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.

Factor VIII (fVIII) 1 is an essential component of the intrinsic pathway of blood coagulation, since genetic deficiency in fVIII results in a coagulation disorder known as hemophilia A and occurs in 1 per 5000 males. In the intrinsic pathway, activated fVIII (fVIIIa) functions as a cofactor for the serine protease factor IXa, and their membrane-bound complex (Xase complex) activates factor X to factor Xa (1). Factor Xa subsequently participates in activation of prothrombin into thrombin, the key enzyme of the coagulation cascade.FVIII is a glycoprotein (ϳ300 kDa, 2332 amino acid residues) consisting of three homologous A domains, two homologous C domains, and the unique B domain, which are arranged in the order of A1-A2-B-A3-C1-C2 (2). Prior to its secretion to plasma, fVIII is processed intracellularly to a series of Me 2ϩ -linked heterodimers produced by cleavage at the B-A3 junction (3) and by a number of additional cleavages within the B domain (2). These cleavages generate a heavy chain (HCh) consisting of the A1 (residues 1-336), A2 (residues 373-740), and B domains (residues 741-1648), and a light chain (LCh) composed of the domains A3 (residues 1690 -2019), C1 (residues 2020 -2172), and C2 (residues 2173-2332).In circulation, most of fVIII is bound to vWf, which confers from physiological concentrations of the proteins, which are ϳ1 (4) and ϳ50 nM (5), respectively, and a high affinity (0.2-0.5 nM) of their interaction (6, 7). Binding to vWf prevents fVIII from premature interaction with components of Xase complex and is also required for maintenance of the normal fVIII level in plasma (8), since vWf deficiency in both humans (8, 9) and animals (10, 11) leads to a secondary deficiency of fVIII.We have recently shown that fVIII catabolism from its complex with vWf in vitro and in vivo is mediated by low density lipoprotein receptor-related protein (LRP) (12). LRP, a member of the low density lipoprotein receptor family (13), is responsible for plasma clearance of lipoprotein remnants, serine proteinases, and their complexes with inhibitors (serpins) (13,14). LRP is most prominent in liver on hepatocytes, and in vasculature it is presented on the surface of smooth muscle cells, fibroblasts, and macrophages (15). Besides fVIII, LRP mediates the clearance of a number of other proteins involved in blood coagulation and fibrinolysis, such as factors IXa (16) and Xa (17, 18), plasminogen activators, and their complexes with plasminogen activator inhibitor (19 -21). A unique place among LRP ligands belongs to 39-kDa receptor-associated protein (RAP), which binds to LRP with a high affinity (K d ϭ 4 nM) and efficiently inhibits binding and endocytosis of all known LRP ligands (22).The sites of fVIII involved in interaction with LRP were localized within the A2 domain residues 484 -509 (12) and

To cite this article: Ovanesov MV, Ananyeva NM, Panteleev MA, Ataullakhanov FI, Saenko EL. Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost 2005; 3: 321-31.Summary. Exposure of tissue factor (TF)-bearing cells to blood is the initial event in coagulation and intravascular thrombus formation. However, the mechanisms which determine thrombus growth remain poorly understood. To explore whether the procoagulant activity of vessel wall-bound cells regulates thrombus expansion, we studied in vitro spatial clot growth initiated by cultured human cells of different types in contact pathway-inhibited, non-flowing human plasma. Human aortic endothelial cells, smooth muscle cells, macrophages and lung fibroblasts differed in their ability to support thrombin generation in microplate assay with peaks of generated thrombin of 60 ± 53 nmol L , 218 ± 55 nmol L )1 and 407 ± 59 nmol L )1 (mean ± SD), respectively. Real-time videomicroscopy revealed the initiation and spatial growth phases of clot formation. Different procoagulant activity of cell monolayers was manifested as up to 4-fold difference in the lag times of clot formation. In contrast, the clot growth rate, which characterized propagation of clotting from the cell surface to plasma, was largely independent of cell type (£ 30% difference). Experiments with factor VII (FVII)-, FVIII-, FX-or FXI-deficient plasmas and annexin V revealed that (i) cell surface-associated extrinsic Xase was critical for initiation of clotting; (ii) intrinsic Xase regulated only the growth phase; and (iii) the contribution of plasma phospholipid surfaces in the growth phase was predominant. We conclude that the role of TF-bearing initiator cells is limited to the initial stage of clot formation. The functioning of intrinsic Xase in plasma provides the primary mechanism of sustained and far-ranging propagation of coagulation leading to the physical expansion of a fibrin clot.

Technologies in molecular biology have greatly advanced the knowledge regarding the origin of haemophilia A and the physiology of the factor VIII (FVIII) protein. A variety of different mutations in the FVIII gene have been identified and their effects on the FVIII protein described. It has been shown that the frequency of haemophilia A is due to a high mutation rate predominantly in male germ cells. A significant proportion is originating de novo in early embryogenesis from somatic mutations, a finding that has implications for genetic counselling. The life-cycle of the FVIII protein and its structure-function relationships are continuously clarified. Most recently it has been shown that FVIII clearance from the circulation is mediated by the low-density lipoprotein receptor-related protein (LRP) and cell-surface heparan sulphate proteoglycans (HSPGs). These findings raise hope for novel recombinant FVIII molecules with prolonged half-life that may improve therapies for haemophlia A.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

To cite this article: Panteleev MA, Ananyeva NM, Greco NJ, Ataullakhanov FI, Saenko EL. Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex. J Thromb Haemost 2005; 3: 2545-53.Summary. Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nM of thrombin, platelets with elevated binding capability constituted 4% of total platelets and were responsible for the binding of 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation (80% and 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.

This article summarizes achievements of basic research and their implementation in clinical treatment of one of the most common inherited bleeding disorders hemophilia A, which is caused by genetic deficiency of coagulation factor VIII (FVIII). We discuss the structure of FVIII, its major interactions in the intrinsic pathway of blood coagulation, and the catabolism of FVIII. We also discuss achievements in the contemporary clinical practice of treatment of hemophilia A. Replacement therapy has substantially improved by development of purification and virus inactivation procedures, allowing preparation of safe and effective therapeutic plasma-derived FVIII concentrates. We give special attention to the principles used in the development of contemporary recombinant FVIII products, which do not inherit a potential risk for viral or prion transmission. Development of FVIII inhibitory antibodies is the major complication of FVIII replacement therapy. We summarize the accumulated knowledge regarding epitopes of FVIII inhibitors and mechanisms by which they inactivate FVIII and discuss approaches to overcome the effects of inhibitors and to prevent their formation by induction of immunotolerance. We also analyze the main concepts and scientific priorities in the gene-therapeutic approach for treatment of hemophilia A.

Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.