Prothrombin activation by immobilized thrombin]

The ability of thrombin, immobilized on BrCN-activated Sepharose 4B, to split prothrombin, was studied. Immobilized thrombin retained up to 70% of its esterase activity and about 5% of its coagulating activity; it was also found to induce partial proteolysis of prothrombin. Two products of prothrombin degradation isolated, i.e. P1 (m. w. 50.000-52.000) and P2 (m. w. 22.000-24.000), did not show either the thrombin or the prothrombin activities. P1 was converted into thrombin under the action of tripsin or Factor Xa. The rate of conversion was considerably increased after addition of Factor V, thromboplastin and Ca2+ ions. Intravenous administration of P1 to rats resulted in changes in the coagulating system of blood, which may be probably indicative of the stimulation of the anticoagulating system. P2 possessed no thrombogenic activity.     

Histone H4 Promotes Prothrombin Autoactivation

Recent studies have documented the ability of prothrombin to spontaneously convert to the mature protease thrombin when Arg-320 becomes exposed to solvent for proteolytic attack upon mutation of residues in the activation domain. Whether prothrombin autoactivation occurs in the wild-type under conditions relevant to physiology remains unknown. Here, we report that binding of histone H4 to prothrombin under physiological conditions generates thrombin by autoactivation. The effect is abrogated by mutation of the catalytic Ser-525 and requires the presence of the Gla domain. Fluorescence titrations document direct binding of histone H4 to prothrombin with an affinity in the low nm range. Stopped flow data and luminescence resonance energy transfer measurements indicate that the binding mechanism obeys conformational selection. Among the two conformations of prothrombin, collapsed and fully extended, histone H4 binds selectively to the collapsed form and induces a transition toward a new conformation where the distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 increases by 13 Å. These findings confirm the molecular plasticity of prothrombin emerged from recent structural studies and suggest that different conformations of the inter-kringle linker domain determine the functional behavior of prothrombin. The results also broaden our mechanistic understanding of the prothrombotic phenotype observed during cellular damage due to the release of histones in the blood stream. Prothrombin autoactivation induced by histone H4 emerges as a mechanism of pathophysiological relevance through which thrombin is generated independently of activation of the coagulation cascade.     

Prothrombin kringle-2 activates cultured rat brain microglia

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-kappaB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.     

Prothrombin activation on phospholipid membranes with positive electrostatic potential

The conversion of prothrombin into thrombin, which is a crucial reaction in hemostatic plug formation, is greatly stimulated by phospholipids plus calcium ions. It has been proposed that phospholipid surfaces which promote blood coagulation should have a negative surface charge [Bangham, A. D. (1961) Nature (London) 192, 1197-1198]. However, the experiments that led to this proposal were carried out with one kind of anionic phospholipid (dicetyl phosphate). Here we report that membranes, which contain phosphatidylserine (PS) as the anionic phospholipid, can be made positively charged by incorporation of stearylamine and still exhibit almost full procoagulant and prothrombin-converting activity. This suggests that electrostatic forces contribute negligibly to the binding of coagulation factors to PS-containing membranes. Introduction of stearylamine in membranes containing phosphatidyl-beta-lactate (PLac) causes considerable inhibition of their prothrombin-converting activity. Since PLac and PS only differ by the presence of an amino group in the polar head group, the much higher procoagulant activity of PS-containing vesicles is indicative of an important function of the amino group of PS in the interaction with coagulation factors. We propose that the association of coagulation factors with PS-containing membranes results from complex formation between Ca2+ ions and ligands supplied by the protein and by PS molecules. The ability to form such a complex may well explain why cell membranes with PS have such excellent procoagulant properties.