Inhibition of intrinsic Xase by protein S: a novel regulatory role of protein S independent of activated protein C.

OBJECTIVE: Protein S is a vitamin K-dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. METHODS AND RESULTS: We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)-deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)-mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the K(M) for factor X activation by fIXa in the absence of fVIIIa and both k(cat) and K(M) in the presence of fVIIIa. CONCLUSIONS: From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C-independent way.

Persistent factor VIII-dependent factor X activation on endothelial cells is independent of von Willebrand factor.

Endothelial cells are able to support the activation of coagulation factor X by activated factor IX in the presence of its cofactor, factor VIII. We have previously reported that this reaction is persistent on endothelial cells, but transient on activated platelets and phospholipid vesicles when activated factor X (Xa) is used as activator of factor VIII. Aim of the present study was to explore the influence of von Willebrand factor and that of the factor VIII activator, either factor Xa or thrombin, on the decay of factor X activation on the endothelial cell surface. Kinetics of factor X activation on human umbilical vein endothelial cells was compared with that on phospholipid vesicles employing purified coagulation factors from plasma as well as recombinant factor VIII variants. Employing factor Xa as factor VIII activator, rate constants for decay of membrane-bound factor X activation were consistently low on endothelial cells (0.02 min) as compared with phospholipid vesicles (0.2 min). Activation of factor VIII by thrombin resulted in two-fold increased decay rates. In the presence of excess of von Willebrand factor over factor VIII, decay rates were not significantly changed. Factor VIII variants with and without a Tyr to Phe substitution, which abolishes high-affinity binding to von Willebrand factor, displayed the same factor X activation decay kinetics. Although previous studies have shown that von Willebrand factor modulates factor VIII activation and stabilisation, this apparently does not affect the progression of factor X activation at the endothelium.

The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII.

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.

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies.

BACKGROUND: Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. OBJECTIVES: To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. METHODS: Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. RESULTS: We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 pm FVIIIa (i.e. 16 mU mL(-1) or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. CONCLUSION: Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.

Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex.

Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXa-fVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membrane-bound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-microm phospholipid vesicles (25/75 phosphatidylserine/phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 +/- 5000 (33 200 +/- 14 100), 20 000 +/- 4500, and 30 500 +/- 1300 binding sites per vesicle with apparent K(d) values of 76 +/- 23 (71 +/- 5), 1510 +/- 430, and 223 +/- 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 +/- 370, 20 +/- 5 nm) and fX (12 630 +/- 690, 14 +/- 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 +/- 67, 23 +/- 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 +/- 0.12 and an intrinsic K(m) value of 8.0 +/- 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.

Role of the C2 domain of factor VIIIa in the assembly of factor-X activating complex on the platelet membrane.

Optimal rates of factor X (FX) activation require binding of factor IXa (FIXa), factor VIII(a) [FVIII(a)], and FX to activated platelet receptors. To define the FVIIIa domains that mediate platelet interactions, albumin density gradient washed, gel-filtered platelets (3.5 x 10(8)/mL) activated by the thrombin receptor peptide, SFLLRN (25 microM), were incubated with 125I-labeled FVIII C2 domain, or 125I-FVIIIa, or 125I-FVIII((LC)), or peptides from the C2 domain region, with or without anti-C2 domain monoclonal antibodies (MoAb), ESH4 or ESH8. FVIIIa (Kd approximately 1.7 nM), FVIII((LC)) (Kd approximately 3 nM), and the C2 domain (Kd approximately 16 nM) all interacted with approximately 700-800 binding sites/platelet. Unlike FVIIIa, the C2 domain did not respond to the presence of excess EGR-FIXa (45 nM) and FX (1.5 microM) with enhanced binding stoichiometry and affinity. Both the MoAb ESH4 and a synthetic peptide corresponding to FVIII residues 2303-2332 (epitope for FVIII MoAb, ESH4) inhibited FVIIIa binding to platelets, whereas MoAb ESH8 and a C2 domain peptide corresponding to residues 2248-2285 (epitope for the FVIII MoAb, ESH8) failed to inhibit FVIIIa binding. Thus, a major platelet-binding site resides within residues 2303-2332 in the C2 domain of FVIIIa, and an additional site within residues 2248-2285 increases the stoichiometry and affinity of FVIIIa binding to activated platelets only in the presence of FIXa and FX but does not directly mediate FVIIIa binding to the platelet surface.

Targeting tissue factor as an antithrombotic strategy.

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of cell-surface protein, such as tissue factor (TF). TF is exposed to the flowing blood as a consequence of vascular injury induced, for instance, by PTCA, or by spontaneous rupture of an atherosclerotic plaque. Expression of TF may also be induced in monocytes and endothelial cells in several conditions such as sepsis and cancer, causing a more generalized activation of clotting. In addition to its essential role in hemostasis, TF may be also implicated in several pathophysiological processes, such as intracellular signaling, cell proliferation, and inflammation. For all these reasons, TF has been the subject of intense research focus. Many experimental studies have demonstrated that inhibition of TF:factor VIIa procoagulant activity is a powerful inhibitor of in vivo thrombosis and that this approach usually results in a less-pronounced bleeding tendency compared with other "more classical" antithrombotic interventions. Alternative approaches may be represented by antibodies directed against TF, by transfection of the arterial wall with natural inhibitors of the TF:factor VIIa complex, such as the TF pathway inhibitor, or with catalytic RNA (ribozyme), which could inhibit the expression of the TF protein by the disruption of cellular TF mRNA. All these approaches seem particularly attractive because they may result in complete inhibition of local thrombosis without incurring potentially harmful systemic effects. Further studies are warranted to determine the efficacy and safety of such approaches in patients.

Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons.

Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 x 10(9) CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 x 10(10) CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.

Region of factor IXa protease domain that interacts with factor VIIIa: analysis of select hemophilia B mutants.

Essential to hemostasis is the interaction of factor IXa with factor VIIIa. Recent studies indicate that helix-330 in the protease domain of factor IXa provides a critical binding site for factor VIIIa. Although weaker interactions cannot be ruled out, a primary role of the EGF1 domain of factor IXa in this context may be to serve as a spacer in properly positioning the factor IXa protease domain for optimal interaction with factor VIIIa. The role of the Gla domain, as well as of the EGF2 domain of factor IXa, in binding to factor VIIIa is not clear. The region of factor VIIIa that interacts with the protease domain of factor IXa is quite possibly located in the A2 domain. Furthermore, it should be noted (Table 1) that the corresponding helix residues in factor VIIa bind to tissue factor, and, in factor Xa, they are involved in binding to factor Va. Thus, a common function of this helix (162 in chymotrypsin numbering) in several blood coagulation proteases may be to serve as an anchoring point for the respective cofactor.

Activation and disturbance of blood haemostasis following strenuous physical exercise.

Physical exercise activates blood coagulation and enhances fibrinolytic activity. To investigate whether these activations of blood coagulation and fibrinolysis are balanced post-exercise and during the period of recovery, 11 moderately active young men were examined immediately after a standardised cycle ergometer test and during the 24 h period of recovery. Blood samples were obtained at rest, immediately after exercise, and 2, 6 and 24 h after exercise. All post-exercise values were corrected for any change in plasma volume. Exercise induced a significant increase in factor VIII activity and this occurred with a significant shortening of activated partial thromboplastin time. A concomitant enhancement of tissue plasminogen activity resulted in significant increases in tissue plasminogen activity antigen and total fibrin/fibrinogen degradation products, and a significant decrease in tissue plasminogen activator inhibitor-1 activity. Increases in coagulation and fibrinolytic activity changed in parallel during exercise. However, during recovery, while the increase in factor VIII activity post-exercise persisted 2 and 6 h into recovery, fibrinolytic activity demonstrated a sharp fall. It is concluded that whereas the enhanced fibrinolytic activity during exercise appears to counterbalance the increase in blood coagulability, this haemostatic balance is not maintained during recovery. This perturbed blood haemostasis could constitute an enhanced risk for coronary artery thrombosis and may contribute to exercise-related cardiovascular events.

A mathematical model for the spatio-temporal dynamics of intrinsic pathway of blood coagulation. I. The model description.

We developed and analyzed the mathematical model of the intrinsic pathway based on the current biochemical data on the kinetics of blood coagulation individual stages. The model includes eight differential equations describing the spatio-temporal dynamics of activation of factors XI, IX, X, II, I, VIII, V, and protein C. The assembly of tenase and prothrombinase complexes is considered as a function of calcium concentration. The spatial dynamics of coagulation was analyzed for the one-dimensional case. We examined the formation of active factors, their spreading, and growth of the clot from the site of injury in the direction perpendicular to the vessel wall, into the blood thickness. We assumed that the site of injury (in the model one boundary of the space segment under examination) becomes a source of the continuous influx of factor XIa. In the first part, we described the model, selected the parameters, etc. In the second part, we compared the model with experimental data obtained in the homogeneous system and analyzed the spatial dynamics of the clot growth.

Depolymerized holothurian glycosaminoglycan with novel anticoagulant actions: antithrombin III- and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor VIIIa complex and heparin cofactor II-dependent inhibition of thrombin.

The inhibition mechanism of a polysaccharide anticoagulant, depolymerized holothurian glycosaminoglycan (DHG), was examined by analyzing its effects on the clotting time of human plasma depleted of antithrombin III (ATIII), of heparin cofactor II (HCII), or of both heparin cofactors. The effect exerted by this agent on the activation of prothrombin and factor X in purified human components were also examined and all effects were compared with those of other glycosaminoglycans (GAGs). The capacity of DHG to prolong activated partial thromboplastin time was not reduced in ATIII-depleted, HCII-depleted, HCII-depleted, or ATIII- and HCII-depleted plasma, whereas its capacity to prolong prothrombin time and thrombin clotting time was reduced in HCII-depleted plasma. DHG inhibited the amidolytic activity of thrombin in the presence of HCII with a second order rate constant of 1.2 x 10(8) (mol/L)-1 min-1. These results indicated that DHG has two different inhibitory activities, one being an HCII-dependent thrombin inhibition and the other an ATIII- and HCII-independent inhibition of the coagulation cascade. The heparin cofactors-independent inhibitory activity of DHG was investigated in the activation of prothrombin by factor Xa and in the activation of factor X by tissue factor-factor VIIa complex or by factor IXa. DHG significantly inhibited the activation of factor X by factor IXa in the presence of factor VIIIa, but not in the absence of factor VIIIa. The interaction between DHG and factors IXa, VIIIa, and X was investigated with a DHG-cellulofine column, on which DHG had strong affinity for factors IXa and VIIIa. These findings show that the heparin cofactors-independent inhibition exhibited by DHG was caused by inhibition of the interaction of factor X with the intrinsic factor Xase complex, probably by binding to the factor IXa-factor VIIIa complex.

Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.

A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage.

Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site-directed DNA-mediated mutagenesis. Thrombin-activated human recombinant-derived factor VIII was isolated in an active form by passage over Mono-S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer.

Human platelet factor V is crosslinked to actin by FXIIIa during platelet activation by thrombin.

Although it has been established that factor V (FV) becomes associated irreversibly with the platelet cytoskeleton after stimulation with thrombin, the chemical nature of this complex is not known. Factor V has recently been demonstrated to be a substrate for factor XIIIa and to form factor V oligomers. We now show that thrombin-activated 125I-FV specifically links to a single protein (43 kDa) of the solubilized platelet membrane in a reaction which requires Ca++ and factor XIIIa. In a purified system, FV, activated by thrombin, forms covalently linked high molecular complexes with 125I-actin catalyzed by factor XIIIa. The site of crosslinking of actin was the factor V fragments, 150 kDa (connecting peptide, C1) and its parent molecule 200 kDa (B). Using radiolabeled actin and unlabeled FV, factor XIIIa catalysed the formation of both homopolymers and heteropolymers. Unlabeled actin was found to compete with radiolabeled FV as a substrate for FXIIIa. To evaluate the biological significance of the crosslinking of factor V to actin, intact platelets were treated with B10 (monoclonal antibody to C1), or monospecific polyclonal antibodies to actin or FXIII. After stimulation with thrombin, the cytoskeleton (material insoluble in Triton X-100) showed markedly decreased 125I-FV in the crosslinked complexes. FV coagulant activity associated with platelet cytoskeleton was also diminished following incubation with an antibody to actin, factor XIII, or B10. These data suggest that FV, through the C1 domain, is crosslinked to actin in the cytoskeleton of thrombin-treated platelets. Activated factor XIII may play a role in plasma FV-platelet interaction as well as the expression of FV derived from the alpha-granules on the cytoskeleton during platelet stimulation.