Blood coagulation in vivo is a spatially nonuniform, multistage process: coagulation factors from plasma bind to tissue factor (TF)-expressing cells, become activated, dissociate, and diffuse into plasma to form enzymatic complexes on the membranes of activated platelets. We studied spatial regulation of coagulation using two approaches: 1), an in vitro experimental model of clot formation in a thin layer of plasma activated by a monolayer of TF-expressing cells; and 2), a computer simulation model. Clotting in factor VIII- and factor XI-deficient plasmas was initiated normally, but further clot elongation was impaired in factor VIII- and, at later stages, in factor XI-deficient plasma. The data indicated that clot elongation was regulated by factor Xa formation by intrinsic tenase, whereas factor IXa was formed by extrinsic tenase on activating cells and diffused into plasma, thus sustaining clot growth. Far from the activating cells, additional factor IXa was produced by factor XIa. Exogenously added TF had no effect on the clot growth rate, suggesting that plasma TF does not contribute significantly to the clot propagation process in a reaction-diffusion system without flow. Addition of thrombomodulin at 3-100 nM caused dose-dependent termination of clot elongation with a final clot size of 2-0.2 mm. These results identify roles of specific coagulation pathways at different stages of spatial clot formation (initiation, elongation, and termination) and provide a possible basis for their therapeutic targeting.

Mortality and disabilities as outcomes of cardiovascular diseases are primarily related to blood clotting. Optimization of thrombolytic drugs is aimed at the prevention of side effects (in particular, bleeding) associated with a disbalance between coagulation and anticoagulation caused by systemically administered agents. Minimally invasive and efficient approaches to deliver the thrombolytic agent to the site of clot formation are needed. Herein, we report a novel nanocomposite prepared by heparin-mediated cross-linking of urokinase with magnetite nanoparticles (MNPs@uPA). We showed that heparin within the composition evoked no inhibitory effects on urokinase activity. Importantly, the magneto-control further increased the thrombolytic efficacy of the composition. Using our nanocomposition, we demonstrated efficient lysis of experimental clots in vitro and in animal vessels followed by complete restoration of blood flow. No sustained toxicity or hemorrhagic complications were registered in rats and rabbits after single bolus i.v. injection of therapeutic doses of MNPs@uPA. We conclude that MNPs@uPA is a prototype of easy-to-prepare, inexpensive, biocompatible, and noninvasive thrombolytic nanomedicines potentially useful in the treatment of blood clotting.

Recombinant factor VIIa (rFVIIa) is used for treatment of hemophilia patients with inhibitors, as well for off-label treatment of severe bleeding in trauma and surgery. Effective bleeding control requires supraphysiological doses of rFVIIa, posing both high expense and uncertain thrombotic risk. Two major competing theories offer different explanations for the supraphysiological rFVIIa dosing requirement: (1) the need to overcome competition between FVIIa and FVII zymogen for tissue factor (TF) binding, and (2) a highdose-requiring phospholipid-related pathway of FVIIa action. In the present study, we found experimental conditions in which both mechanisms contribute simultaneously and independently to rFVIIa-driven thrombin generation in FVIIdeficient human plasma. From mathematical simulations of our model of FX activation, which were confirmed by thrombingeneration experiments, we conclude that the action of rFVIIa at pharmacologic doses is dominated by the TF-dependent pathway with a minor contribution from a phospholipid-dependent mechanism. We established a dose-response curve for rFVIIa that is useful to explain dosing strategies. In the present study, we present a pathway to reconcile the 2 major mechanisms of rFVIIa action, a necessary step to understanding future dose optimization and evaluation of new rFVIIa analogs currently under development. IntroductionThe use of a recombinant factor VIIa (rFVIIa) product, NovoSeven, which is licensed for the treatment of hemophilia patients with inhibitory Abs against factor VIII (FVIII) or factor IX (FIX), has proven to be safe and efficacious, but its dosing remains problematic. [1][2][3][4] The recommended dosing schedule is a supraphysiological dose of 90 g/kg every 2-3 hours until hemostasis is achieved, producing an approximately 250-fold increase above basal plasma concentrations of FVIIa (0.1 to 25nM). 5 Not only does such a treatment regimen incur a high cost, but ineffective drug responses and thrombotic complications have also been reported. [6][7][8][9] However, our current understanding of the mechanism of rFVIIa action is unclear, limiting our ability to optimize the safety and cost of treatment. 10,11 Off-label use of rFVIIa for nonhemophilia patients at similar high doses indicates that the high dose required for hemophilia treatment cannot be explained by deficiencies of FVIII or FIX alone. 5,12 FVIIa is a weak enzyme and its activity requires either of 2 cofactors, tissue factor (TF) or negatively charged phospholipids. 13 Disagreement over which of the 2 cofactors explains the high pharmacologic dose of the drug has led to TF-and phospholipiddependent theories of rFVIIa action. Although these mechanisms may appear to be nonexclusive, the 2 theories support opposing approaches to dose adjustment.The TF-dependent mechanism suggests that the hemostatic effect of rFVIIa is mediated by its binding to TF expressed on cell surfaces at the site of injury, 14 forming the extrinsic tenase complex, which activates factor X (FX), leading to thrombin genera...