The role of membrane patch size and flow in regulating a proteolytic feedback threshold on a membrane: possible application in blood coagulation.

Positive feedback controls in proteolytic systems are characterized by thresholds which are regulated by the concentration of the initial stimulus and the kinetic parameters for enzyme generation and inhibition. Significant complexity is added when a positive feedback is localized on a membrane in contact with a flowing medium, such as seen in the early steps of blood coagulation. A partial differential equation model of an archetypal feedback loop is examined in which a proteolytic enzyme catalyzes its own formation from a zymogen on a membrane in contact with a flowing medium. As predicted from prior solution-phase and membrane-phase analyses, the threshold conditions for activation of the system are regulated by the kinetics of enzyme generation and inhibition and by the density of reactant-binding sites on the membrane; but the present analysis also establishes how the feedback threshold is controlled by the flow rate of the adjacent medium and the physical size of the membrane patch on which the feedback loop is localized. For given systems of particular kinetic properties, lower flow rates or larger active patches of membrane can result in the activation threshold being exceeded, whereas higher flow rates or smaller membrane patches can prevent initiation. In addition to numerical simulation, a simplified non-flowing model is analyzed to formulate an approximate mathematical statement of the dependence of the minimum activatable patch size on the kinetic and other parameters.

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.

Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including nonpathogenicity and potential for long-term transgene expression from either integrated or episomal forms. We have generated and characterized two B-domain-deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIdelta756-1679) or Thr761 to Asn1639 (fVIIIdelta761-1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD-fVIII of the predicted size in both lysates and supernatants (Mr approximately 155 kD for fVIIIdelta756-1679 and Mr approximately 160 kD for fVIIIdelta761-1639) after transient transfection into COS-1 cells. Functional fVIII quantification appeared maximal using fVIIIdelta761-1639, as evaluated by Coatest and clotting assay (98+/-20mU/ml/1x10(6) cells and 118+/-29 mU/ml/1x10(6) respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIdelta761-1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1-1) promoter, and demonstrated VIII activity approximately 30% of that seen using CMV promoter. This BDD-fVIII (rAAV(pHU1-1) fVIIIdelta761-1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS-1 cells using a maximally-achievable MOI approximately 35 (Coatest 15 mU/ml; clotting assay 25+/-20 mU/ml/1x10(6) cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.

The soluble recombinant form of a binding protein/receptor for the globular domain of C1q (gC1qR) enhances blood coagulation.

The gC1qR is a ubiquitously expressed, 33 kDa cellular protein which recognizes the globular domains of C1q. Recent evidence suggests that the gC1qR also serves as the Zn(++)-dependent endothelial cell binding site for factor XII and high-molecular-weight kininogen, and activates intrinsic coagulation and kinin pathways in purified systems. In addition, activated lymphocytes have been reported to release soluble gC1qR. Thus, the present study investigated the procoagulant potential of soluble gC1qR in human plasma using the recombinant protein (rgC1qR). rgC1qR supported a dose-dependent shortening of extrinsic coagulation using the prothrombin time in the presence of diluted (1/50-1/500) thromboplastin. Maximum enhancement of the prothrombin time resulted in shortening of the clotting time from 78.8 +/- 0.4 s to 68.5 +/- 0.6 s (mean +/- SD, n = 8) in the presence of 50 micrograms/ml (1.5 mumol/l) rgC1qR. rgC1qR also enhanced the intrinsic pathway of coagulation evaluated in the absence of activators of the contact system, as demonstrated by a shortening of the plasma recalcification time from 348 +/- 66 s to 140 +/- 23 s (n = 4). rgC1qR, however, had no effect on intrinsic coagulation in the presence of undiluted kaolin or ellagic acid, and under these conditions failed to shorten the activated partial thromboplastin time of factor VIII or factor-IX-deficient plasma. rgC1qR further failed to affect thrombin and factor Xa generation assayed using chromogenic substrates, and did not enhance thrombin-induced conversion of fibrinogen to fibrin. Interestingly, the procoagulant activity of the rgC1qR was measurable in either factor-XII- or factor-XI-deficient plasma, suggesting that it was not exclusively focused on the contact system of coagulation. Although the mechanism of action of gC1qR on blood coagulation remains obscure, the data suggest a potential role for this protein in hemostatic and thrombotic events.

Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation.

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37 degrees C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane.

Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases.

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

Initiation of the tissue factor pathway of coagulation in the presence of heparin: control by antithrombin III and tissue factor pathway inhibitor.

Activation of factor X by both the unactivated tissue factor:factor VII complex (TF:VII) and the activated tissue factor:factor VIIa complex (TF:VIIa) has been studied in the presence of tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and heparin. At near-plasma concentrations of TFPI, ATIII, and factor X, factor X activation that occurs in response to TF:VII is essentially abolished in the presence of heparin (0.5 micromol/L). This effect requires both inhibitors, acting on different targets: (1) ATIII, which in the presence of heparin blocks the activation of TF:VII, and (2) TFPI, which inhibits the TF:VIIa that is generated. In the absence of ATIII, TFPI alone with heparin reduces but does not abolish factor X activation. Conversely, in the absence of TFPI, ATIII + heparin reduces but does not abolish TF:VIIa generation and allows continuing activation of factor X. These results indicated that when the unactivated TF:VII complex is the initiating stimulus, heparin-dependent reduction in the rate and extent of factor X activation requires both ATIII and TFPI. In contrast, if TF:VIIa is used to initiate activation, only TFPI is involved in its regulation.

Mathematical analysis of activation thresholds in enzyme-catalyzed positive feedbacks: application to the feedbacks of blood coagulation.

A hierarchy of enzyme-catalyzed positive feedback loops is examined by mathematical and numerical analysis. Four systems are described, from the simplest, in which an enzyme catalyzes its own formation from an inactive precursor, to the most complex, in which two sequential feedback loops act in a cascade. In the latter we also examine the function of a long-range feedback, in which the final enzyme produced in the second loop activates the initial step in the first loop. When the enzymes generated are subject to inhibition or inactivation, all four systems exhibit threshold properties akin to excitable systems like neuron firing. For those that are amenable to mathematical analysis, expressions are derived that relate the excitation threshold to the kinetics of enzyme generation and inhibition and the initial conditions. For the most complex system, it was expedient to employ numerical simulation to demonstrate threshold behavior, and in this case long-range feedback was seen to have two distinct effects. At sufficiently high catalytic rates, this feedback is capable of exciting an otherwise subthreshold system. At lower catalytic rates, where the long-range feedback does not significantly affect the threshold, it nonetheless has a major effect in potentiating the response above the threshold. In particular, oscillatory behavior observed in simulations of sequential feedback loops is abolished when a long-range feedback is present.

Calcium stimulation of procoagulant activity in human erythrocytes. ATP dependence and the effects of modifiers of stimulation and recovery.

The human erythrocyte membrane is generally considered to have no procoagulant activity. The normal membrane is characterized as having an asymmetric distribution of phospholipid species such that negatively charged and aminophospholipids are predominantly located on the inner leaflet of the membrane bilayer. Elevation of cytoplasmic Ca2+ in erythrocytes produces an assortment of biochemical and structural responses that include diminished phospholipid asymmetry and an elevation in procoagulant activity. Maintenance of the normal asymmetric distribution of phospholipid species is believed to be largely mediated by a phospholipid translocase mechanism. We have utilized a recently developed single-step kinetic assay of procoagulant activity to investigate the mechanisms of Ca2+ stimulation of procoagulant activity and recovery from the procoagulant state upon removal of Ca2+. This study demonstrated that stimulation of procoagulant activity by elevated cytoplasmic Ca2+ is greatly diminished in ATP-depleted erythrocytes. Phospholipid translocase inhibitors failed to fully inhibit recovery from the procoagulant state after removal of Ca2+. The data indicate that recovery of endogenous lipid from a procoagulant cofiguration may not be entirely mediated by the phospholipid translocase. Additionally, the data are inconsistent with the phospholipid translocase mediating the Ca(2+)-induced elevation of procoagulant activity, although the involvement of other protein(s) is indicated.

Kinetics of the inhibition of factor Xa and the tissue factor-factor VIIa complex by the tissue factor pathway inhibitor in the presence and absence of heparin.

The inhibition by tissue factor pathway inhibitor (TFPI) of its two target enzymes--factor Xa and the tissue factor-factor VIIa complex (TF:VIIa)--has been studied under near-physiological reactant concentrations and conditions. Over a TFPI range of 0-1 nM, the rate of inhibition of factor Xa, in the presence of Ca2+ and anionic phospholipid vesicles at 37 degrees C, was proportional to TFPI concentration, giving an association rate, k1, of 0.96 x 10(9) M-1 min-1. Factor Xa inhibition did not proceed to completion, the reaction attaining a near-equilibrium that was dependent on the TFPI concentration. The estimated dissociation rate of the TFPI:Xa complex, k-1, was independent of TFPI concentration, with a mean value of 0.02 min-1. The resulting calculated value of K1, the apparent dissociation constant for the initial step, is 21 pM. Slow decay of the remaining factor Xa in such incubations, detectable after attainment of the rapid initial near-equilibrium, confirmed the two-step mechanism proposed by Huang et al. (1993) [J. Biol. Chem. 268, 26950-26955], but did not permit determination of a rate constant for the second step. Omission of anionic phospholipid had no significant effect on either k1 or k-1. A high-molecular-weight fraction of heparin, at saturating levels (> or = 0.05 unit/mL, congruent to 25 nM), increased k1 2-fold, with no detectable effect on k-1. The second stage of TFPI action was studied by preformation of the TFPI:Xa complex, and its incubation with the TF:VIIa complex in the presence of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)

Mathematical analysis of a proteolytic positive-feedback loop: dependence of lag time and enzyme yields on the initial conditions and kinetic parameters.

A model of a proteolytic positive-feedback loop, similar in general terms to feedback loops that occur in blood coagulation and other systems, has been examined by both explicit and numerical analysis. In this loop, modeled as a closed system, each enzyme (E1, E2) catalyzes the formation of the other from its respective zymogen (Z1, Z2), and both enzymes are subject to irreversible inhibition. The system shows three major characteristics. (1) No significant Z1 or Z2 activation occurs unless the combination of initial conditions and kinetic parameters is above a threshold level. This threshold occurs when the product of the enzyme generation rates equals the product of their inhibition rates. When the formation-rate product is less than the inhibition-rate product, there is no response: E1 and E2 generation is minimal and the lag time is effectively infinite. Conversely, when the generation-rate product exceeds the inhibition-rate product, explosive formation of both E1 and E2 is seen. For responses exceeding the threshold, the following obtain. (2) The lag time in E1 and E2 generation is a highly nonlinear function of the zymogen concentrations and the enzyme generation and inhibition rates. In contrast, there is a simple logarithmic relationship between the lag time and the initial trace concentration of the enzyme that is responsible for initiating the system; in this model, E1. (3) The extent of Z1 and Z2 activation is similarly a nonlinear function of the conditions and parameters but is independent of the initiating trace level of E1.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.

Interaction of feedback control and product inhibition in the activation of factor X by factors IXa and VIII.

A simple numerical model of the activation of factor X by factors IXa and VIII has been constructed in order to identify and examine the major controls that operate in a nonflowing system in the presence of (1) inhibitors of factor Xa and (2) feedback activation of factor VIII by factor Xa. The model confirms, and allows parameter estimation for, (1) the control of factor Xa yield by factor VIIIa decay; (2) the control of generation-curve area by the rate of factor Xa inhibition; and (3) the reduction in the factor VIIIa decay rate in the presence of factor IXa. Beyond confirmation of existing data, the model also predicts that below a definite, but very low, threshold level of factor IXa (less than or equal to 10 pM), minimal feedback activation of factor VIII will occur. The concentration of factor IXa at which the threshold is observed in simulations is dependent on the rate of inhibition of factor Xa.

Analysis of the generation and inhibition of factor Xa. Area under generation curves is independent of enzyme generation rate.

The activation of factor X in the presence of antithrombin has been studied in order to determine the parameters that control the area under the resulting factor Xa generation curve. Generation curves were analyzed using a model containing three parameters: the total generation of factor Xa, Emax; the rate of factor Xa generation, expressed as a first-order rate constant, kappa 1; and the rate of inhibition, expressed as another first-order rate constant, kappa 2. Using factor IXa-VIIIa to activate factor X, we found the area under the generation curve to be proportional to Emax, which was varied by varying the factor IXa concentration, and inversely proportional to kappa 2, which was varied by varying the antithrombin concentration. With this activator, however, kappa 1 varied in parallel with Emax, resulting in a correlation between integrated area and kappa 1. In order to determine whether Emax or kappa 1, or both, was a controlling parameter, similar activations were done with varying concentrations of the factor X-activating enzyme of Russell's viper venom. With this activator it was possible to vary Emax and kappa 1 independently, again at varying antithrombin concentrations. These results showed the integrated area to be proportional to Emax and inversely proportional to kappa 2, as before, but independent of the activation rate, kappa 1. In this system, therefore, the area under the factor Xa generation curve is controlled by the amount of factor Xa generated and its rate of inhibition but is independent of the rate of factor Xa generation.

The use of acetylated factor X to prevent feedback activation of factor VIII during factor X activation: a tool for kinetic studies.

The modification of human factor X by 2-sulfo-N-succinimidyl acetate was investigated and shown to produce a factor X species which, when activated, has no activity toward factor VIII. Acylation of factor X (0.9 microM) was carried out in the presence of 1 mM calcium at different reagent concentrations and pH values at 22 degrees C for time courses up to 1 h. Optimal modification was achieved using 0.3 mM reagent at pH 8.0 for 30 min. The modified zymogen, acetylated factor X, is activated at full rates by factor IXa/VIIIa and by the factor X-activating protein of Russell's viper venom. The activated product, acetylated Xa, has an enhanced amidolytic activity (110%) but has almost no detectable clotting activity (0.1%). More importantly, we have shown that acetylated Xa, in contrast to native Xa, does not activate factor VIII. This allows accurate quantitation of factor VIII activation without complications due to positive feedback reactions. We have demonstrated this in an examination of the activation of factor VIII by factor IXa.

A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X.

Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

Deficiency of plasma protein S, protein C, or antithrombin III and arterial thrombosis.

Protein C and protein S are vitamin K-dependent coagulation factors that together act as an anticoagulant, and antithrombin III is a plasma protein that inhibits several activated factors in the coagulation cascade. Although deficiencies of any of these three proteins have been associated with deficiencies of these factors. We report one patient with a protein S deficiency, another with a protein C deficiency, and a third with an antithrombin III deficiency, each of whom who had extensive arterial thrombosis. We suggest that deficiencies of these proteins may constitute risk factors for arterial thrombosis.