The kinetics of blood coagulation

The kinetics of the blood coagulation system have been formulated and an expression obtained for the “prothrombin time” in terms of the concentrations of the components of the system. A linear plot of data obtained from plasma dilution curves gives the values of the parameters of the system, and yields a mathematical method of comparing relative thromboplastin potencies. The analytical expressions given lead to the proper choice of thromboplastin potency and plasma dilution for minimum error in the clinical determination of prothrombin. National Institute of Health.     

Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism

Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.     

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.     

Nonlinear effects in kinetics of blood coagulation

A linear mathematical model of the kinetics of humoral system of hemocoagulation is presented in the works by Levin (1966), Moro a. Brakhucha-Reyd (1969), Martoran a. Moro (1974). Present paper considers a nonlinear mathematical model of the intrinsic path of hemocoagulation which takes into account the effect of positive feed back which is related to the action of factors Va and VIIIa as co-factors. It is found that the intrinsic path of hemocoagulation normally functions as an amplifier cascade, if the wall damage is above some threshold value. When the damage is below the threshold value the intrinsic path of hemocoagulation does not function.     

Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa

The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.     

Tolerance and threshold in the extrinsic coagulation system

This paper focus on the quest for mechanisms that are able to create tolerance and an activation threshold in the extrinsic coagulation cascade. We propose that the interplay of coagulation inhibitor and blood flow creates threshold behavior. First we test this hypothesis in a minimal, four dimensional model. This model can be analysed by means of time scale analysis. We find indeed that only the interplay of blood flow and inhibition together are able to produce threshold behavior. The mechanism relays on a combination of raw substance supply and wash-out effect by the blood flow and a stabilization of the resting state by the inhibition. We use the insight into this minimal model to interpret the simulation results of a large model. Here, we find that the initiating steps (TF that produces together with fVII(a) factor Xa) does not exhibit threshold behavior, but the overall system does. Hence, the threshold behavior appears via the feedback loop (in that fIIa produces indirectly fXa that in turn produces fIIa again) inhibited by ATIII and blood flow.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.     

Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation

We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.     

Capsular polysaccharide induction of apoptosis by intrinsic and extrinsic mechanisms

A purified microbial capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces Fas ligand (FasL) upregulation on macrophages and, as a consequence, apoptosis of lymphocytes. The mechanisms that lead to lymphocyte apoptosis in both in vitro and in vivo systems were investigated by cytofluorimetric analysis and Western blotting experiments. Caspase 8 cleaves caspase 3 in two different pathways: directly as well as indirectly by activation of Bcl-2 interacting domain, which initiates caspase 9 cleavage. Therefore, the caspase 8 and caspase 9 pathways cooperate in an amplification loop for efficient cell death, and noteworthily we provide evidence that they are both activated in one single cell. Furthermore, both activation of GXM-mediated caspase 8 and apoptosis were also found in in vivo systems in an experimental model of murine candidiasis. Collectively, our data show that GXM-induced apoptosis involves, in a single cell, a cross-talk between extrinsic and intrinsic pathways. Such a finding offers opportunities for the therapeutic usage of this polysaccharide in appropriate clinical settings for taming T-cell responses.     

The kinetics of milk coagulation: IV. The kinetics of the gel-firming process

A mechanistic kinetic model of gel firmness development during milk gel formation is presented. The model correctly accounts for the influence of enzymatic kappa-casein hydrolysis on the rate of firmness development in renneted milk gels. The model used is based on two first-order reactions occurring in series. The first reaction is enzymatically controlled and corresponds to the formation of gel crosslink sites by kappa-casein hydrolysis. The second reaction is nonenzymatic and corresponds to the process of crosslink formation and depletion of active sites. The model successfully predicts gel firmness development in the temperature range 31-45 degrees C for a variety of initial enzyme concentrations.     

Low-molecular heparin complex formation with the blood coagulation proteins--thrombin and fibrinogen]

It has been found that low molecular heparin (LMH) forms complexes with fibrinogen and thrombin. The formation of the heparin-fibrinogen and heparin-thrombin complexes has been testified by cross-linked electrophoresis. The reaction of complex formation was carried out at variable weight ratios of the components, i.e. 1:3 and 1:6, respectively. This complex causes lysis of unstabilized clots of fibrin. All these complexes manifested a slight anticoagulative activity.     

Genetic mechanisms of age regulation of protein C and blood coagulation.

Blood coagulation activity in humans increases with age. We previously identified two genetic elements, age-related stability element (ASE; GAGGAAG) and age-related increase element (AIE; unique stretch of dinucleotide repeats), which were responsible for age-related stable and increasing expression patterns, respectively, and together recapitulated normal age regulation of the human factor IX (hFIX) gene. Here we report the age-regulatory mechanisms of human anticoagulant protein C (hPC), which shows an age-stable pattern of circulatory levels. The murine protein C gene showed an age-related stable expression pattern in general agreement with that of the hPC. Through longitudinal analyses of transgenic mice carrying hPC minigenes, the hPC gene was found to have a functional age-related stability element (hPC ASE; CAGGAAG) in the 5'-upstream proximal region but was found to lack any age-related increase element. Three other ASE-like sequences present in the hPC gene, GAGGAAA and (G/C)AGGATG, also bound nuclear proteins but were not active in the age regulation of the hPC gene. Functional hPC ASE and hFIX ASE were apparently generated through convergent evolution, and hFIX ASE can fully substitute for the hPC ASE in conferring age-related stable expression pattern of the hPC gene. In the presence of the hPC ASE, hFIX AIE can convert the age-stable expression pattern of the hPC gene to a hFIX-like age-related increase pattern. These results support the universality of ASE and AIE functions across different genes. Clearance of hPC protein from the circulation was not significantly affected by age. We now have established the basic mechanisms responsible for the age-related increase of blood coagulation activity.     

Identification of extrinsic blood coagulation pathway inhibitors from the tick Ornithodoros savignyi (Acari: Argasidae)

The salt BaSO(4) selectively adsorbs two proteins from crude Ornithodoros savignyi salivary gland extract. They co-purify during reversed-phase HPLC, but can be separated by hydrophobic-interaction chromatography. Their molecular masses are 9333 and 9173Da. The 9.3kDa protein was designated BSAP1 and the 9.1kDa protein BSAP2. Their amino acid compositions show significant differences, in particular the presence of seven and eight cysteine residues in BSAP1 and BSAP2, respectively. The proteins do not contain gamma-carboxyglutamic acid, hydroxyproline, or hydroxylysine. The proteins do not inhibit the intrinsic coagulation cascade, but inhibit the extrinsic pathway. The observed inhibition is not due to inhibition of factor VII. Both proteins bind to membranes. BSAP1 binds neutral and negatively charged membranes more strongly than BSAP2. Its affinity for negative membranes is, however, much lower than for neutral membranes. In contrast, BSAP2 binds both membranes equally strongly. The binding of the proteins to the membranes was significantly lowered upon pre-incubation with Ca(2+).     

The endothelial protein C receptor supports tissue factor ternary coagulation initiation complex signaling through protease-activated receptors

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.     

RANKL induces components of the extrinsic coagulation pathway in osteoclasts

Prothrombin is converted to thrombin by factor Xa in the cell-associated prothrombinase complex. Prothrombin is present in calcified bone matrix and thrombin exerts effects on osteoblasts as well as on bone resorption by osteoclasts.We investigated whether (1) osteoclasts display factor Xa-dependent prothrombinase activity and (2) osteoclasts express critical regulatory components upstream of the prothrombinase complex.The osteoclast differentiation factor RANKL induced formation of multinucleated TRAP positive cells concomitant with induction of prothrombinase activity in cultures of RAW 264.7 cells and bone marrow osteoclast progenitors.Expression analysis of extrinsic coagulation factors revealed that RANKL enhanced protein levels of factor Xa as well as of coagulation factor III (tissue factor). Inhibition assays indicated that factor Xa and tissue factor were involved in the control of prothrombinase activity in RANKL-differentiated osteoclasts, presumably at two stages (1) conversion of prothrombin to thrombin and (2) conversion of factor X to factor Xa, respectively.Activation of the extrinsic coagulation pathway during osteoclast differentiation through induction of tissue factor and factor Xa by a RANKL-dependent pathway indicates a novel role for osteoclasts in converting prothrombin to thrombin.     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation.

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.