from Streptomyces griseus. The degradation products were purified by gel-filtration, DEAE-cellulose chromatography and electrophoresis, resulting in nine glycopeptides, eight of which contained aspartic acid and one--serine. The other amino acids were found only in trace amounts. The glycopeptides were shown to contain hexoamines, mannose, galactose and sialic acid. The oligosaccharide chains form a sequence of structurally similar variants. The individual microheterogeneity of canine fibrinogen with respect to carbohydrate chains was detected. A comparison of the carbohydrate composition of fibrinogen and glycopeptides suggests the presence of four carbohydrate chains in the protein molecule.

In the process of dog prothrombin hydrazinolysis 1 M of glycine is relased in 1 M of protein. It was concluded that glycine is the C-terminal amino acid of the prothrombin.

After degradation of canine prothrombin by the complex of Streptomyces griseus proteases four glycopeptides were obtained. Each of them contained aspartic acid, hexosamines, mannose, galactose and sialic acids. Canine prothrombin contains two or three carbohydrate chanins, which are bound to aspartic (asparagine) residues. Microheterogenity of the carbohydrate chains of canine prothrombin was found.